Cancer of The Uterine Cervix [304978]

Cancer of The Uterine Cervix”

Cytology, Histopathology, Clinical & Laboratory Aspects

Text & Colour Atlas

Dr. MM Kamal

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Cancer of The Uterine Cervix”

Cytology, Histopathology, Clinical & Laboratory Aspects

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A [anonimizat]. [anonimizat], Ph.D. (1883-1962). [anonimizat](Euboea). [JKB1] Dr. George Papanicolaou graduated in medicine from the University of Athens in 1904, acquired a doctorate in philosophy in natural sciences from the University of Munich and served in the Greek Army during the Balkan wars. Here, he came in contact with the Americans and with the hope of better prospects of launching a scientific career, the Papanicolaou couple shifted to New York 1913and began research on the oestrous cycles of mammals, using cellular samples from the vagina of guinea pigs. Later he extended his work to humans and inevitably, received vaginal smears from women suffering from cervical cancer. This is how he stumbled upon cancer cells. He was not searching for them –instead, he observed and recognized them serendipitously.

As is true for many important scientific observations, this diagnostic examination was poorly received initially. Papanicolaou’s contemporaries saw no reason to change from diagnostic biopsy of the relatively accessible cervix to the smear technique. It was not until Papanicolaou received the support of Joseph Hinsey, the Chairman of Anatomy at Cornell in 1939, that he laid aside his endocrinologic studies and began full-time pursuit of the smear method for cancer detection. As a result of Papanicolaou’s collaboration with Herbert F Traut, a pathologist with a special interest in gynecologic material, seminal publications in 1941 & 1943 launched the era of development and expansion of Cytopathology. This era saw the advent of screening for cervical cancer. In 1954, Papanicolaou published his magnum opus, the comprehensive Atlas of Exfoliative Cytology. The Pap test fulfilled the need for a simple and inexpensive method of examining large numbers of prospective cancer bearers that existed in all populations around the globe. Though formally retired from Cornell University in 1951, Papanicolaou’s activity continued undiminished until he died in 1962, at the age of 77 yrs.

Papanicolaou’s contribution to the field of cytopathology was two-fold: he recognized the importance of wet fixation and he began systematically to accumulate examples of cancer cells in vaginal smears. These sketches of normal and abnormal vaginal and cervical cells (Fig 1 ) have stood the test of time

Figure 1: Normal and abnormal cervicovaginal cells

and are as complete. They are both the foundation and the methodology for meaningful cytologic literature.

The woman behind this man was his wife Andromahi Mavrogeni who was a long time assistant in his laboratory. The fact that the Papanicolaou’s test has been modified so little during the greater part of its existence is surely a tribute to the insight and inventiveness of this scientist couple. Awards and accolades naturally followed as a tribute to their untiring efforts but the highest honor came in when the scientific community at large decided to name cervical cytology as ‘The Pap smear’.

Disciples of Papanicolaou’s smear examination assimilated his teachings and inaugurated their studies. Several future cytopathologists visited his laboratory and many remained for protracted periods, including Leopold Koss, George Wied, and James Reagan. These stalwarts have played a major role in developing cytopathology as a subspecialty of pathology and also in maintaining high standards in cytopathology by their educational and research activities. In 1983, the town of Kymi along with the Greek government and the University of Athens celebrated the 100th birth anniversary of Papanicolaou. The United States similarly honored its famous citizen in 1978 and 1984 Cyprus issued a stamp depicting both Drs. Mr. and Mrs. Papanicolaou. (Fig 2 )

Figure 2: United States postage stamp issued in 1980 honoring Dr.G. N. Papanicolaou and the Pap test

The revolutionary discovery of the “The Pap smear” in the early 20th century and its role in diagnosing & preventing cancer cervix, began to arouse worldwide medical attention. A massive cytology screening project using the Pap smear was initiated in 1956 in Jefferson County that lasted for 16 years. When the records of this screening program suggested that the death rate dropped from 23.7 to 10.2/lac, the WHO announced, in 1969, that cancer cervix was a preventable disease. Unfortunately, there is no significant change in death rates among the populations of the relatively unscreened women in the developing world.

“Preventable but yet not prevented” is the reality today !!! Despite its accomplishments, the Pap test is an endangered species. Will it go the way of the dinosaur, or can we prevent the extinction of this remarkable investigative device?

[JKB1]This needs to be checked, ma’am

References

1. Kline TS: The Papanicolaou Smear- A Brief Historical Perspective And Where We[JKB1] Are Today. Arch Pathol Lab Med- Vol 121, march 1997.205 -209[JKB2] .

2. Hinsey JC. George Nicholas Papanicolaou, May 13, 1883 – February 19, 1962. Acta Cytol. 1962;6:483–486.

3. Winfred[JKB3] Gray====

4. Siang Yong Tan, Yvonne Tatsumura. George Papanicolaou (1883–1962): Discoverer of the Pap smear. Singapore Med J 2015; 56(10): 586-587 doi: 10.11622/smedj.2015155

[JKB1]Couldnot find this reference

[JKB2]

[JKB3]Ill add this once the corrections are made.

Chapter 2II

Cancer Cervix – Epidemiology & Disease Burden

AUTHORS

Sharmila  PIMPLE

Professor, Department of Preventive Oncology, Tata Memorial Centre.

Gauravi  MISHRA

Assos.Professor, Department of Preventive Oncology, Tata Memorial Centre.

Introduction:

Cervical cancer is the fourth most common cancer affecting women worldwide, after breast, colorectal, and lung cancers,  with 528 000 new cases every year. It is also the fourth most common cause of cancer death (266 000 deaths in 2012) in women worldwide. Globally one in 70 women developed cervical cancer between birth and age 79 years.  [1,2]

Almost 70% of the global burden falls in areas with lower levels of development, and more than one-fifth of all new cases are diagnosed in India, the second-most populous country in the world.1,2 It is the second most commonly diagnosed cancer and the third leading cause of cancer death among females in less developed countries. [3] The Age Standardized Incidence Rates (ASIRs) and Age Standardized Death Rates (ASDRs ) per 100 000 in 2013 were higher in developing countries vs developed countries (ASIR, 15.70 vs 9.58; ASDR, 8.32 vs 3.96). [4]

Incidence rates are highest in sub-Saharan Africa, Latin America and the Caribbean, and Melanesia and lowest in Western Asia, Australia/New Zealand, and Northern America.[3]  In 2013, incidence rates per 100 000 were the lowest in Australasia  (ASIR, 6.83; ASDR, 2.65), North Africa and Middle East (ASIR, 7.23; ASDR, 3.19) and high-income North America (ASIR, 7.26; ASDR, 2.84), and the highest in Oceania (ASIR, 58.4; ASDR, 26.49), eastern sub-Saharan Africa (ASIR, 31.5; ASDR, 25.57), and western sub-Saharan Africa (ASIR, 30.2; ASDR, 22.3).[2]

The large geographic variation in cervical cancer rates can be explained by a lack of access to effective screening in public health care settings and further to services that facilitate early detection and treatment in less developed regions of the world. Non-existent or inadequate screening and limited access to the standard treatment options that are often unaffordable has led to a majority of cases being detected in the advanced stages of the disease in these regions.[5]

Time Trends In Cervical Cancer

In several western countries, where organized cytology-based screening programs have long been established, cervical cancer rates have decreased by as much as 65% over the past four decades. In Norway, cervical cancer incidence rates decreased from 18.7 per 100,000 in 1970 to 9.6 per 100,000 in 2011.  [6]

In Finland, cervical cancer incidence rates decreased from 21.1 in 1966 to 7.3 in 2007 [7]. Rates have also decreased in high-risk areas, including China, Taiwan, Korea, in part due to improved screening activities and socioeconomic conditions [8-12], although the decreases in proportionate terms were much smaller compared with those in western countries.

National Cancer Registry Programme (NCRP)  established under the Indian Council for Medical Research (ICMR)   represents the government-supported population-based national cancer registry  (PBCR) network across major cities and regions with in the country which collects the vital information to project the magnitude, trends and patterns of cancer in the country.

Cervix cancer incidence shows wide variations across India with Aizawl PBCR, North East India  (24.3) registering the highest Age-Adjusted Incidence Rate followed by Barshi,  Maharashtra (19.5) and Bangaluru (18.9). [13]

An ICMR led study (1990 -2003) and another linear Regression model study (1982-2003)  that evaluated trends in cervical cancer for cervix, showed a decreasing trend in the age-adjusted incidence rate for cervical cancer in all PBCR’s except rural PBCR of Barshi in western Maharashtra. The registry of Bangalore and Delhi showed more than 2% mean annual percentage (MAPC%) change, whereas for the rest of the registries a MAPC of 1-2% was observed for cervical cancers in the ICMR study. [14,15]

In the recent  Population-Based Cancer Registry (PBCR)  report on Time trends in Cancer Incidence rates for 1982-2010, India,  all the urban PBCR’s at Banglore( APC: -2.0%), Bhopal (-1.3%), Chennai (-3.5%), Delhi (-3.0%), Mumbai (-1.8%) including the rural population-based cancer registry of Barshi (-2.4%) reflected a negative  Annual Percentage Change(APC) for cervical cancer showing a statistically significant decrease in incidence rates of cervical cancer. 13 [16] . This could be reflective of the socio-economic development of the regions,  with improved access to genital hygiene since none of the registry areas had an organized cervical cancer screening programs in the region.

In contrast to the favourable trends reported above, cervical cancer rates have been increasing among younger generations in several countries, including Finland, the United Kingdom, Denmark, and China.[17]  This unfavorable trend is thought to reflect increases in HPV prevalence from changing sexual behaviors. 14-16 [17-19] The exceptionally low overall cervical cancer rates in the Middle East and other parts of the developing world are thought to reflect the low prevalence of HPV infections due to societal disapproval of extramarital sexual activity.  [20]

EPIDEMIOLOGY OF HUMAN PAPILOMA VIRUS IN  CERVICAL CANCER

HPV infection is a sexually transmitted disease. Infection with high-risk HPV infection  ( hrHPV) is now viewed as a necessary precondition for the development of all cervical cancer and precancerous intraepithelial lesions and is one of the most common sexually transmitted infections worldwide [21]

Persistent infection with about 15 hrHPV types is the major risk factor for cervical cancer, with HPV-16 and HPV-18 infections accounting for about 70% of the total cases. [22] In a  pooled analysis of 11 case-control studies, HPV types 16, 18, 45, 31, 33, 52, 58, and 35 accounted for 95 percent of the squamous-cell carcinomas positive for HPV DNA. [23]

Human papillomavirus 16 constitutes approximately more than one-half of cervical cancers. Concurrent multiple (type) infections are common and more than one-fourth of the HPV infections that occur constitutes multiple HPV types.[24] Progression of HPV infection to cervical cancer occurs through successive steps from HPV transmission, viral persistence, the progression of these persistently infected cells to precancer and invasion. [5]

RISK FACTORS FOR HPV INFECTION AND CARCINOGENESIS:

Several risk factors for contracting HPV infection and its further progress to cervical carcinogenesis has been established. High parity, long-term use of oral contraceptive pills, tobacco consumption,  co-infection with other sexually transmitted agents, lifestyle factors such as multiple sexual partners, younger age at first sexual intercourse, immunosuppression and diet have been identified as the co-factors most likely to influence the risk of acquisition and progression from cervical HPV infection to high-grade cervical precancers to invasive cancers.[25,26]

IARC pooled analysis suggests that genital HSV-2 infection may act in conjunction with HPV infection to modestly increase the risk of invasive cervical cancer the effect been likely to be mediated by the induction of inflammatory responses. Two-fold increased risk for the presence of antibodies to Chlamydia trachomatis (OR_/2.1; 95% CI, 1.1_/4.0) was also demonstrated  [27,28] High parity has been associated with cervical carcinogenesis in many case /control studies. In the IARC-pooled analysis for the role of parity, the risk increased linearly with an increasing number of full-term pregnancies. [29]

High parity is known to increase the risk of cervical cancer since parity and pregnancy-associated hormonal changes maintain the transformation zone on the ectocervix for longer durations facilitating the direct exposure to HPV and other cofactors. Pregnancy-induced hormonal changes are also likely to influence the immune response to HPV infection, persistence and progression. [29,30,31]

The strongest evidence for a role of oral contraception ( OC ) use in HPV carcinogenesis derives from the large pooled analysis of the International Agency for Research on Cancer (IARC) studies.  The study demonstrated a strong dose-response relationship with increasing years of use.[32] OC induced hormone-related mechanisms have been reported to affect the progression from premalignant to malignant cervical lesions most likely due to promoting the integration of HPV DNA into the host genome and thereby resulting in deregulation of E6 and E7 expression. [26,33]

The effects of tobacco smoking have been reported in many case-control studies. Consistent increased risk of cervical cancer with increasing exposure to tobacco smoking was established with intensity and duration of smoking. [26,34] Nicotine and tobacco-specific carcinogens have been detected in the cervical mucus of smokers pointing towards the likely effect of tobacco-related carcinogens exerting a direct mitogenic effect causing DNA damage and aiding the development of cervical precancer and cancer. [35,36]

Another prospective study has also shown evidence of the fact that smokers maintain cervical HPV infections for significantly longer durations and take a longer time to clear an oncogenic infection than women who never smoked.[37] Exposure to tobacco is hypothesized to compromise effective host local immune response against viral infections since tobacco smoking has been shown to reduce the number of Langerhans’ cells and other markers of immune function.[38]

Other possible factors include diet, in particular diets poor in fruits and vegetables.[39] An international expert panel and a recent systematic review have provided conclusive evidence that diets high in vegetables and fruits, carotenoids, vitamin C and vitamin E were possibly protective and that folate and retinol possibly had no relationship with invasive cervical cancer. [40]

Infection by HIV constitutes a risk factor for infection as well as for neoplastic progression, in particular during the immunosuppression periods. [25]  HIV-positive women have consistently been shown to be at increased risk of cervical premalignant lesions when compared with their HIV-negative counterparts,  the association is stronger for women with low CD4/T-lymphocyte count. [41] Women infected with both HIV and HPV were also found to be a much higher risk of premalignant cervical lesions than women infected with either of the two viruses separately. [42]    A meta-analysis investigating the association of genital HPV infection and HIV acquisition showed overall, individuals with genital HPV infection, irrespective of the oncogenic HPV type, had twice the risk of acquiring HIV (OR, 1.96; 95% CI, 1.55 to 2.49). [43]

Unpublished data from the IARC series of case-control studies showed that associations between various cofactors and cervical carcinogenesis are not modified by specific genotypes. [26]

HPV  viral cofactors such as the viral type, the persistence of the infection, the viral load per cellular unit, as well as the integration of the viral DNA into the cellular DNA are other major determinants of progression to cervical precancers and invasive cancers. Most cervical HPV infections though are transient and cleared by cell-mediated immunity within 1–2 years of exposure. [44]  The median time to clearance of HPV infections is 6–18 months.[45] There is no defined period for determining the persistence of the HPV infection however Infections lasting more than about 1 year to 2 years are likely to pose a greater risk for progression to premalignant and malignant cervical disease.[46]

The detailed natural history and pathogenesis will be discussed in the chapters ahead.

The burden of cervical HPV infection and type distribution

In a five continents meta-analysis among women with normal cytology findings, HPV infection prevalence (all types) varied widely, from as high as 21% in Africa and 16% in Latin America and the Caribbean to 9% in Asia and 5% in Northern America. [47]

Overall HPV prevalence in women with normal cervical cytology was estimated to be 10·4% (95% CI 10·2–10·7). 48 The five most common HPV types in HPV-positive women worldwide were HPV16, HPV18, HPV31, HPV58, and HPV52, representing 50% of all HPV infections. HPV16, HPV18, and HPV31  respectively were the three most common types in studies using MY09/11, GP5+6+, and other PCR primers. HPV16 was observed with a prevalence of 2·5% followed by 0·9% for HPV18. [48]

In all world regions, HPV prevalence was highest in women younger than 35 years of age, declining to a plateau in middle age. In Africa, the Americas, and Europe, a clear second peak of HPV prevalence was observed in women aged 45 years or older.[47,48]  At the ages of peak sexual activity, the prevalence of  HPV infections in the female population is estimated around  40%, with an annual infection rate of 10–15%. The prevalence decreases after the age of 30 years to around 5–10%. The infections by HPV 16 are those that present the most prolonged longevities with average persistence values of 16 months in some studies. [49]

World HPV prevalence for most common  five top hrHPV types among  women with normal cytology  findings worldwide were reported to be    HPV 16: 55.4 (95% CI; 55.0-55.8),   HPV 18: 14.6 (95% CI; 14.3-14.9) , HPV  45: 4.8 (95% CI; 4.6-5.0) , HPV 33: 4.2 (95% CI; 4.1-4.4)   HPV 58: 3.8 (95% CI; 3.7-4.0) and HPV 31: 3.5 (95% CI; 3.4-3.7)   respectively. [50]

Based on India studies performing HPV detection tests among normal women, about 5.0% of women in the general population are estimated to harbor cervical hrHPV infection at a given time and 82.7% of invasive cervical cancers are attributed to HPVs 16 or 18. Prevalence (%) of HPV 16 and/or HPV 18 among women with Low-grade cervical lesions (LSIL/CIN-1)was  28.2% and, with High-grade cervical lesions (HSIL/CIN-2/CIN-3/CIS) was 62.8%.[49]

Epidemiological studies with  HPV typing have estimated HPV-16/18 to account for 70% of all cervical cancers worldwide. The  HPV-16/18 fraction is reported to be slightly higher in more developed (72–77%) than in less developed (65–72%) regions. [51]

The reported HPV-16/18 fraction in various squamous intraepithelial lesions is about 41–67% in the high-grade squamous intraepithelial lesion (HSIL), 16–32% in the low-grade squamous intraepithelial lesion (LSIL) and 6–27% in atypical squamous cells of undetermined significance (ASCUS). After HPV-16/18, the six most common HPV types were found to be the same in all world regions, namely 31, 33, 35, 45, 52 and 58 and these account for an additional 20% of cervical cancers worldwide.[51]

HPV16 and HPV18 were consistently the two most common types identified in each decade and showed a stable adjusted-Relative Contribution(RC)  from 1940 to 2007. The RC of other HPV types too did not show variation over time.[52]

References

1. Incidence/mortality data: Ferlay J, Soerjomataram I, Ervik M, Dikshit R, Eser S, Mathers C, Rebelo M, Parkin DM, Forman D, Bray, F (2013). GLOBOCAN 2012 v1.0, Cancer Incidence and Mortality Worldwide: IARC CancerBase No. 11 [Internet]. Lyon, France: International Agency for Research on Cancer. Available from http://globocan.iarc.fr.

Prevalence data: Bray F, Ren JS, Masuyer E, Ferlay J (2013). Global estimates of cancer prevalence for 27 sites in the adult population in 2008. Int J Cancer, 132(5):1133–1145. http://dx.doi.org/10.1002/ijc.27711 PMID:22752881)

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M Schiffman, PE Castle, J Jeronimo, AC Rodriguez, Sholom Wacholder. Human papillomavirus and cervical cancer. Lancet 2007; 370: 890–907)

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Ahmedin Jemal, Melissa M. Center, Carol DeSantis, et al. Global Patterns of Cancer Incidence and Mortality Rates and Trends Cancer Epidemiol Biomarkers Prev 2010;19:1893-1907. Published OnlineFirst July 20, 2010.

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Gustafsson L, Ponten J, Bergstrom R, Adami HO. International incidence rates of invasive cervical cancer before cytological screening. Int J Cancer 1997;71:159–65

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Moreno V, Bosch FX, Munoz N, Meijer CJ, Shah KV, Walboomers JM, et al. Effect of oral contraceptives on risk of cervical cancer in women with human papillomavirus infection: the IARC multicentric case-control study. Lancet 2002;359:1085–92.

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Giulian AR, Sedjo RL, Roe DJ, Harri R, Baldwi S, Papenfuss MR, et al.Clearance of oncogenic human papillomavirus (HPV) infection: effect of smoking (United States). Cancer Causes Control 2002;13:839–46.

Poppe WA, Ide PS, Drijkoningen MP, Lauweryns JM, Van Assche FA. Tobacco smoking impairs the local immunosurveillance in the uterine cervix. An immunohistochemical study. Gynecol Obstet Invest 1995;39:

Garcia-Closas R, Castellsague X, Bosch X, Gonzalez CA. The role of diet and nutrition in cervical carcinogenesis: A review of recent evidence. Int J Cancer 2005.

World Cancer Research Fund. Food, nutrition and the prevention of cancer. A global perspective. Washington, DC: American Institute for Cancer Research, 1997. 301–8

Xavier Castellsague, F. Xavier Bosch, Nubia Munoz. Environmental co-factors in HPV carcinogenesis. Virus Research 2002; 89: 191-/199.

La Ruche, G., You, B., Mensah-Ado, I., Bergeron, C., Montcho, C., Ramon, R., et al., Human papillomavirus and human immunodeficiency virus infections: relation with cervical dysplasia_/neoplasia in African women.  Int. J. Cancer 1998;76:480-/486.

Pascale Lissouba, Philippe Van de Perre, Bertran Auvert. Association of genital human papillomavirus infection with HIV acquisition: a systematic review and meta-analysis. Sex Transm Infect 2013;89:350–356.

Stanley M. Immune responses to human papillomavirus. Vaccine 2006; 24 (suppl 1): S16–22.

Plummer M, Schiff man M, Castle PE, Maucort-Boulch M, Wheeler CM. A 2-year prospective study of HPV persistence among women with ASCUS or LSIL cytology. J Infect Dis 2007;195: 1582–89.

Bulkmans NW, Berkhof J, Bulk S, et al. High-risk HPV type-specific clearance rates in cervical screening. Br J Cancer 2007; 96: 1419–24.

Bruni L, Diaz M, Castellsague X, Ferrer E, Bosch FX, de Sanjose S. Cervical human papillomavirus prevalence in 5 continents: a meta-analysis of 1 million women with normal cytological findings. J Infect Dis.2010;202:1789-1799.

Silvia de Sanjosé, Mireia Diaz, Xavier Castellsagué, Gary Cliff ord, Laia Bruni, Nubia Muñoz, F Xavier Bosch. Worldwide prevalence and genotype distribution of cervical human papillomavirus DNA in women with normal cytology: a meta-analysis. Lancet Infect Dis 2007; 7:453–59

Woodman CB, Collins S, Winter H, Bailey A, Ellis J, Prior P, et al. Natural history of cervical human papillomavirus infection in young women: a longitudinal cohort study. Lancet 2001;357:1831–6.

Bruni L, Barrionuevo-Rosas L, Albero G, Aldea M, Serrano B, Valencia S, Brotons M, Mena M, Cosano R, Muñoz J, Bosch FX, de Sanjosé S, Castellsagué X. ICO Information Centre on HPV and Cancer (HPV Information Centre). Human Papillomavirus and Related Diseases in the World. Summary Report 2015- 04-08. [19th October 2015]

Gary Clifford, Silvia Franceschi, Mireia Diaz, Nubia Munoz, Luisa Lina Villa.HPV type-distribution in women with and without cervical neoplastic diseases. Vaccine 2006;24: S3/26-34.

Laia Alemany, Silvia de Sanjos, Sara Tous, Wim Quint, Carlos Vallejos, Hai-Rim Shin, et al. Time trends of human papillomavirus types in invasive cervical cancer, from 1940 to 2007 Int. J. Cancer 2014;135:88–95.

Natural History and Pathogenesis of Genital HPV Infection

Contents:

Introduction

Historical Aspects

HPV And Human Disease

HPV Virology

Genomic Organization Of HPV

Natural History of Genital HPV Infection

Pathogenesis Of HPV Infection

Host Immune Factors

Other Factors

Abbreviations:

HPV – Human Papilloma Virus, ORF – Open Reading Frame, LSIL- Low-Grade Squamous Intraepithelial Lesion, HSIL – High-Grade Squamous Intraepithelial Lesion

Introduction

Invasive squamous cell carcinoma of the cervix is still the commonest malignant tumor of the female genital tract in most countries and the most frequent neoplasm among women in many of them. [1]. Widespread use of cervical cytological screening programs has resulted in a dramatic decrease in the incidence rates of cervical cancer in countries like the United States. No other form of cancer, better documents, the remarkable effects of screening, early diagnosis and curative therapy on mortality rates than does cervical cancer. The precancerous lesions of the cervix have received a great deal of attention due to the easy accessibility of cervix to visual inspection, and tissue sampling for morphological as well as molecular diagnostics and therapeutic procedures. The role of Human Papilloma Virus as a causative agent in cervical precancerous lesions and cancer and it has been firmly established both epidemiologically and biologically[2]. Although HPV infection is essential, it is not sufficient to develop cancer, and many other factors such as multiparity, host immune response, hormonal factors, cigarette smoking also have some definite contribution. Epidemiological studies performed over the past three decades have proved that invasive cervical cancer is a multistep process involving a precursor pre-invasive stage that is closely linked with HPV infection.[2][3]

Historical Aspects

An Austrian Gynaecologist, Schauenstein was the first one to mention the histological similarity between the non-invasive and the invasive cervical cancer (1908), but the term precancerous lesions of the cervix were suggested first by Schottlander and Kermauner in 1912. Schiller, in 1920 mentioned the detailed analysis of histological patterns of various preinvasive and invasive cervical cancer forms. Robert Meyer and Hinselmann worked separately for early diagnosis of cervical cancer.

The credit of introducing a device for magnificent inspection of cervix i.e., colposcope goes to Hinselmann.[4] In 1941, an innovative smear technique for vaginal cytology (later known as the "Pap smear") was introduced. J. Ernst Ayre, while working in Florida, (1951) first described and illustrated squamous epithelial cells with a perinuclear "halo" in the smears of the uterine cervix. He described them as halo cells. He believed that some long-standing inflammation might be responsible for these peculiar changes. The term "koilocytic atypia" was introduced by Koss and Durfee for abnormal squamous cells in patients with cervical cancer even before the association between HPV and cervical cancer was known.

The first evidence of the link between HPV and cervical cancer was the electron microscopic demonstration of viral particles in genital condylomata acuminata. Zur Hausen and Munoz were awarded a Nobel prize for their dedicated extensive work on HPV. The last two decades have seen a sudden outburst of studies and publications related to the association between HPV and cervical cancer. Various immunohistochemical techniques, Southern blot analysis, in situ hybridization and PCR amplification techniques proved this association which is now a well-established fact today.[5]

Human Papilloma Virus and Human disease

HPV is associated with a variety of clinical conditions that range from innocuous lesions to cancer. HPV is implicated not only in the causation of cervical cancer, but more than 10 % of all human cancers are said to be associated with it. Till today more than 150 different types of HPV have been described and new types are regularly added to the list. HPV is epitheliotropic and site-specific. It is broadly classified into two types, cutaneous and mucosal or genital. HPV sometimes referred to as the "wart virus" has been recognized for many years as the cause of ordinary skin warts and condylomas. Skin warts although very common, are benign, self-limiting and in general resolve spontaneously within 1 to 5 years. They are transmitted by direct contact with infected tissue or indirectly by contact with virus-contaminated objects. Common sites for these papillomas and warts are oral cavity, respiratory mucosa specially larynx, trunk and sometimes conjunctiva [6]. Anogenital warts are most common amongst sexually active individuals. Anogenital cancers are the most important diseases associated with HPV infection. More than 90% of invasive cervical cancers are also associated with Human Papilloma Virus Infection.

Table 1 enlists various HPV types and their clinical manifestations.

HPV   virology

Human Papillomavirus is a small circular double-stranded DNA virus of the papovaviridae family and containing approximately containing 7900 base pairs. Mature viral particles are icosahedral (containing 20 faces) and can be visualized with electron microscopy. HPV genome reveals a well-conserved general organization. The viral genome is functionally divided into two regions, 1. The noncoding upstream regulatory region (URR) and 2. The coding region is represented as an open reading frame or ORF.

The noncoding region does not code for proteins but contains elements required for regulation of gene expression, DNA replication and its packaging into virus particles.[6,7]  The coding region or the ORF can be divided into an early and late region based on the specific schedule in the course of productive infection. The so-called early genes are expressed shortly after infection and before the onset of DNA replication. Products of these genes’ mediate functions like replication and expression of viral DNA and transformation of the host cell. There is a total of seven early genes from E1 to E7. They are numbered according to their size; the higher the number, the smaller the corresponding ORF. Expression of the early gene products determines whether the HPV infection is active or latent or may lead to malignant transformation. The late genes code for structural proteins of viral particles and are activated during the final stages of the viral cycle.[8] The L1 gene codes for the major capsid protein and L2 gene codes for the minor capsid protein.

Table 2:  HPV and functions of genes.[9]

a.

b.

Figure 1. Schematic representation of the genomic organization of HPV and b.  the Open Reading Frame [7]

Natural History of Genital HPV infection:

Genital HPV infection is a venereally transmitted infection with a lifetime risk of about 50 to 80 % in women. Most of the infections (75%) clear spontaneously within 12 to 36 months with an effective immune response. About 40 different genotypes of HPV are known to cause the genital HPV infection. They are further divided into low risk and high-risk types depending on their propensity to progress to precursor lesions for cervical cancer. Low risk types are associated with low grade lesions such as cervical condylomas and CIN I or LSIL (low grade squamous intraepithelial lesion). Although 15 genotypes of high-risk HPV are known, type 16 & 18 are the most common. HPV though essential, is not enough to cause cervical cancer and different cofactors such as tobacco, multiparity, local host immune response, presence of other sexually transmitted diseases and hormonal factors have also proved their definite role. HPV virus particles are perfectly adapted to their natural host tissue, the differentiating epithelial cells of the skin or mucosa and exploit the cellular machinery for their purposes. It cannot infect or enter the mature superficial squamous epithelial cells that line the ectocervix, vulva, and vagina. Establishing infection at this site requires cuts, breaks, and micro-abrasions to gain the virus access to the basal layer of the epithelium via interaction with receptors like alpha 6 integrins. The cervix with its relatively large area of immature squamous epithelium in the transformation zone is particularly vulnerable to HPV infection as it lacks the typical micro ridges present on mature squamous epithelial cells. Normally the transitional zone coincides with the external Os, but in pregnancy, at puberty, and in some contraceptive users, the position of Os and transformation zone may not coincide. This exposes the tissues in the endocervical canal to the vagina. This undergoes metaplasia as a protective response, and it is during this metaplastic process the epithelium is most vulnerable to viral entry. [10] HPVs are undergoing a complete life cycle only in fully differentiated squamous epithelium. The existence of the viral genome in the infected cell is central to the life cycle of papillomaviruses and their associated pathologies.  After entry into the basal cell, further steps in the viral life cycle depend on whether it is a low risk type of virus or high risk type.

In low risk group, the virus remains circular not attached to chromosomes, termed as episomal, whereas in high risk group the viral DNA gets incorporated into the host cell DNA hence known as integrated. Both forms can multiply in the nuclei of the host cell.

Pathogenesis in Low-Risk Type HPV

This group of viruses establishes their small double-stranded DNA genome as a circular extra-chromosomal element or episome in the nucleus infected cells. (Fig 2) two types of infection can occur. 1. Latent infection and 2. Productive infection.

Fig2. (11)

In normal squamous epithelium, cells in the basal layers are dividing as stem cells or transit-amplifying cells. After division, one of the daughter cells migrates upwards and begins to undergo terminal differentiation, while the other remains in the basal layer as a slow-cycling self-renewing population. Following the entry of HPV into the suprabasal layer through micro-abrasions, the viral genome replicates automatically with each division of the cell. The Papillomavirus DNA replication is dependent upon the DNA synthesis machinery of the host cell. The factors necessary for viral replication are – a) the viral DNA sequence to be multiplied, b) DNA polymerase enzyme and c) replication factors. The viral DNA sequence is provided by the early genes of HPV. The replication factors and the DNA polymerase enzyme are available in the nucleus of the dividing basal cell. Viral copies are formed automatically without disturbing the host cell DNA when the cell divides. Thus, the viral multiplication is seen in low copy number. This infection is called as a latent infection. In this type, no tissue changes are seen on light microscopy.  [12]                                                                                                                                                             Another type of infection is the productive or permissive type of infection. In this, the epithelial cells containing a low copy number of virus particles differentiate and move upwards in the epithelium.  The problem for the virus is that the necessary cellular DNA polymerase and replication factors are only available in dividing cells; however, the virus replicates in nondividing cells. To solve this problem, HPV encodes proteins that in the context of the viral life cycle, reactivate cellular DNA synthesis in non-cycling cells, inhibit apoptosis and delay the differentiation program of the infected keratinocyte, creating an environment permissive for viral DNA replication. [13] Now the virus can produce large copies of viral DNA. As the cell further moves upwards, late genes are expressed that code for structural proteins making capsid. The viral DNA is packaged with the capsid and they are released automatically when the superficial cells are shed off. These cells containing a large number of viral particles are nothing but koilocytes. The perinuclear zone in the koilocytes is the storage space for the virus; hence they are increasingly seen over the superficial layers. This is a productive viral infection seen mostly with type 6 and 11 and clinically appearing condylomas and warts due to the proliferation of maturing cells. This infection automatically regresses within 1 to 2 years and has no malignant potential.

fig. 3: The HPV Life Cycle [14]

Mechanism in High Risk Viruses

High risk HPV types can be distinguished from low risk HPV types by the structure and function of the E6 and E7 products. In infection with a high risk type of viruses (16,18 etc.), viral DNA is integrated into the host genome. Viral DNA integration into the host genome is found in all cases of cervical carcinoma and their metastasis., this viral DNA integration is in itself a mutation with consequences both on the viral and cellular genome.  Integration of viral genome disrupts or deletes the E2 region resulting in loss of its expression; hence interfering its function of down-regulation of transcription of E6 and E7. This leads to increased expression of E6 and E7 genes.  The products of E6, E7 genes deregulate the host cell growth cycle by binding and inactivating two important tumor suppressor genes p53 and Rb.  The E6 gene product selectively binds p53 and causes its degradation;  as a consequence, the normal activities of p53 which govern cell cycle arrest in G1, apoptosis and DNA repair are impaired. This allows the accumulation of additional mutations. Whereas the E7 gene product binds to Rb which disrupts the complex between Rb and E2F-1 and makes the factor E2F1 available for transcription of proteins required for entry of the cell into S phase of the cell cycle. The outcome is the stimulation of cellular DNA synthesis and cell proliferation. Next, the E5 gene products induce an increase in mitogen-activated protein kinase activity, thereby enhancing cellular responses to growth and differentiation factors. This results in continuous proliferation and delayed differentiation of the host cell. [15,16]

Fig. 4 – Mechanism in High Risk Viruses [17]

Cellular Transformation:

The inactivation of p53 and Rb gene proteins can give rise to an increased proliferation rate and genomic instability. Therefore, the host cell makes more and more damaged DNA that cannot be repaired, leading to transformed cancerous cells.   In addition to the effects of activated oncogenes and chromosome instability, potential mechanisms contributing to transformation include methylation of viral and cellular DNA, telomerase activation, hormonal and genital factors. [18] It is plausible that high risk HPV infection occurs early in life, may persist and in association with other factors promoting cell transformation, may lead to a gradual progression to more severe disease.  Accumulation of mutations is essential for progression towards malignancy and invasion.

Genetic Predisposition:

Genetic predisposition has a great contribution in cervical cancer. Heritability could affect many factors contributing to the development of cervical cancer including susceptibility to HPV infection, the ability to clear HPV infection and the time required for the development of the disease. Certain haplotypes such as DOA1, DQB1 are commonly seen in cervical cancer patients; which may be associated with the less effective triggering of an immune response against HPV infected cells.  Thus, HLA haplotypes is an important factor in the initial stage as it influences the point between reversion and progression of HPV induced lesion. [19.20]

Hormonal Factors:

The role of estrogen in cervical carcinogenesis is significant. There is definite evidence of increased cancer risk in women who are long-term users of oral contraceptives containing synthetic estrogen. High parity is also suggested to increase the risk of squamous cell carcinoma of the cervix among HPV-positive women, with repeated exposure to elevated levels of estrogen during pregnancy.  Estrogen is physiologically implicated in the eversion of the columnar epithelium onto the ectocervix as well as the formation of the transformation zone.  It is known that the cervical transformation zone featuring squamous metaplasia beside the squamo-columnar junction is highly susceptible to HPV infection and gives rise to most cervical cancers. Aromatase, an enzyme responsible for the conversion of androgen to estrogens is overexpressed in cervical cancer patients. The tumour suppressor gene BRCA1 is reported to repress the estrogen-induced transcriptional activity of the estrogen receptor. Thus estrogen, aromatase, and estrogen-responsive genes may be potential risk factors for the initiation and further development of cervical cancer.

Host Immune Response

Host immune response plays an important role in the regression or reversion of the lesions. The major line of defense against the virus is cell-mediated Immunity. therefore, conditions associated with impaired CMI such as transplant recipients, HIV disease show a high risk of acquisition and progression of HPV infection. HPV infections with high-risk viral types, the persistence of HPV infection and the presence of squamous intraepithelial lesions are more common in immunocompromised patients than in immuno-competent women.  On entry of the virus through cuts in the epithelium, the antigen-presenting cells under the epithelium catch the antigen and cause stimulation of cytotoxic, memory and helper T cells. In the majority of cases of genital HPV infection, the HPV is cleared by effective CMI and complete remission is achieved. The time for clearance ranges from months to years depending on the immune response.  Poor genital hygiene impairs natural mucosal barriers hence may result in persistent or progressive infection. HPV has developed several mechanisms for evading immune surveillance. The majority of these mechanisms are related to evading innate immunity and delaying the adaptive immune response. Other factors are the characteristics of the viral site of infection and the effects of viral oncoproteins. The most important factor that favour the virus to escape from these immune mechanisms is the absence of a lytic phase hence no cell injury hence no inflammation. There is no viremia during HPV infection. The immune system recognizes the virus only in the presence of capsid which is formed in superficial layers of the epithelium where they escape from APC. The release of viral proteins causes inhibition of interferon thus further favouring the viral progression.

Sexual Factors:

The risk of contracting genital HPV infection and cervical cancer is influenced by sexual activity. multiple sexual partners, multiparity and onset of sexual activity at an early age have an increased risk of HPV infection.  Additional association with other sexually transmitted diseases increases the chances of HPV infection.  Coinfections with other viruses such as Herpes Simplex type II, Cytomegalovirus also may play a role in the initiation of cervical cancer.

Viral Factors:

The type of HPV variant and the viral load directly correlates with the severity of the disease. HPV variants differ in biological and chemical properties and pathogenicity. The oncogenicity of specific HPV variants appears to vary geographically and with the ethnic origin. Infections with multiple variants of viruses can also occur. with multiple infections, at least one is of high risk type [21]. Although previous studies do mention about the association of Herpes Simplex Virus Infection in cervical cancer, it is not proved by molecular and advanced diagnostic techniques [22].

Other Factors:

Cervical cancer risk also seems to be independently influenced by other variables including cigarette smoking and alcohol consumption and diet. Local immune suppression induced by smoking and the mutagenic activity of cigarette components may contribute to the persistence of HPV or to malignant transformation like that seen in the lung.

References

Rosai J. Rosai and Ackerman’s Surgical Pathology. 10th edition Missouri: Elsevier; 2011(page numbers)

2. Arends MJ, Buckley CH and Wells M, Etiology, pathogenesis and pathology of cervical neoplasia. J Clin Pathol 1998; 51: 96-103

3. Ambros RA, Kurman RJ, Current concepts in the relationship of Human Papillomavirus Infection to the Pathogenesis and classification of precancerous lesions of the uterine cervix, Semin Diagn Pathol 1990 Aug; 7(3): 158-72

4. Epidermoid carcinoma of the uterine cervix and the related precancerous lesions; in Koss' Diagnostic Cytology and Its Histopathologic Bases, 5th Edition by Editors: Koss, Leopold G.; Melamed, Myron R. Copyright ©2006 Lippincott Williams & Wilkins

5. Hajdu SI, The link between koilocytes and human papillomaviruses, Ann Clin Lab Sci      Autumn 2006; 36: 4: 485-487

6. Gomez DT, Santos JL; Human Papillomavirus infection and Cervical Cancer: Pathogenesis and Epidemiology; in Communicating Current Research and Educational Topics and Trends in Applied Microbiology, 2007, 680-688*

7. Zekani J, Sirotkovic M, Skerlev M; Oncogenic aspects of HPV Infections of the Female Genital Tract.www.researchgate.net/publication/221914064

8. Xinrong Hu, Molecular Pathogenesis of Cervical Carcinoma, Analysis of clonality, HPV sequence Variations and Loss of Heterozygosity 2001 Dissertation for the Degree of Doctor of Philosophy (Faculty of Medicine) in Pathology presented at Uppsala University in 2001 )

9.  Graham SV Human Papillomavirus: Gene Expression, Regulation and Prospects for Novel Diagnostic Methods and Antiviral Therapies; Future Microbiol. 2010;5 (10):1493-1506.

10. Arends MJ, Buckley CH, Wells M Etiology, Pathogenesis and Pathology of Cervical Neoplasia J Clin Pathol 1998; 51: 96-103

11.Rebecca V, Chapter 7- Biology with Brendle at Guilford College WWW.studyblue.com

12. Narisawa SM, Ki yono T; Basic mechanisms of high-risk HPV induced carcinogenesis; Virology Division, National Cancer Centre Research Institute 2007

13.Münger K, Howley PM; Human Papillomavirus immortalization and transformation functions  Virus Res. 2002 Nov;89(2):213-28.

14. Castillo A, HPV infection and carcinogenesis in the upper aero-digestive tract Colomb. Med. v.42 n.2 Cali abr./jun. 2011

15. Yashinouchi M, Hongo K, Nakamura J, Kodoma S, Itoh H, Sakai H, et al; Jour clinical microbio 37; 3514; 1999

16. Sarijanen SM, Sarijanen KJ, New Concepts on the Role of Human Papillomavirus in Cell Cycle Regulation; Annals of Medicine 31, 175; 1999

17.  M. El Mzibri, M. Attaleb, R. Ameziane El Hassani, M. Khyatti, L. Benbacer, M. M. Ennaji Chapter 12; Evaluation of p53, p16INK4a and E-Cadherin Status as Biomarkers for Cervical Cancer Diagnosis

18. Park TW1, Fujiwara H, Wright TC,  Molecular biology of cervical cancer and its precursors.Cancer. 1995 Nov 15;76(10 Suppl):1902-13.

19. Lazo PA, The molecular genetics of cervical carcinoma; British Journal of cancer 80;2; 1999; 2008-2018

20. Magnusson PKE; Paul Lichtenstein P, and Gyllensten UB Heritability of cervical tumors International J. Cancer 88:698–701, 2000.

21. Wiley J and sons: HPV-induced Oncogenesis Rev. Med. Virol. 2009; pgs. 19-113

22.  Vopr Virusol.Posevaia TA, Shubladze AK; Etiological role of the herpes simplex virus in causing cervical cancer].1979 Nov-Dec; (6):611-5.

Normal Cervical Cytology

The Bethesda System for Reporting Cervical Cytology

Introduction

George Papanicolaou introduced cervical cytology to the world.  His landmark publication in collaboration with H. F. Traut ‘Diagnosis of Uterine Cancer by the Vaginal Smear’ in 1943  paved the way to diagnose Uterine cervical lesions with the help of a simple and effective method. [1]

Then In 1951, J. Ernest Ayre first described and illustrated squamous epithelial cells with a perinuclear “halo” in smears of the uterine cervix.[ 2]

Then  Richart in 1966 introduced the CIN (Cervical Intraepithelial Neoplasia) classification with CIN I, CIN II and CIN III based on the tissue architecture.[3]

As the knowledge of cervical carcinogenesis improved, it became imperative to unify the different terminologies and to effectively communicate with the clinicians to optimize the management of patients.  Thus a meeting was convened with experts from all over the world at the National Institutes of Health in Bethesda, Maryland, the USA  in 1988.  As an outcome of this workshop, the TBS reporting for cervical cytology was introduced in 1991 to provide a uniform system of terminology for reporting with clear guidelines for the management of these lesions. [4]

The first edition of TBS introduced the term SIL- Squamous Intraepithelial Lesion –as a potential precancerous lesion, which is characterized by a high spontaneous regression rate and the lack of predictable progression of SIL to invasive cancer. The Bethesda System replaced 3 levels of CIN ( Cervical Intraepithelial Neoplasia )   with 2 levels, LSIL ( Low Grade Squamous Intraepithelial Lesion ) and HSIL ( High Grade Squamous Intraepithelial Lesion ), which could be used to describe any squamous abnormality of the lower genital tract.  [4]

The evolution of scientific knowledge, technological advances and corresponding changes in the clinical management of Cervical Cancer and its precursor lesions have entailed revisions in the Bethesda system over some time. Two workshops were conducted in 1991 and 2001 respectively and two editions of the Bethesda Atlas were published in 1994 and 2004 as an outcome of these workshops.

The Bethesda system has helped in the standardization of terminology for Pap smears, has initiated significant research in the biology and cost-effective management for human papillomavirus-associated anogenital lesions, and, also led to a uniform worldwide recommendation of clinical management for these lesions. [5]

The latest revision has been done in 2014 with the help of an appointed task force. This group of renowned cytopathologists conducted an internet-based open comments survey with participation from the international cytopathologist community. The feedback was compiled, reviewed and resulted in the 2014 edition of TBS and its accompanying Atlas.

Throughout this period, three guiding  principles have been followed by The Bethesda System :

a. The classification terminology of the Cervical Smear report should be uniform and reproducible in different laboratories across the world but at the same time be flexible to suit the local population needs.

b. The Cervical smear report should give clinically appropriate and relevant information to the treating clinician.

c. The terminology used in the report  must be updated periodically to reflect the  current understanding of Cervical cancer[6]

The 2014 Bethesda System for Reporting Cervical Cytology[7]

The system has 5 components of a Pap smear report- Specimen Type, Adequacy, General category, Interpretation, Adjunctive testing. Two additional components may be added where applicable- Computer-assisted interpretation of Pap smear and Educational Notes and Comments appended to the cytology report.

1. Specimen Type

Conventional smear ( Pap smear ) /  Liquid-based preparation / other

Specimen Adequacy

Satisfactory for evaluation ( describe presence/absence of endocervical / transformation zone components and any other quality indicators- partially obscuring blood, inflammation, etc. )

Unsatisfactory for evaluation …… ( specify the reason )

Specimens rejected / not processed (specify reason )

Specimens processed and examined, but unsatisfactory for evaluation of epithelial abnormality because of ( specify reason )

General categorization ( optional )

Negative for Intraepithelial lesion or malignancy

Other: Refer  Interpretation/result ( explained later )

Epithelial cell abnormality:  Refer Interpretation/result.  Specify squamous/glandular as appropriate ( explained later )

Interpretation / Result

Negative for Intraepithelial lesion or malignancy- ( NILM ) when there is no cellular evidence of Neoplasia, this term should be stated in the General categorization section or Interpretation / Result section of the report- -whether or not there are organisms or other non-neoplastic findings.

Non Neoplastic Findings- ( optional to report )

Non neoplastic cellular variations –

-Squamous Metaplasia

-Keratotic changes

-Tubal Metaplasia

-Atrophy

-Pregnancy associated changes

Reactive cellular changes associated with

-Inflammation including Repair

–Lymphocytic Cervicitis

-Radiation

-Intrauterine Contraceptive Device ( IUD )

Glandular cell status Post- Hysterectomy

Organisms

-Trichomonas vaginalis

-Fungal organisms morphologically consistent with Candida spp.

-Shift in flora suggestive of bacterial vaginosis

-Bacteria morphologically consistent with Actinomyces spp.

-Cellular changes consistent with Herpes Simplex virus

– Cellular changes consistent with Cytomegalovirus

Other

Endometrial cells  ( in a woman > 45 years of age )

( specify if negative for Squamous Intraepithelial Lesion )

Epithelial Cell Abnormalities

Squamous Cell

-Atypical squamous cells

-of undetermined significance ( ASC-US )

-cannot exclude HSIL ( ASC- H )

– Low Grade Squamous Intraepithelial Lesion ( LSIL )

( encompassing HPV / Mild dysplasia/ CIN 1 )

-High Grade Squamous Intraepithelial Lesion ( HSIL )

( encompassing Moderate and Severe dysplasia/ CIS / CIN 2 and CIN 3 )

–with features suspicious for invasion  (if suspected )

– Squamous cell carcinoma

Glandular Cell

-Atypical

-Endocervical cells ( NOS or specify )

-Endometrial cells ( NOS or specify )

-Glandular cells ( NOS or specify )

-Atypical

-Endocervical cells ( Favor Neoplastic )

-Glandular cells ( Favor Neoplastic )

-Endocervical Adenocarcinoma in situ

-Adenocarcinoma

-Endocervical

-Endometrial

-Extrauterine

-NOS

Other Malignant Neoplasms ( specify )

Adjunctive testing-  Brief description and report

Computer-assisted interpretation of Cervical Cytology-  specify device and result ( where applicable )

Educational Notes and Comments appended to Cytology Report -optional Concise suggestions, consistent with clinical follow up guidelines may be mentioned ( if considered necessary ).

Detailed explanatory notes  (Report )

A brief explanation about the adequate smear and normal physiological cytology findings is given below.

Specimen Adequacy: Any smear with abnormal cells is by definition satisfactory for evaluation.

Cellularity-  In both conventional and LBC preparations, a minimum threshold of 5000 cells is generally accepted as adequate cellularity but can be lowered to 2000 cells in atrophic, post hysterectomy (vaginal vault ) and post-therapy smears. Normal cellularity is usually 8000-20000 cells.

A rough guide to estimate the number of cells

Thinprep – 50 cells / 10x field in 100 fields, 1600 cells/ 10x field in 50 fields

Surepath- 118 cells / 10x field in 42 fields, 676 cells / 10x field in 118 fields

Conventional smear- compare with “reference images”.  If a reference image has 1000 cells in a 4x field, the smear should have at least 8 such fields to estimate the cellularity as 8000 cells.

Endocervical / Transformation zone component-  In both conventional and LBC preparations, an adequate TZ sample needs at least 10 well preserved endocervical/squamous metaplastic cells, singly or in clusters.

Obscuring substances like inflammatory exudates, blood or lubricants may be stated in the report in conventional smears, they usually do not cause a problem in LBC smears.

Non Neoplastic findings- Negative for Intraepithelial Lesion or Malignancy

This category includes normal cells and reactive responses of the epithelial cells to inflammation, specific infective organisms as well as hormone levels. TBS states that reporting these specific findings is optional.

But in the Indian context, this category forms the majority of Pap smear reports. Also, most of these patients are symptomatic and they can be offered specific diagnoses as well as treatment. Thus a strong case is to be made for specific mention of the findings in this category, to realize the full potential of the Pap test.

( Reactive changes- squamous metaplasia, hyper and parakeratosis, tubal metaplasia, Atrophy, Pregnancy-related changes, Hormonal changes, Repair, Radiation, IUD and microorganisms.)

Normal epithelial cells – preparation specific criteria

Conventional- plenty of background material present.

LBC- Clean background, glandular structures may assume a 3-dimensional shape, appear cellular and hyperchromatic, nucleoli may be more prominent.

Squamous Metaplasia-  Indicates the ongoing response to noxious stimuli. Important because of D/D with HSIL / ASC-H.

Hyperkeratosis and Parakeratosis –  importance depends on nuclear features. If nuclei are pyknotic-it is usually a reactive process, rarely may hide an underlying malignancy. If nuclei are enlarged/pleomorphic- indicate HPV infection or malignancy.

Tubal Metaplasia- may be mistaken for endocervical neoplasia.

Atrophy-  indicate decreased hormonal levels consonant with age, post surgery/chemo/radiation, premature menopause, postpartum state.

Pregnancy-related changes-  navicular forms of intermediate cells seen.

Decidual cells-  indicate pregnancy / post partum state / abortion.

Cytotrophoblasts-  derived from placenta, seen in late pregnancy, postpartum period. Single cells with scanty vacuolated cytoplasm, large, dark nucleus, even chromatin. Background hemorrhagic, inflammatory exudate.

Syncytiotrophoblasts- derived from the fusion of cytotrophoblasts, seen in late pregnancy, postpartum period. Large cells with > 50 nuclei, irregular nuclear contours, cytoplasm tapers at one end.

Arias –Stella's reaction- seen in pregnancy or patients on hormonal therapy.  Glandular cells- single or in clusters, a variable amount of vacuolated cytoplasm, large hyperchromatic nuclei with grooves, pseudo inclusions, smudgy chromatin, multiple prominent nucleoli.  The background shows inflammatory exudates, may show leukophagocytosis.

RCCI- ( including Repair )- can be seen in squamous, columnar or metaplastic epithelium. The most common category of inter-observer variation. Criteria are somewhat subjective. Usually, cells are seen in groups/sheets. It indicates a trauma followed by a reparative process.

Lymphocytic Cervicitis- background of the reactive lymphoid population. Usually seen with Chlamydia trachomatis infection.

Radiation-induced changes-   patients who have received radiotherapy for carcinoma cervix or rarely for other malignancies can be monitored for recurrence by Pap smear. These changes resemble dysplastic changes and may persist indefinitely.  Important to differentiate from viable malignant cells that indicate nonresponse or relapse of malignancy.

RCC associated with IUD-  exfoliated glandular cells may show changes resembling adenocarcinoma. Also, 25% of cases may be associated with Actinomyces infection. Removal of IUD and follow up is recommended.

Glandular cells status post hysterectomy- rarely seen, originate from glandular rests. Need correlation with clinical findings to rule out malignancy.

Further categories of Cervical Cytology in TBS  will be dealt with more extensively in subsequent chapters of this book.

The TBS 2014 includes a chapter on  Anal cytology which was also included in the 2001 Bethesda atlas.  New insights in Anal cancer epidemiology are explained along with morphological findings.

A separate chapter on  Adjunctive Testing has been added to reflect the advances in HPV testing methods and immunocytochemistry methods.

Automation in Cervical Cytology screening has led to the introduction of ‘location-guided screening' devices. A chapter titled ‘Computer-Assisted Interpretation’ provides an overview of current systems in this field.

The use of  Educational Notes and Comments as a part of the Pap smear report is optional.  An entire chapter discusses the use of comments and it is recommended that these comments should be clear, concise and should be based on evidence.

A new chapter on Risk Assessment in Cervical Cancer is a valuable addition which discusses the impact of cervical cytology report on patient management.

The Bethesda Interobserver Reproducibility Study (BIRST-II)  re-emphasizes the degree of interobserver and interlaboratory variability in cervical cytology and histology interpretation.  Future editions of TBS await the results of this ongoing study.

The accompanying Bethesda 2014 Web Atlas is an invaluable tool for all students and practitioners of cervical cytology reporting. Its universal and easy accessibility has helped all cytologists to improve their quality of reporting.[ 7]

Indian Scenario- TBS

TBS for reporting Cervical Cytology is designed and focused to detect and treat precursor lesions of cervical carcinoma as part of a population screening program.

In terms of absolute numbers, Indian women bear the greatest burden of the disease as more than a quarter of new cases and cervical cancer deaths in the world occur in India alone. [8, 9]

But in low resource settings, like those in India and other developing countries, Pap smear screening programs are not widely available and Pap smears are taken whenever patients present with Gynaecological complaints in Outpatient Departments of Public and Private hospitals. So, Pap smear forms an integral part of the comprehensive health care of women in India and other similar countries.

Symptomatically most of the patients present with leucorrhea, low backache, pain in the abdomen, irregular bleeding, dyspareunia, etc. Thus many conditions like infections, inflammation, repair, malnutrition including Folic acid deficiency are detected on a cervical smear besides the occasional epithelial abnormalities and carcinomas.

( SIL  1.3 % to 3.23% as compared to RCCI ( Reactive Cellular changes associated with Inflammation ) (19.6% to 38.3% to 91.3% ) 10, 11,12,13

The diagnosis and consequent treatment of all these conditions form a large proportion of Pap smear indications besides the detection of SIL and carcinoma.

Conclusions

TBS has succeeded in achieving a lot towards reporting on Cervical Cytology to date. TBS offers a systematic way of reporting a cervical scrape smear and also gives the treating physician a consultation about the treatment strategies.  It has led to the standardization of reports and facilitated the collection and analysis of data across laboratories worldwide.

TBS 2014 is accompanied by Bethesda 2014 Web Atlas which offers a high-quality educational resource for all interested students.

In keeping with the technological advances, TBS 2014 has 2 chapters-

Chapter 9: Adjunctive Testing and Chapter 10: Computer-Assisted Interpretation

which help the students familiarize themselves with the latest developments in diagnostic and management practices. [7]

The TBS task force is committed to periodic appraisal and updating of this system.

References

Papanicolaou GN, Traut HF. "The diagnostic value of vaginal smears in carcinoma of the uterus". American Journal of Obstetrics and Gynecology. 1941;42:193.

Ayre JE. Cancer Cytology of the Uterus. Grune Stratton, New York, 1951

Richart RM: Natural history of cervical intraepithelial neoplasia. Clin Obstet Gynecol 10: 748, 1967

National Cancer Institute Workshop. The 1988 Bethesda System for reporting cervical/vaginal cytologic diagnoses. JAMA 1989,262:931-34

Kumar, Vinay; Abbas, Abul K.; Fausto, Nelson; & Mitchell, Richard N. (2007). Robbins Basic Pathology (8th ed.). Saunders Elsevier. pp. 718–721. ISBN 978-1-4160-2973-1.

The Pap Test and Bethesda 2014  Nayar R. Wilbur D.C. Acta Cytologica

2015;59:121-132

The Bethesda System for Reporting Cervical Cytology- Definitions, Criteria and

Explanatory notes: 3rd Edition,  Ritu Nayar, David C. Wilbur, Switzerland, Springer International Publishing, 2015.

8. Global Inequalities in Cervical Cancer Incidence and Mortality are Linked to Deprivation, Low Socioeconomic Status, and Human Development Gopal K. Singh, Romuladus E. Azuine, DrPH, RN1; M  Siahpush, International Journal of MCH and AIDS Volume 1, Issue 1, 2012, Pages 17-30

A meta-analysis of social inequality and the risk of cervical cancer. Seema Parikh, Paul Brennan, and Paolo Boffetta, International  Journal of cancer: 105, 687– 691 (2003)

Studies on the Epidemiology of Cervical Cancer in Southern Assam. Satya B. Paul, Basant K. Tiwary, Arun Paul Choudhury, Assam University Journal of Science & Technology: Biological and Environmental Sciences Vol. 7 Number I 36-42, 2011

A comparative study of cervical smears in an Urban Hospital in India and a population-based screening program in Mauritius. Kaustubh Mulay et al,  Indian journal of pathology and microbiology 52:1: 34-37. 2009

Cervicovaginal cytology: Clinicopathological and Social Aspect of Cervical Cancer Screening in Rural (Maharashtra) India. Dhiraj Nikumbh et al, International Journal of health sciences and research. 1(2) : 125-132. 2012

14. Prevalence of cervical dysplasia in western Uttar Pradesh. Kamna Gupta et al, Journal of Cytology 30 (4) 257-262. 2013

The Inflammatory Smear

The Borderline lesions

ATYPICAL SQUAMOUS CELLS (ASC) – ASC-US and ASC-H Dr.Jayasree.K

Introduction

The term Atypical Squamous Cells (ASC) was introduced in The Bethesda System (TBS) to designate equivocal cytologic changes that may reflect a squamous intraepithelial lesion (SIL).

Evolution of terminology

Papanicolaou in 1941 introduced the numerical terminology which consisted of Class I to V7. (Table 1).The present terminology ASC comes under Papanicolau class II which is a mixture of reactive changes to koilocytosis and atypia. Later it was found that class II was inadequate to accommodate the diversity of benign lesions. Then the concept of precursor lesions has emerged. Reagan in 1961 introduced the term “Dysplasia” for the intraepithelial changes12. This created a wide variety of terminologies like keratinized dysplasia, non-keratinized dysplasia, metaplastic dysplasia (each with mild, moderate and severe subclasses). This classification was more subjective and thus the clinician found its application difficult from a management point of view.

Richard in 1966 introduced CIN (Cervical Intraepithelial Neoplasia) classification with CIN I, CIN II and CIN III based on the tissue architecture13. But the term neoplasia for low grade CIN was objected by experts.

Then Meisels in 1976 recognized manifestations of genital HPV infection which are reflected in the smear as koilocytosis and are considered as less than true dysplasia. Subsequently, HPV DNA was identified in the majority of cervical neoplasm and HPV was considered as a key link in the pathogenesis of cervical cancer 13. Koilocytotic changes were seen associated with dysplasia/CIN. So the separation of HPV effect as a separate entity from dysplasia / CIN became biologically invalid.

In 1988 a meeting was convened with experts from all over the world at Bethesda, USA to discuss various terminologies. A common terminology that helps in the management was emerged, called "The Bethesda System"(TBS) 6, 7. TBS was modified in 1991, 2001 and recently in 20141, 3,11,14,15. A comparison of various classification systems is given in Table 2.

Atypical squamous cells (ASC)

Definition

ASC refers to cytologic changes suggestive of Squamous Intraepithelial Lesion (SIL), which are qualitatively or quantitatively insufficient for a definitive interpretation. ASC category was developed to designate the interpretation of an entire specimen, not individual cells, because atypia in individual cells remains a highly subjective and, hence, variable interpretation5. The interpretation of ASC requires that the cells in question should be showing squamous differentiation, increased nuclear-cytoplasmic ratio and minimal nuclear chromasia, chromatin clumping, irregularity, smudging, or multinucleation.4

ASC-US and ASC-H

According to TBS 1991 atypical squamous cells were categorized into Atypical Squamous Cells of Undetermined significance (ASC-US) which is further qualified ASC-US favor reactive, ASC-US favor SIL and ASC-US NOS. Later this was revised in 2001 to ASC-US and ASC-H (Atypical Squamous Cells- cannot exclude high grade SIL) 3. In the 2014 Bethesda System, ASC continues to be included under squamous epithelial cell abnormality, with subcategorization as “atypical squamous cells – undetermined significance” (ASC-US) and “atypical squamous cells – cannot exclude a high-grade squamous intraepithelial lesion” (ASC-H). ASC-US refers to changes that are suggestive of LSIL but which are insufficient for a definitive interpretation as such. The ASC-H category was developed to highlight the minor subset of ASC considered suspicious for a cancer precursor lesion [HSIL]. This dichotomous reporting terminology for atypia is in keeping with the 2-tiered reporting scheme for HPV-related squamous lesions which is based on our current understanding of the natural history of HPV-related infections – low-grade changes represent largely transient HPV infection, and high-grade morphology represents a precancerous lesion.

The category of atypical squamous cells (ASC) is the most prevalent of all abnormal cervical cytology interpretations. In contrast to ASC-US, which is a common cytologic interpretation that has been extensively characterized in large studies, ASC-H interpretations are relatively uncommon. Based on HPV biology and behavior of pre-invasive HPV associated squamous lesions, the focus is aimed at the detection and treatment of HSIL 8, 9. A low grade change due to HPV infection represents transient HPV infection (ASC-US) whereas high grade morphology represents precancerous lesion (ASC-H) 16. Updated guidelines published in October 2007 give a greater emphasis for high risk HPV testing 2,10,16,17. HPV testing is not recommended for primary screening due to concerns regarding specificity. However, HPV testing is useful as a secondary test, following an ASCUS cytology test, for triage to colposcopy. The ASCUS/LSIL Triage Study (ALTS) which began in 1997 also consider high risk HPV testing (hrHPV) as the most cost-effective triage test 18, 19. New algorithms are proposed on special populations like adolescents and pregnant women. Occasionally a specimen with cytologic features that lie between LSIL and HSIL is encountered. In such cases, 2014 Bethesda recommends an interpretation of ASC-H in addition to LSIL interpretation 11, 12, 13. This indicates that definite LSIL is present as well as some cells that suggest the possibility of HSIL.

Numerous non-neoplastic conditions may produce cytologic changes that raise consideration for an ASC designation, including inflammation, air-drying (inherent to the conventional Pap smear ), atrophy with degeneration, hormonal effects, and other artifacts. In many instances, the process that resulted in the ASC interpretation remains undefined, even following a diagnostic workup. In screening programs representative of the US population, approximately 40–50 % of women with ASC are infected with high-risk/oncogenic types of human papillomaviruses (HPV) [ 3 – 5 ].TBS book pg 104== The degree of apparent atypia in reactive cells may also be exaggerated by active smearing. These issues result in the artifacts that make it difficult for the cytotechnologist and pathologist to adequately visualize the nuclear details of the cells in question, resulting in a less than a definitive diagnosis.
The immediate wet fixation that is used in the Thin Prep process may allow the pathologist and cytotechnologist to better differentiate between cellular changes due to reactive conditions, definitive squamous atypia, and dysplastic lesions. This is not to say that ASC interpretations are eliminated by the adoption of the Thin Prep Pap Test, but the laboratory staff has the opportunity to refine their morphologic criteria, eliminating certain "ASC" cases arising from poor preparation, and better-identifying cases that may harbor abnormality.

ASC-US

It must be emphasized that this category is not a waste paper basket. Before using this category, cytologic findings should be carefully reviewed and judiciously evaluated and then those features that are most consistent with benign reactive changes may be classified as “negative for intraepithelial lesion or malignancy” whenever possible. The 3 prerequisites in the interpretation of ASC are firstly the cells should be squamous, secondly the nuclear to cytoplasmic ratio should be increased, and thirdly one must be able to see minimal nuclear changes which may include hyperchromasia, chromatin clumping, irregularity, smudging, and/or multinucleation. Abnormal-appearing nuclei are a prerequisite for the interpretation of ASC.

The epithelial cells that are generally incorporated in the ASC-US category are the mature squamous and squamous metaplastic cells exhibiting changes that are minimal and fall short of a diagnosis of LSIL or HSIL. The nuclei are slightly enlarged and this enlargement is compared to the normal nucleus of the same cell type. The nuclei may show slight nuclear membrane irregularities but are often smooth. The cytologic findings of cytoplasmic and nuclear changes associated with HPV infection (perinuclear halos/koilocytes) warrant an interpretation of SIL. However, when the changes are incomplete or are suggestive of koilocytosis (e.g., cytoplasmic halos closely resembling koilocytes but with minimal nuclear abnormalities) are generally designated as ASC-US [ 10 ] pg 105.

When dealing with the metaplastic squamous cells, one needs to make sure that the irregularities are not due to a vacuole pushing the nucleus into the aberrant shape. The chromatin pattern is finely granular and evenly distributed. Chromocenters or nucleoli are generally inconspicuous or absent unless a reactive process is occurring in conjunction with the atypia, at which point, the differential diagnosis of reactive needs to be considered depending upon the presence or lack of the other criteria. With either conventional or liquid-based preparations, an ASC interpretation may arise from any of the several different cellular changes, including but not limited to, squamous atypia, atypical squamous metaplasia, and atypical parakeratosis. The criteria for ASC-US differ in different types of cells 5.

1. ASC-US with “Mature” Intermediate Type Cytoplasm

Nuclear enlargement is 2.5-3 times that of a normal intermediate nucleus, (approximately 35 mm 2 ) or twice the size of a squamous metaplastic cell nucleus (approximately 50 μm 2 ) but the chromatin is finely granular and evenly distributed without significant hyperchromasia. The nuclear membrane is smooth and regular. There may be variations in nuclear size. The normal-appearing intermediate cells that are present on a slide provide an appropriate source of comparison for assessing whether the nuclear size and appearance meet criteria for ASC-US or SIL.

Fig 1: ASC-US in mature intermediate type cytoplasm – Nuclear changes are more marked than reactive, less than LSIL. Pap x400

Fig==A 23-year-old woman. Intermediate cells with nuclear enlargement ×2−3 that of the normal intermediate squamous cell nucleus. There is partial haloing of cytoplasm with slight nuclear crenation and hyperchromasia, that do not meet the diagnostic criteria for LSIL. A repeat cervical cytology showed similar findings. hrHPV DNA test was positive.

Fig.

When in doubt regarding the nature of cells reactive (NILM) or ASC-US, the age of the patient and previous Pap smear reports or reports of hrHPV test, if any, need to be reviewed. The prevalence of ASC-US declines with increasing age in the screening population, as does the prevalence of hrHPV DNA (including genotypes 16 and 18) [ 13 ]. ASC-US cytology in younger women is more prevalent and more often reflective of an HPV-related lesion than in older women [ 13 ].

2. ASC-US in Atrophy

The Pap smears from women during the peri and postmenopausal phase commonly show mild bland nuclear enlargement that is a common cause for ASC over utilization. In this age group, problems like recovering of scanty material from the overall dry lower genital tract and its lining cells and later artifacts related to smearing of these inherently dry cells ( Conventional Pap smear ) generally lead to overdiagnosis of ASC. Changes of mild nuclear enlargement without significant hyperchromasia or nuclear irregularity are not generally associated with HPV-related disease.

An atrophic smear with cells showing nuclear enlargement with concomitant hyperchromasia marked irregularities in nuclear contour in the form of pleomorphism- spindle or tadpole cells or chromatin distribution, can be a diagnostic dilemma both related to ASC-US or ASC-H or beyond like HSIL. The interpretation of HSIL may be difficult because of the lack of maturity (and hence high nuclear to cytoplasmic ratio) of the parabasal cells. In low-risk scenarios, it may be practical firstly to categorize such atypia as ASC-US rather than ASC-H and also advise adjunctive hrHPV testing to avoid overtreatment. Secondly, a short course of local application of oestrogen (1gm estrogen cream local application for one week), followed by a repeat Pap smear a week after completing the regimen will be useful to clarify the diagnosis. The ‘hormonally deaf’ abnormal epithelial cells will persist and will stand out against the normal parabasal cells undergoing maturation and differentiation.

Fig 2: ASC-US Atrophy- Parabasal cells with enlarged, smudged nucleus. Pap X400

3. ASC-US in metaplastic squamous cells

Inflammation and repair go hand in hand. The NILM smears often display squamous metaplastic cells. Round or ovoid cells that are approximately one-third the size of superficial cells and therefore resemble large metaplastic or small intermediate cells may also be classified as ASC-US. In mature squamous metaplasia, nuclear size is increased. However, in immature squamous metaplasia, the increased nuclear-cytoplasmic ratio is more important than nuclear enlargement. Metaplastic cells showing some degrees of cellular overlap, dyscohesion, anisonucleosis, and/or loss of nuclear polarity may be designated as “atypical repair” which is a degree beyond “reactive and reparative” changes seen in a NILM smear. In favor of a reactive process is the generally fine

granularity of the chromatin pattern and the fact that most nuclei show prominent nucleoli. An atypical repair can be classified under the ASC-US category when the nuclear abnormalities are at the lower end of the spectrum. Cytologic features that are favouring the possibility of invasive carcinoma, especially in high-risk patients, should be placed in the ASC-H category. The incidence of subsequent SIL among women with atypical repair has been reported to range from 25 to 43 % in high-risk population groups; however, the incidence of SIL in a more diverse population is much lower (5.2 %) [ 18 ].

ASC-H Dark single cells

Fig 3: ASC-US (Mature metaplastic cells). Pap X 250

Fig 4: ASC-US (Immature Metaplastic Cells with nuclear atypia). Pap X 250

4. ASC-US in parakeratotic squamous cells

Cells are either single or in three-dimensional clusters. They have dense eosinophilic or orangeophilic cytoplasm, cellular pleomorphism, some with an increased nuclear-cytoplasmic ratio, irregular nuclear contour, and dark irregularly distributed chromatin.

Fig 5: Atypical parakeratotic squamous cell.Pap X 250A 21-year-old woman.

Fig==Thick cohesive sheets of cells with focal nuclear enlargement, orangeophilic cytoplasm, poorly formed cytoplasmic vacuoles, and binucleation. In this case, the follow-up biopsy was LSIL (CIN1)

5. ASC – US due to compromised specimen

Poor fixation, obscuring material or drying artifacts in perimenopausal and postmenopausal women compromise visualization and evaluation of cellular changes. In such cases, a repeat smear or other follow up should be emphasized in the report.

Summary of morphologic criteria of ASC-US (Table 3)

1. Cells present predominantly singly and in few sheets===fig

2. Nuclei are 2.5 to 3 times the size of a normal intermediate cell nucleus or 1.5 to 2 times the size of a normal metaplastic cell nucleus fig===

3. There is a slight increase in nuclear to cytoplasmic ratio fig==

4. Nuclear membranes are smooth to slightly irregular fig==

5. Chromatin is finely granular and evenly distributed. fig==

6. Nucleoli/chromocenters are inconspicuous or absent. fig==

7. HPV cytopathic effect and/or binucleation may suggest HPV

change, but fall short of LSIL diagnosis fig==

8. Bi-nucleation accompanies HPV but also can be from a reactive

process. fig==

9. Atypical parakeratosis may present with minimal cellular

pleomorphism, a slight increase in nuclear size and vesicular

chromatin fig==

10. Atypical metaplastic cells with slightly increased N/C ratio, slight nuclear membrane irregularities, slight hyperchromasia fig==

Differential Diagnosis of ASC-US:

1. Pseudokoilocytosis due to reactive change as in Trichomonas infection, artifact or glycogen.

2. Perimenopausal changes. like a generalized increase in the size of intermediate cells. increased nuclear size with minimal hyperchromasia without an increase in the N/C ratio.

The burden of disease in ASC-US

Individual's risk of High grade SIL is 15-30%

Approximately 33 – 50% of HSIL occurs in women with ASC Pap results

However, an individual's risk of cancer is only about 1:1000

ASC-US: Quality Control and Quality Assurance

ASC is a diagnosis of exclusion

In a normal population, it should not exceed 5%.

ASC-US: SIL ratio should not be more than 2:1

By ALTS data: 50% of ASC-US show HPV positivity

Quality Assurance

Calculation of ASCUS/SIL ratio

Correlation of ASCUS cases with HPV typing

Review by second cytotechnologist and/or cytopathologist

Correlation of ASCUS cases with the results of colposcopically directed biopsy

HPV positive rates and ASC-US

Most recent HPV Q-Probe data conclude that a hrHPV positive rate of 43.7%.

In other studies the median rate is reported as 34.1% to 50.6%, depending on the age of the population studied.

Management of ASC-US

The preferred approach for evaluation of women ages 25 or older with ASC-US cytology is testing for high-risk types of HPV with triage of women who test positive to colposcopy.

The use of HPV testing to triage further evaluation of ASC-US is the most effective strategy for detecting high-grade premalignant disease or cervical cancer. Reflex HPV testing has the additional advantage that it saves the patient from the inconvenience of a second visit for HPV testing. This is possible only when cervicovaginal cells are collected in an LBC vial. This vial can be immediately sent for HPV DNA testing.

Repeat cytology in one year is a reasonable alternative to HPV triage if HPV testing is not available. Colposcopy is performed if repeat cytology shows ASC-US or a more severe cervical abnormality.

For women ages 21 to 24 years with ASC-US cytology, repeat cytology at 12 months rather than reflex HPV testing is advised.

ASC-H

ASC-H is a designation reserved for the minority of ASC cases (< 10%), in which the cytology changes are suggestive of HSIL16. Only equivocal specimens specifically worrisome of HSIL should be distinguished from the bulk of ASC. Cases classified as ASC-H are associated with a higher predictive value for detecting an underlying CIN2 or CIN3 than ASC-US. This reporting category should be used carefully and sparingly. It warrants immediate colposcopy to exclude the presence of HSIL. ASC-H does not represent a single biologic entity. It subsumes changes related to oncogenic HPV infection and neoplasia as well as findings that suggest the possible presence of CIN and rare carcinoma. Studies show 35- 50% of women with ASC are infected with high risk HPV (hrHPV), and the remaining non-infected women are not at increased risk. The nuclear changes are mainly noted in the immature metaplastic cells.

Cytomorphology

In ASC-H, atypical cells are usually sparse. Only a few cells with features of high grade dysplasia will be present. ASC-H is defined as “changes suggestive of / or cannot rule out a high grade squamous intraepithelial lesion”.The cells are quantitatively or qualitatively insufficient for a definitive interpretation”. ASC-H usually affects immature squamous metaplastic cells. The cells are arranged either singly or in loose cohesive groups or cohesive overcrowded groups. The patterns are

Small cells with high N/C ratios: “Atypical (Immature) Metaplasia”

Immature squamous metaplasia is one of the most common mimics of HSIL. An interpretation of ASC-H is appropriate, especially when only rare abnormal cells with “metaplastic” cytoplasm and high nuclear to cytoplasmic ratio are present. Cells occur singly or in small fragments of less than 10 cells. Sometimes cells may stream in mucus. ASC-H cells demonstrate nuclear enlargement at least 1.5 times to 2.5 times of metaplastic cells, high N: C ratio, coarse chromatin pattern as well as some degree of hyperchromasia, abnormal nuclear shapes and nuclear membrane irregularity: however the nuclear changes fall short of a definitive diagnosis of HSIL (Table 4). Sometimes cells may show all features of HSIL, but the number of cells to make a definitive diagnosis of HSIL may not be sufficient.

Crowded Sheet Pattern

This pattern consists of a crowded group of cells containing nuclei with loss of polarity. Cellular and nuclear details are difficult to visualize. In such a situation, the features in favor of squamous over glandular differentiation are the streaming of nuclei within the cluster, dense cytoplasm, polygonal shape and fragments with sharp linear edges. Vigorous scraping with sampling devices, brushes, and brooms, may represent an avoidable cause of thick cell fragments. The tips of the sampling device may also scrape cells from the glandular crypts in cases with cervical erosions. In this situation, the differential diagnosis of hyperchromatic crowded cell groups includes reactive or neoplastic endocervical cells. The thickness of the cluster makes it difficult to determine if the cells are reactive or neoplastic. In an inflammatory or reactive process, the cells within the cluster often include acute inflammatory cells. And secondly, the presence of nucleoli is more indicative of a reactive than neoplastic process.

In Liquid-Based Preparations, the ASC-H cells are usually sparse. The cells are very small and seen mostly singly. The nuclei of these cells are around two to three times the size of neutrophils. The N/C ratio and nuclear irregularity are variable. Rare metaplastic cells with dense cytoplasm and nuclear enlargement with hyperchromasia are present in a background of scattered acute inflammation. Degeneration of nuclei may be the source of confusion. But note that degenerated nuclei, in the absence of a bona fide SIL, are often irregular or hyperchromatic, but the irregularities tend to involve the entire nuclear outline, imparting a wrinkled appearance, and the chromatin is smudgy (Fig. 4.26 ).

Differential Diagnosis

Repair- Identification of prominent nucleoli is more typical of repair. In “atypical repair”, cells can be immature metaplastic cells or glandular cells. They differ from the typical “repair” by the presence of considerable nuclear crowding, loosely cohesive groups, uneven chromatin distribution, anisonucleosis, irregular nuclear contours, and irregular nucleoli. The differential diagnosis is invasive carcinoma. The lack of single atypical cells and clean background would favor a reactive process.

Atrophy- Application of topical oestrogen for one week, followed by repeat smear after one week will result in the maturation of cells. The presence of frank cellular necrosis favor neoplasm.

Radiation change- Typical benign radiated cells show a proportionate nuclear and cytoplasmic enlargement. Degenerative changes are seen in both cytoplasm and nucleus.

Nonneoplastic entities that may be interpreted as ASC-H include histiocytes, degenerated endometrial cells, atrophic parabasal cells, and cells in patients wearing an intrauterine device.

single dark cells that cannot be resolved any further but very few =ASC-H

Fig 6: ASC-H -Scanty cellularity Pap X 400

Fig 7: ASC-H – Scanty cells with high N/C ratio-similar to HSIL Pap X 400

Fig 8: ASC-H – Hyperchromatic crowded groups. Pap X 250

Management guidelines

The recommended management of ASC-H is colposcopy10. Management of women with ASC-H following colposcopy that does not result in histologic diagnosis of CIN2 or more severe lesion should be individualized based on a review of all pathologic or clinical findings. A careful review is required before treating women with ASC-H who does not have histologically diagnosed CIN2 or worse. In contradistinction to a definitive interpretation of HSIL, immediate treatment without colposcopy is not possible.

The burden of Disease in ASC-H

ASC-H is associated with a higher risk of oncogenic HPV DNA detection and greater risk of underlying CIN2 or worse (30-40%) compared to ASC-US (10-15%).

Conclusion

ASC is the product of limitations of light microscopy and diagnostic capabilities. It provides a comfort/ discomfort level. ASC tends to decrease with experience and interestingly will increase with a lack of experience and fear of missing abnormal lesions.

There are many criticisms for the ASC category. The major one is its subjective and inconsistent applications. Low inter-observer and intraobserver reproducibility are evidenced in many studies. It is the most controversial term of TBS. But studies have shown that if we eliminate ASC-US, the LSIL rate will increase. So there is a strong reason to retain the ASC category. ASC category conveys the uncertainty of Gynecologic cytology. As cervical carcinogenesis is a continuum process there is no sharp dividing line between different categories. Now ASC-US is well accepted and widely used by cytologists and is much familiar to clinicians.

Table 1: CIN-Terminology-Evolution

PAPANICOLAOU – CLASS I-V

REAGAN – DYSPLASIA- Mild, Moderate, Severe, CIS

RICHART (1968) – CIN I, II, III

TBS (1988) – ASCUS, LSIL, HSIL

REVISED TBS (1991) – ASCUS (favor reactive, neoplastic or NOS), LSIL, HSIL

MODIFIED TBS (2001) – ASC-US, ASC-H, LSIL, HSIL

Table 2: Classification of SIL

Dysplasia/CIS* CIN# Bethesda

Mild CIN I LSIL °

Moderate CIN II

Severe CIN III HSIL∞

*CIS-Carcinoma in situ CIS

# CIN- Cervical Intraepithelial Neoplasia

°LSIL- Low grade Squamous Intraepithelial Lesion

∞ HSIL- High grade Squamous Intraepithelial Lesion

Table 3: ASC-US

Table 4: ASC-H

References:

1. Barbara A, Crothers, D O.The Bethesda System 2001: Update on Terminology and Application. Clinical Obs. and Gyn. 2005; 48:98-107.

2. Barbara.S. Apgar, Anne L Kittendore, Catherine M Bettcher, Jean Wong, Amanda J Kaufman. Update on ASCCP Consensus Guidelines for Abnormal Cervical Screening Tests and Cervical Histology. Am Fam Physician 2009;80(2):147-155.

3. Diane D Davey. Bethesda 2001: Science, Technology, and Democracy Join Forces for Patient Care. Diagnostic Cytopathology 2002; 26:135-136.

4. Diane Solomon, Ritu Nayar. The Bethesda System for Reporting Cervical Cytology: Definitions, Criteria. Second edition. Springer-2004, Chapter 4; 67-87.

5. Diane Solomon, William J Farble, G Peter Vooijs, David C Wilbur, N Sreedevi Amma, Robert John Collins, Diane D Davey, Bryon Kenyan Knight, Ronald D Luff, Alexander Meisels, James Navin, Dorothy L Rosenthal, Torril Sauer, Mark Stoler, Harry Z Suprun, Kazuhiro Yamauchi. ASCUS and AGUS Criteria-IAC Task Force Summary. Acta Cytologica1998; 42:16-24

6. Koss LG. The New Bethesda System for reporting results of smears of the uterine cervix. J Natl Cancer Inst 1990; 82(12):988-991

7. Leopold G. Koss (Editor), Myron R. Melamed (Editor) Koss' Diagnostic Cytology and Its Histopathologic Bases, 5th Revised edition r 2005;

8. Manos MM, Kinney WK, Hurley LB. Identifying women with cervical neoplasia: Using Human Papillomavirus DNA testing for equivocal Papanicolaou results. JAMA 1999; 281:1605-1610.

9. Mark H Stoler. Testing for human papillomavirus: data-driven implications for cervical neoplasia management. Clin Lab Med 2003; 23:569-583

10. Massad LS, Einstein MH, Huh WK. Updated Consensus Guidelines for the Management of Abnormal Cervical Cancer Screening Tests and Cancer Precursors. Obstet Gynecol 2013; 121; 829-846.

11. Michael R. Henry.The Bethesda System 2001: an update of new terminology for gynecologic cytology. Clin Lab Med 2003; 23:585-603

12. Reagan J W, Fu YS: Histologic types and prognosis of cervical cancer of the uterine cervix: Int.J Radiat Oncol.Biol.Phys 1979; 5:1015.

13. Richard RM: Cervical Intraepithelial Neoplasm. Pathology Annual 1973; 3:301-328

14. Ritu Nayar, David C Wilbur. The Pap test and Bethesda 2014.Acta Cytologica 2015; 59:121-132.

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16. Ritu Nayar, David C Wilbur (Eds). The Bethesda System for Reporting Cervical Cytology. 3rd Edition, 2015; Springer: 103-134

17. Schiffman MH, Bauer HM, Hoover RN, et al. Epidemiologic evidence showing that human papillomavirus infection causes most cervical intraepithelial neoplasia. J Natl Cancer Inst 1993;85:958–64.

18.Schiffman M, Adrianza ME. ASCUS-LSIL Triage Study: design, methods, and characteristics of trial participants. Acta Cytol 2000; 44:726-742.

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Michael R. Henry

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Cytology of SILs and Invasive Ductal carcinoma

Cytology of Adenocarcinoma

Radiation Changes

Last terminology for Diagnosis of SILs

Cervical Cancer: Histological Types & Morphology – sent 15th march 2016

Dr. R Ravi Dr. Rasika Gadkari

Introduction

Cervix is the distal tubular portion of the uterus. Cervix protrudes into the vaginal vault and thus anterior and posterior fornices are formed. The lowermost opening is external os, from where endocervix begins as a narrow canal up to internal os or isthmus, from where the endometrial cavity begins. Space between uterus and rectum is the pouch of Douglas. Immediately posterior and inferior to urinary bladder is the anterior part of the cervix. The endocervix is lined by columnar mucinous epithelium and in continuity, with vaginal mucosa, the ectocervix is lined by squamous epithelium. The region where there is a transition from squamous to columnar epithelium in the cervix is the transformation zone. Most of the cervical epithelial neoplasm and their precursors occur at the transformation zone. Thus the lining epithelium is the squamous, columnar and metaplastic squamous epithelium. Squamous epithelium comprises of superficial layer, intermediate layer and basal layer.The one cell thick basal layer has high nucleocytoplasmic ratio which progressively decreases from basal to superficial layer.

FULL FORM According to ICD-O codes Squamous tumors and precursors in cervix are as follows.Table1

Table1: ICD-O Code of Cervical Cancer

Squamous cell carcinoma- An Invasive carcinoma composed of Squamous cells of varying differentiation.(1)SUPERSCRIPT

Cervical intraepithelial neoplasia (CIN) may be found on the surface or edge of tumor particularly in smaller growths. may arise between invasive carcinoma & CIN involving crypts. Similarly invasion can not be excluded when neoplastic squamous epithelium shows CIN II and CIN III changes AND  underlying stroma is not present.

Histopathology: Most frankly / FRANK invasive squamous cell carcinomaS of cervix infiltrate as network of bands with stroma. There are islands of squamous cells rounded,angular and spiked. everal grading systems have been proposed depending upon the type and differentiation of predominant cells.(2).The most commonly used classification is modification of Borders (3),in which squamous cell carcinoma is divided into differentiated, moderately differentiated and poorly differentiated. Cervical stroma in the tumor is usually infiltrated by lymphocytes and plasma cells. A markedly eosinophilic response (4 ), a foreign body-type giant cell reaction is occasionally seen.

Types  –

1)Keratinizing type  – Squamous cell carcinoma composed of whorls of squamous cells with keratin & epithelial pearls. Intercellular bridges and cytoplasmic is usually seen. The nuclei show atypia, hyperchromatism and are large with coarse chromatin. Mitosis may not be frequent and are usually seen in less differentiated cells.

2Keratininzing SHOULD THE WORD TYPE COME EVERYWHERE – Tumor cells ARE composed of recognizable squamous cells. Cells show individual cell keratinization and intercellular bridges however keratin pearls are not seen. Obvious cellular atypia IS seen and mitosis is usually numerous.

3) Basaloid – Tumor IS composed of nests of immature basal types of squamous cells. The cytoplasm is scanty. Some foci of keratinization may be seen, however, keratin pearls are usually absent. When seen in vulva association with HPV type 16 is frequently encountered. (5,6).This is an aggressive tumor ……???with basaloid features.

4)Verrucous –This is a highly differentiated squamous cell carcinoma with the hyperkeratotic undulating warty surface. Tumor invades the stroma with a pushing border. Nuclei show minimal atypia.HPV infection is usually not seen. The tumor tends to recur locally, after excision. However, usually, they do not metastasize. The tumor is distinguished from condyloma by THE presence of broad papillae and lack of fibrovascular core SEEN in verrucous carcinoma and correlation with clinical presentation.?? CLARIFY CLI PRESENTATION FEATURES OF BOTH

5) Warty-  This has a warty – GIVE MEANING OR synonyms LIKE DRY HORNY surface and cellular features of HPV infection. High risk HPV DNA is typically detected. (7).AND IS also known as condylomatous squamous cell carcinoma.

5) Papillary –There are broad papillae with connective tissue stroma showing features SIMILAR TO THAT OF CIN. The superficial biopsy may not reveal evidence of invasion, however, the underlying tumor is a typical squamous cell carcinoma. The tumors are usually positive for HPV type 16. This differs from warty type by inconspicuous keratinization and lack of cellular features of HPV infection. (8)

6) Lymphoepithelioma like-This tumor is strikingly similar to the nasopharyngeal tumor by the same name. The tumor is composed of poorly defined islands of undifferentiated cells, in a dense lymphocytic background. Tumor cells have uniform vesicular nuclei with prominent nucleoli and mildly eosinophilic cytoplasm. Cells are arranged in a syncytial pattern with indistinct cell border and immunohistochemistry shows POSITIVITY FOR MARKERS LIKE cytokeratin positive and Lymphoid cells show T cell markers Indicating a cell-mediated immune response.MODIFY  Using PCR on frozen tissue from these tumors,  EBV was not identified in cases from the USA, however,  EBV DNA was identified in 11 out of 15 lymphoepithelial carcinomas in Taiwan. HPV DNA was uncommon suggesting that EBV DNA may play a role in the etiology of the Asian population.(9 ,10)

7) Squamotransitional carcinoma –Rarely transitional cell carcinomas of the cervix are described. The tumor may be pure transitional or may have a squamous component. Detection of HPV type 16, presence of allelic losses at chromosome 3p with infrequent involvement of chromosome 9 suggest that tumor is more closely related to squamous carcinoma than to primary urothelial / UROTHELIUM .(11, 12 ). Furthermore these tumors are more likely to express cytokeratin7 than CK20 which suggests only histological rather than an immunophenotypic resemblance to the transitional epithelium.

8) Early invasive Squamous cell carcinoma  WHO definition  A Squamous cell carcinoma with early stromal invasion the extent of which has not been precisely defined and a low risk of lymph node metastasis. A synonym is Micro invasive squamous cell carcinoma. FOLLOWING features increase the likelihood of identifying early invasion.

i) Extensive  CIN III

ii) Widespread expansile  AND & deep extension into endocervical crypts.

iii) Luminal necrosis and intraepithelial squamous maturation. Historically criteria for a diagnosis of microinvasive carcinoma were based on the depth of invasion varying from 3 to 5 mm. It mostly equates with FIGO 1A. Pooled data indicates that a maximum depth of invasion of 3 mm or less is associated with a risk of lymph node metastasis (<1% )REFERENCE. On the other hand invasion of 3 to 5 mm is associated with an overall risk of lymph node metastasis in 2% and a recurrence rate of 4%. Microinvasive carcinoma is usually associated with stromal edema, desmoplasia, and lymphocytic response. Immunohistochemistry with CD31 and CD34 may add in the reorganization of lymphatic and vascular involvement. (1)A term Superficially Invasive Squamous Cell Carcinoma Is coined in LAST –FULL FORM terminology for these lesions

Uncommon carcinomas and Neuroendocrine tumors  (1)

WHO definition – Epithelial tumors of uterine cervix other than those of squamous and glandular types ARE INCLUDED IN THIS CATEGORY.

Adenosquamous carcinoma –   Carcinoma composed of glandular &AND squamous elements. Both elements show atypical features. Mucin secreting cells WHEN SEEN in an otherwise routine squamous cell carcinoma…. ?? like mucoepidermoid carcinoma. Tumors do not behave differently from conventional squamous carcinoma. Routine mucin staining is not recommended. (1) PL DESCRIBE CLASSICAL ARCHITECTURAL PATTERNS THAT MAY BE SEEN

Poorly differentiated tumors resembling poorly differentiated squamous carcinoma but with many mucin-producing cells& lacking keratinization or intercellular bridges should be diagnosed as poorly differentiated adenocarcinoma. (WHO)

Glassy Cell carcinoma – It is a variant of Poorly differentiated adenosquamous carcinoma It accounts for1-2%of all cervical cancers. The tumor occurs in young people, grows rapidly, develops frequent metastasis and responds poorly to radiotherapy However chemotherapy may be promising (1 1863). Tumor lacks ER PR.(2 132)Tumor cells are large with distinct cell borders and a ground glass cytoplasm. Prominent eosinophilic infiltration is present in the stroma helps to separate the tumor from non-keratinizing squamous cell carcinoma. (13)

Adenoid cystic carcinoma – The most common tumor occurring in patients over 60 years of age. Most present with postmenopausal bleeding and have a mass on pelvic examination. Histopathologically it is similar to its counterpart in the salivary gland. The characteristic cystic spaces are filled with slightly eosinophilic hyaline basophilic mucin and are surrounded by palisading epithelial cells. In contrast to the salivary gland counterpart, these tumor cells show more nuclear pleomorphism, high mitosis & necrosis. (8, 49 )Immunostain for basement membrane component (collagen type) and laminin are strongly positive(1918). The majority of cases studied stain for S100 and HHF35, suggest myoepithelial differentiation. Differential diagnosis is small cell carcinoma, adenoid basal carcinoma, and non-keratinizing squamous cell carcinoma. Histogenitically basaloid squamous carcinoma and adenoid basal carcinoma are part of the morphological spectrum of basaloid cervical neoplasm. (10,59).Circumstantial evidence suggests that adenoid basal carcinoma may be a precursor of adenoid cystic carcinoma. Prognosis is poor with tumor recurring locally frequently and showing metastasis

Adenoid Basal carcinoma (Definition WHO) –A cervical carcinoma in which, rounded generally well-differentiated nests of basaloid cells, show focal gland formation or sometimes central squamous differentiation. The patient is usually more than 50 years old and has a clinically detectable abnormality of cervix. Often tumor is discovered incidentally.

Histopathology – Small nests of basaloid cells almost always beneath or arising from CIN/Small invasive squamous carcinoma(14). Cells are small with scanty cytoplasm & are arranged in cords nests & focal glandular or squamous differentiation.

Prognosis Tumor is low grade and rarely metastasizes.

Neuroendocrine tumors – This group includes Carcinoid, Atypical carcinoid, Large cell neuroendocrine carcinoma and small cell carcinoma.

Carcinoid – Generally benign showing characteristic organoid arrangements as observed in other sites. The degree of nuclear atypia and mitosis, both typical and atypical, are important to diagnose carcinoid and atypical carcinoid.

Atypical carcinoid – Carcinoid with cytological atypia and increased mitosis (5-10 hpf) and foci of necrosis.

Small cell carcinoma – Accounts for 1-6% of cervical cancer. The cells are small, show characteristic nuclear molding with high N: C ratio  Five-year survival rate is 14 to 39%. (15,16 )

Large cell Neuroendocrine carcinoma.- This tumor shows focal adenocarcinomatous differentiation.(17 ,18,) Tumor cells are large with abundant cytoplasm, large nuclei, prominent nucleoli, and frequent mitosis. Tumors are aggressive and appear to have the same outcome as small cell carcinoma. (19)

Neuroendocrine differentiation is demonstrated in all these tumors by chromogranin A, Synaptophysin,& neuron-specific enolase. A variety of hormones like calcitonin, gastrin, serotonin may be present but clinically insignificance is limited. (20)

Undifferentiated carcinoma  – This is a carcinoma lacking specific differentiation. Differential diagnosis includes poorly differentiated squamous carcinoma, adenocarcinoma, glassy cell carcinoma & large cell neuroendocrine carcinoma.

Mesenchymal tumors

Leiomyosarcoma    – A Malignant tumor composed of smooth muscle. Macroscopically soft fleshy often with areas of hemorrhage and necrosis. The myxoid variant has a typical gelatinous appearance.

Histopathologically spindle cell hypercellular tumor showing interlacing fascicles, diffuse marked nuclear atypia, high mitotic activity, and atypical mitosis. Infiltrative border and vascular invasion are frequently seen. Epitheloid, myxoid and xanthomatous types have been reported.

At least two of the three criteria i.e. marked nuclear atypia, a mitotic rate higher than 10/ hpf and tumor necrosis are required for the diagnosis of leiomyosarcoma (21). A low mitotic count is typical of the myxoid variant. Antibodies to smooth muscle actin and or desmin may be used to demonstrate smooth muscle cell differentiation. These tumors should be differentiated from postoperative spindle cell nodules, diagnosis of which depends largely on the history of recent surgery.

Endometroid stromal sarcoma (low grade)-A sarcoma arising outside of uterine fundus composed of cells resembling endometrial stromal cells. The tumor may arise from cervical endometriosis and must be distinguished from stromal endometriosis and endometrial stromal sarcoma which has infiltrated cervix.

Undifferentiated Endocervical sarcoma – An endocervical sarcoma lacking stromal and other specific differentiation. (20,13,24)The tumor is composed of spindle or stellate cells with scanty cytoplasm, ill-defined cell border, oval and hyperchromatic nuclei. Tumor cells are arranged in fasciculated, storiform or sheet-like pattern. Prominent vascular pattern typical of endometrial stromal sarcoma is not seen. Stromal proliferation tends to encircle endocervical glands.

Sarcoma botryoides – Tumor composed of cells with round, oval or spindle-shaped nuclei some of which show differentiation towards skeletal muscle fibers. Macroscopically these tumors are usually polypoid and are pedunculated or sessile. .They have a glistening surface and soft consistency. The cut section is smooth, myxoid with areas of hemorrhage. Typically there is a dense cambium layer composed of closely packed cells with small hyperchromatic nuclei. Nuclei have open chromatin and inconspicuous nucleoli. The mitotic rate is high. Rhabdomyoblasts may be sparse. Islands of cartilage are frequently seen. (22)

Immunohistochemistry against actin, desmin or myoglobin is positive. Although the first two antibodies are not specific for skeletal muscle differentiation, they are more sensitive than myoglobin. An ultrastructural examination may reveal characteristic rhabdomyoblast differentiation such as thin filaments with z band material. An association with ovarian Sertoli Leydig cell tumor and cervical sarcoma botryoids has been described.

Alveolar Soft Part sarcoma –These are histologically similar to their counterpart elsewhere. Prognosis appears better than its counterpart in other sites. (23)

Angiosarcoma – Malignant tumor cells of which recapitulate the morphological features if endothelium. Neoplastic cells are immunoreactive for CD31, CD34,& Factor VIII related antigen.

Malignant Peripheral nerve sheath tumors (MPNST) are also similar to MPNST  occurring at other sites. These cells are positive for S100 and negative for HMB45, SMA, desmin, and myogenin (1424)

Other Malignant  tumors include Alveolar Rhabdomyosarcoma, liposarcoma, Epithelioid sarcoma, Osteosarcoma, and  Malignant fibrous Histiocytoma

Mixed epithelial and mesenchymal tumors of the cervix resemble those at other sites. These include Carcinosarcoma, Adenosarcoma, Wilms tumor Adenofibroma and adenomyoma

References

Tumors of Breast & Female genital organs, World Health Organization Classification of Tumors 2003.

StickRJ, ZainaR, Bundy BN, Askin FB, Woodward J, FetterB, PaulsonJA, Disaia PJ Stehman F B (1994) Evaluation and comparison of histopathologic grading system of epithelial carcinoma of uterine cervix. Int J GynaecolPathol13:99:108

Briders AC(2002) Squamous cell epithelioma of skin A Study of 256 cases (Ann Surg.73:141-160)

BethwaitePB, HollowayL J, Yeong ML, Thornton A(1993) Effect of tumor associated tissue eosinophilia on survival of women with stage IB carcinoma of uterine cervix.  J Clin.Pathol 46 1016 -1020.

Kurman RJ, TokiT, Schiffman MH,(1993) Basaoloid and wart carcinoma of vulva. Distinctive type of squamous cell carcinoma frequently associated with human papilloma viruses. Am J Surg Pathol17:133-145.

The pathogenesis of carcinoma of the vulva is diverse and includes both human papilloma virus (HPV)-positive and; Toki T, Kurman RJ, Park JS, Kessis T, Daniel RW, Shah KV. … Int J Gynecol Pathol. 1991;10(2):107–125

The pathogenesis of carcinoma of the vulva is diverse and includes both human papilloma virus (HPV)-positive and  Toki T, Kurman RJ, Park JS, Kessis T, Daniel RW, Shah KV. … Int J Gynecol Pathol. 1991;10(2):107–125

Brinck U, JakobC, Bau O, Fuzesi L (2000) Papillary Squamous cell carcinoma of uterine cervix report of 3 cases and a review of its classification, Int J Gynaec Pathol 19:231-235

Tseng C.J,Pao CC,Tseng LH,ChangCT,LaiCH,Soong YK,Hsueh S,jen H,1997.Lymphoepithelioma like carcinoma of uterine cervix associated with Epstein Barr virus & human papillomavirus Cancer 80:91-97

Lopez RiosF, Migueh PS, Bellas C, BallestinC, Harnandez L(2000)Lymphoepithelioma like carcinoma of uterine cervix; a case report studied by PCR for Epstein Barr virus Arch PatholLab Med 124:746-747

Lininger RA, Wistuba I, Gazdar A, Koeng Ctavassoli FA, Anbores-SaavedraJ1998.Human papillomavirus type 16 is detected in transitional cell carcinoma & squamo transitional cell carcinoma of cervix and endometrium  Cancer83 521-527

Maitra A, Wastuba II, Gibbons DGazdar AFAlbores SavendraJ 1999 Allelic loss at Chromosome3pare seen in HPV16 associated with transitional carcinoma of cervix Gynaecol Oncol74 361-368

Lotoki RJ, Krepart GV, Paraskevas M, Vadas G, Heywood M, Fung FK(1992) Glassy cell carcinoma of cervix bimodal treatment strategy.GynaecolOncol44:254-259.

Ferry JA, Scully RE(1988)-Adenoid cystic carcinoma and adenoid basal carcinoma of the uterine cervix. A Study of 49 cases. Am J Surg Path12.134-144

Anon(1999)-Cancer risk in BRCAII mutation carriers, The Breast Cancer  Liknage Cinsirtium H NatlCancer Inst9 1310-1316

BermudezA,VighiS,Garcia A,SardiJ(2001).Neuroendocrine cervical carcinoma a diagnostic and therapeutic challenge. Gynecol Oncol 82:32-39

Cui J, Antiniou AC, Dite GS, Siuthy MC, Venter DJ, Easton DF, Giles GG, Mc CredieMR.HopperJL.2001After BRCA1and BRCA2What next Multifactorial segregation analysis of three generations, population based Australian families affected by female breast cancer AmJHum Genet 68:420-31

Krishnamurthy S, JuhbluthAA, Busam KJ, RosaiJ(1988)-Uterine tumors resembling ovarian sex cord tumors have an immunophenotype consistent  with true sex cord differentiation: An J Surg Pathol.22:1078-1082

Gilks CB, Young RH, Gersell DJ, Clement PB.(1997): Large cell neuroendocrine carcinoma of the uterine cervix. A Clinicopathological study of 12 cases Am J Surg Pathol21:905-914

Seckl MJ, Mulholland PJ, Bishop AE, Teale JD, Ha; es CN, G;aserM, Watkins S, SecklJR(1999): Hypoglycemia due to insulin-secreting small cell carcinoma of Uterine cervix: N Eng J Med.341:733-736

Bell SW, Kempson RL, Hendrickson MR,(1994) Problematic Uterine Smooth muscle neoplasms A Clinicopathologic study of 213 cases. Am J Surg Pathol18:535-558

Daya DL, ScullyRE.(1988): Sarcoma botryoides of the uterine cervix in young women.A clinicopathologic study of 13 cases: Gynaecol Oncol29:290-304

Nielsen GP, Oliva E, Young RH, Rosenberg AE, Dickerson GR, Scully RE,(1995): Alveolar soft part sarcoma of the female genital tract a report of 9 cases and review of the literature. Int J Gynaecol Pathol14:283-292

Adenocarcinoma of the Cervix

Overview of Screening Technologies

Srabani Mittal, Cancer Foundation of India, Kolkata, India

Dipanwita Banerjee, Chittaranjan National Cancer Institute, Kolkata, India

Dr. Ranajit Mandal, Chittaranjan National Cancer Institute, Kolkata, India

Partha Basu, Early Detection and Prevention Section (EDP)/Screening Group (SCR), International Agency for Research on Cancer, Lyon, France

____________________________________________________________________________________

Guiding principles of cervical cancer screening

Screening target age and screening interval

Cancer screening aims to detect preclinical disease in a healthy population through systematic administration of a simple and safe test to all men/women belonging to a specified target age group. Since the primary objective of cervical cancer screening is to detect the premalignant lesions (known as cervical intraepithelial neoplasia; CIN), World Health Organization (WHO) recommended screening of women between 30 to 49 years of age.1At this age the women have the highest possibility of harboring the CIN 2 and CIN 3 lesions that are considered as true cervical cancer precursors or high grade lesions. Screening at younger age detects a large number of low grade lesions that regress of their own and the women are subjected to unnecessary biopsies and treatment. However, screening of HIV positive women should be initiated whenever they are sexually active. The upper age range can be extended to 55 or 60 years of age if resources permit. The frequency of screening in the screen negative women depends on the screening test being used and the resources available within the program. Too frequent screening should be avoided and the minimum interval between two rounds of screening should be five years. Programs with limited resources can extend the interval to 10 years if a highly sensitive screening test (like HPV DNA test) with a very low possibility of missing cases can be used. Even once a lifetime screening around the age of 40 years can reduce cervical cancer incidence by 30%.2 Achieving high coverage (>70%) of the target population rather than frequent screening is the most important determinant of the success of the screening program.

Confirmation of the diagnosis of screen-positive women

Administering the screening test is only a component of cervical cancer screening that involves community mobilization to motivate a large number of women to participate, training of all levels of service providers, ensuring further assessment of screen-positive women and ensuring appropriate treatment and follow up of the screen-detected abnormalities. It is extremely important to ensure appropriate investigations of the screen-positive women to establish the disease and treatment of the screen-detected cases of CIN and cancer. Traditionally colposcopy followed by directed biopsies has been used to confirm the diagnosis in the screen-positive women and subsequent treatment decisions were made based on histology report. However, this multiple visit approach (at least three) is very inconvenient for the women and the compliance is often poor. Several alternate strategies have been recommended and adopted to reduce the number of visits and improve the compliance of women. The positive predictive value of colposcopy is reasonably high to detect CIN 2/CIN 3 diseases and treatment can be done based on colposcopy diagnosis alone. Such a strategy of ‘colposcopy and treat, also known as seeing and treat’ improves compliance to treatment and is convenient to the women though some over-treatment is unavoidable. Over-treatment is acceptable as the treatment methods are simple and safe and the treated women with negative histology will require less intensive follow up. In more basic settings where organizing colposcopy and histopathology is challenging, a simpler approach like direct treatment of the screen-positive women (‘screen and treat’) is also recommended.1This will be further discussed in the subsequent sections.

A second screening test is sometimes recommended for the women positive on the primary screening test. The second test is called the triaging test and the women positive on both the tests are only referred for colposcopy. Such a triaging strategy is used if the primary screening test is less specific and/or if the colposcopy services are insufficient and expensive.

Treatment of women with premalignant disease

To achieve the desired effect of the program to reduce cervical cancer burden, the screen-detected CIN 2/CIN 3 lesions must be treated appropriately. CIN lesions can be treated by ablation or by excision. Ablative techniques are simpler, less expensive, can be performed by trained non-clinicians and have very low complication rates. Two different ablative techniques are available – Cryotherapy and Thermo-coagulation; both of them are equally effective and safe even when performed by nurses3.A major advantage of ablative therapies is that the treatment can be done in primary care or secondary care settings. The excision methods of treatment include large loop excision of the transformation zone (LLETZ) and cold knife conization (CKC). In both the techniques the entire transformation zone is excised and submitted for histopathological evaluation. While LLETZ is much simpler, requires local anesthesia only and has a low complication rate, CKC has to be done under regional or general anesthesia, requires a higher level of technical expertise and has higher complication rates.

All the screen-positive women need to be assessed for suitability of treatment by ablative techniques using the following criteria (with colposcopy or with the naked eye after application of 3-5% acetic acid):

Squamocolumnar junction (SCJ) is fully visible

Type 1 Transformation Zone (TZ)

Lesion occupying less than 75% of cervix

Lesion not extending to endocervix or vagina

There is no suspicion of invasive or glandular disease

If suitable, the lesions may be treated by Cryotherapy or Thermocoagulation without waiting for histopathological verification. Only the women with lesions not suitable for Cryotherapy should be referred for treatment by exciscion.4

Follow up of the women after treatment is also important. Women treated for CIN 2 or CIN 3 have a much higher risk of developing cervical cancer even after 10 years of treatment compared to normal women.5 Post-treatment initial follows up should be done after 1 year either by the screening test or by colposcopy. The protocol for subsequent check-ups varies from program to program. Usually, women are sent back to routine screening if they are normal on 2 or 3 consecutive rounds of yearly screening.

Cervical cancer screening tests

Cervical screening tests such as conventional cytology (Pap smear), LBC, HPV testing, and VIA can detect cervical precancerous lesions in apparently healthy, asymptomatic women.

Cytology

The most widely used cervical screening test is cytology. High-income countries have integrated cytology screening services in medical and public health services and have achieved high coverage through better program organization. This has resulted in substantial declines in cervical cancer incidence and mortality over time6. Some of the middle-income countries in South and Central America and Asia also implemented population-based cervical cancer screening using cytology for a few decades. However, these programs were largely ineffective in reducing cervical cancer burden due to poor coverage with screening, treatment, and follow-up care and lack of quality assurance.7 Majority of the LMICs have neither initiated nor can initiate and sustain quality assured cytology screening programs in their underdeveloped and fragmented health services with several competing priorities, lack of resources and trained manpower. Cytology needs good quality laboratory infrastructure, rigorous monitoring and supervision, highly skilled technicians or pathologists and a good system of recalling the screen positives. All these are very challenging to implement in low resourced countries.

Cytology results are most widely reported using The Bethesda system (TBS); the major categories of 2014 TBS is given in Table 1.8 The threshold for positive cytology for referral for triaging/diagnostic investigations such as colposcopy may be at ASCUS or LSIL in various settings. All women with ASC-H, LSIL or HSIL report on cytology must have a further evaluation with colposcopy. The management options for ASCUS smears are either repeat smear after 6 months or HPV test (triaging) or direct colposcopy.

Table 1: The Bethesda System 2014 – major reporting categories

Cytology smears are prepared by spreading the cervical cell specimen collected using a spatula and cervical brush on to a glass slide which is then fixed and stained using Papanicolaou staining. Cytology is a highly subjective and provider dependent test with varying performance between laboratories and cytologists reading the smears.

High frequency of cervical inflammation in developing countries contributes to a significant amount of inflammatory debris in Pap smear, posing major challenges in interpretation and reporting. The high false-negative rate (14-33%) is largely due to limitations of sampling and smear preparation. Quality assurance in preparing, fixing, staining, reading and reporting the smears is critical for accurate results. The sensitivity and specificity of a single, quality assured Pap smear to detect CIN 2+ lesions is around 60% and 95% respectively.9,10

Liquid-based cytology (LBC)

LBC offers an improved test specimen collection with a lower frequency of unsatisfactory smears, lower debris, and shorter time needed for interpretation compared to conventional cytology. It is the first technical advancement in cervical cytology in more than 50 years. For LBC, the cervical cell specimen is washed into a vial of liquid transport medium and filtered and a random sample is presented as a thin layer on a slide avoiding overlapping of cells. LBC samples may be used for reflex HPV and another molecular testing (for triaging in case of ASCUS report). However, LBC has more or less equivalent sensitivity and specificity as compared to cytology for the detection of CIN 2 or worse lesions.11

HPV testing

HPV testing involves detecting HPV DNA or mRNA of two oncoproteins (E6 and E7) in cervical cell samples collected by pelvic examination or by self- sampling. HPV testing is the most accurate, reproducible and provider-independent cervical screening test. Its sensitivity to detect CIN2+ lesions exceeds 90% and CIN3+ exceeds 95%; it is more sensitive but less specific than cytology12

If available, HPV testing is the most suitable test to screen women above the age of 30 years, since HPV negative women can be left alone for at least 7-10 years because of the high Negative Predictive Value (NPV) of a negative HPV test for CIN3 and cervical cancer13,14. Characteristics of some selected clinical HPV tests are given in Table 2.

Table 2. Characteristics of selected common, commercially available clinical HPV detection tests

HPV tests have been widely evaluated and compared with cytology in several randomized controlled trials. A cluster-randomized trial in India involving around 135,000 women aged 30-59 years demonstrated that following a single round of HPV testing, a significant 53% reduction in the incidence of advanced cancer (stage II+) and a 48% reduction in cervical cancer mortality could be achieved.14 In four European randomized trials involving 176,464 women aged 20-64 years, HPV testing was compared with cytology screening. These women were followed up for a median of 6.5 years and the relative efficacy of HPV- versus cytology-screening for prevention of invasive cancer was compared. HPV testing provided 60-70% greater protection against invasive cervical carcinomas compared with cytology15 Data from the randomized trials in Europe and India14,15 support initiation of HPV based screening and extension of screening intervals to at least 5 years. WHO recommends repeat screening after 10 years in HPV negative women in resource-limited countries16.

Cervical cell samples are collected with a brush in a liquid medium for the HPV test. The test positive women need further evaluation by colposcopy. In some settings HPV positivity may be high and sending all HPV positive women to colposcopy may overburden the services. Reflex cytology testing or genotyping for HPV 16 or 18 may be used to triage HPV positive women before referral for colposcopy; In Low Middle-Income Countries (LMICs), HPV positive women may be triaged by VIA screening in a two-visit ‘screen and treat’ approach.

HPV testing is the most promising test for cervical screening in the future including LMICs, given the inevitability of widespread HPV vaccination leading to a lower prevalence of CIN. In such a scenario, an objective test such as HPV testing will be the most suitable screening test. Already many European countries, Australia and some Latin American countries have introduced the HPV test as the primary screening test. The collection of high vaginal samples by the women themselves (self-collection) is feasible and has almost the same accuracy as that of the provider collected cervical samples.17 Such a strategy can significantly improve the compliance of the women and improve the logistics of the program.

Visual screening tests

Visual inspection with acetic acid (VIA) involves naked eye visualization of the cervix 1 minute after the application of 3-5% dilute acetic acid under bright light. Test results are reported as negative, positive or suspicious of invasive cancer and their criteria18 are given Table 3.

Table 3: Criteria for different VIA test results

A positive test is defined by the appearance of a well-delineated, dense acetowhite area abutting the squamocolumnar junction (SCJ) in the transformation zone (TZ) of the cervix. It is a suitable test in premenopausal women under 50 years of age when the TZ is fully visualized on the ectocervix. Interpretation of tests is difficult in postmenopausal and older women. VIA is not ideal for women above 50 years of age or when the SCJ is not fully visible. Figure 1 shows images of the cervix for different VIA outcome categories.

Figure 1: VIA outcome categories

VIA is a simple, feasible and affordable point of care test, providing immediate results enabling diagnosis and/or treatment to be carried out in the same visit for screen-positive women. A wide range of health professionals including doctors, nurses, midwives, and primary health care workers can be trained to perform VIA after a short period of training. The infrastructural need is minimal and the consumables are universally available.

VIA has been extensively evaluated in a variety of settings. Although the pooled sensitivity and specificity of VIA to detect CIN 2 + lesions were 80% and 92% respectively in a pooled analysis19, these values varied widely in different settings due to the subjective and provider dependent nature of the test, varying quality of reference standard investigations and differing age groups included in the studies. VIA also suffers from high false-positive rates.

VIA screening was followed by a 31% reduction in cervical cancer mortality in a randomized trial in Mumbai20 In a randomized trial in South India, VIA screening was associated with a 25% decline in cervical cancer incidence and a 35% reduction in mortality.21 This evidence culminated in the launching of a population-based VIA screening program in the state of Tamil Nadu in India. A large number of nurses have been trained in providing VIA throughout the primary health centers in the state and 55% of targeted 15 million have been screened through the first round of a VIA screening program during 2012-2014.

The visual tests seem to perform better in HIV-infected women than in the general population due to the increased prevalence of large high-grade lesions.22,23

The safety, acceptability and effectiveness of a single visit ‘screen and treat’ approach (SVA) in which VIA positive women eligible for cryotherapy, are treated in the same setting have been well established24,25,26 Women with precancerous lesions not eligible for Cryotherapy may be referred for loop electrosurgical excision procedure (LEEP) for diagnosis and treatment. The SVA paradigm represents a major innovation for scaling up cervical cancer screening in LMICs and has been adopted by many countries in sub-Saharan Africa and Asia27. Primary care practitioners should utilize every opportunity in routine health care interactions to screen women aged 30 years and above with VIA and investigate and treat screen-positive women. Despite all its limitations, implementing VIA screening in low-income countries provides a realistic approach to building up infrastructure and human resources that may facilitate the introduction of affordable HPV screening in the future.

Visual inspection after application of Lugol’s iodine (VILI) is another simple inexpensive visual screening technique. The test relies on the identification of Iodine negative areas on the cervix after the application of Lugol’s Iodine (Figure 2). VILI has not been widely evaluated and has high false positivity. The consumables are expensive and are not readily available. The test is not yet recommended by the WHO for routine screening.

Figure 2: Bright yellow Iodine negative area after application of Lugol’s Iodine

Management of women with positive screening tests

The ultimate aim of cervical cancer screening is to reduce the disease burden by detecting the precancerous lesions early and managing them appropriately.

Management of women positive on the HPV test

There is no single definitive strategy for the management of HPV positive women. The management approaches generally depend on factors like available resources, logistics, and program guidelines. As cervical cancer is a rare outcome of HR-HPV infections, management strategies are based on an assessment of risk thresholds that identifies the small percentage of women who are likely to develop cervical precancer or cancer. To allow risk stratification and subsequent identification of at-risk women, additional tests (Triage tests) are indicated depending on the availability of the tests and the feasibility of doing it. Triage tests like Cytology, HPV genotyping for HPV 16/18, test for E6/E7 mRNA, p16/Ki-67 dual staining and VIA may be used for risk-based management.

As the majority of the HPV infections are transient, most of the HPV positive women will not have any clinically detectable disease on Colposcopy. Referral of all HPV positive women for Colposcopy will lead to overburdening of the system, particularly in the context of LMICs with limited Colposcopy facility. This approach is also likely to increase the cost of public health programs and should be considered for situations where performing triage tests are not feasible or ensuring follow up of positive women is challenging.

Direct treatment of HPV positive women has been widely promoted, especially in LMICs, to ensure a linkage between screening and treatment and minimize loss to follow up. In settings where colposcopy and histopathology services are not available, WHO recommendation of ‘screen and treat’ strategy has strongly encouraged treatment of HPV positive women by ablative treatment methods like cryotherapy, provide the lesion suits the eligibility criteria for ablation.4 Various management strategies for HPV positive women is summarized in Figure 3.

Management of women positive on VIA test

VIA is a real-time test that allows for making treatment decisions in the same sitting as screening. In the context of LMICs where there is no or limited facility for colposcopy and histology, WHO guidelines for ‘screen and treat’ strategies recommend immediate treatment of VIA positive cases by ablative method if the lesion characteristic fulfills the criteria for ablative treatment.4 In situations where Colposcopy and biopsy facilities are available, the VIA positive women can be referred for evaluation by Colposcopy. Treatment can be done either in the same sitting as Colposcopy (‘See and treat’) or based on histopathology report. The management options for VIA positive women are given in Figure 4.

Management of women with cytological abnormalities

The management options for women with cytological abnormalities are presented in Figure 5 and discussed in details in the chapter 'Role of colposcopy in the management of women with abnormal cytology'

Figure 3: Flowchart on the management of women positive on the HPV test

Figure 4: Flowchart on the management of women positive on VIA test

Figure 5: Flowchart on the management of women with cytological abnormalities

References:

WHO guidelines for screening and treatment of precancerous lesions for cervical cancer prevention;http://apps.who.int/iris/bitstream/10665/94830/1/9789241548694_eng.pdf; accessed on 12th August,2017-08-15

IARC Handbook of Cancer Prevention Volume 10, https://www.iarc.fr/en/publications/pdfs-online; accessed on 12th August,2017-08-15

Dolman L, Sauvaget C, Muwonge R, et al: Meta-analysis of the efficacy of cold coagulation as a treatment method for cervical intraepithelial neoplasia: A systematic review. BJOG 121:929-942, 2014

WHO guidelines: WHO guidelines for screening and treatment of precancerous lesions for cervical cancer prevention WHO, Geneva, Switzerland (2013)

McCredie MR, Sharples KJ, Paul C, Baranyai J, Medley G, Jones RW, et al. Natural history of cervical neoplasia and risk of invasive cancer in women with cervical intraepithelial neoplasia 3: a retrospective cohort study. Lancet Oncol. 2008 May 1;9(5):425–34.

IARC Handbooks of Cancer Prevention. Vol. 10. Cervix Cancer Screening IARC, Lyon, France (2005)

R Murillo et al. Cervical Cancer Screening Programs in Latin America and the Caribbean. Vaccine 26 Suppl 11, L37-L48. 2008 Aug 19.

Nayar R, Wilbur DC (2015) The Pap Test and Bethesda 2014.Acta Cytol 59:121–132

Cuzick J, Myers O, Hunt WC, et al. A Population-based Evaluation of Cervical Screening in the United States: 2008–2011. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology. 2014;23(5):765-773. doi:10.1158/1055-9965.EPI-13-0973.

Nanda K et al. Accuracy of the Papanicolaou test in screening for and follow-up of cervical cytologic abnormalities: a systematic review. Annals of Internal Medicine, 2000, 132: 810-819.

Whitlock EP Vesco KK Eder M Lin JS Senger CA Burda BU Liquid-based cytology and human papillomavirus testing to screen for cervical cancer: a systematic review for the U.S. Preventive Services Task Force.Ann Intern Med2011155687-97, W214-5.

Marc Arbyn, GuglielmoRonco, AhtiAnttila, Chris J.L.M. Meijer, Mario Poljak, Gina Ogilvie, George Koliopoulos, Pontus Naucler, RengaswamySankaranarayanan, Julian PetoVaccineVolume 30, Supplement 5, 20 November 2012, Pages F88-F99

Mark Schiffman, Andrew G. Glass, Nicolas Wentzensen, Brenda B. Rush, Philip E. Castle, David R. Scott, Julie Buckland, Mark E. Sherman, Greg Rydzak, Peter Kirk, Attila T. Lorincz, Sholom Wacholder and Robert D. Burk. A Long-term Prospective Study of Type-Specific Human Papillomavirus Infection and Risk of Cervical Neoplasia Among 20,000 Women in the Portland Kaiser Cohort Study Cancer Epidemiol Biomarkers Prev July 1, 2011 (20) (7) 1398-1409; DOI:10.1158/1055-9965.EPI-11-0206

Sankaranarayanan R, Nene BM, Shastri SS, Jayant K, Muwonge R, Budukh AM, Hingmire S, Malvi SG, Thorat R, Kothari A, Chinoy R. HPV screening for cervical cancer in rural India. New England Journal of Medicine. 2009 Apr 2;360(14):1385-94.

Ronco G, Dillner J, Elfström KM, Tunesi S, Snijders PJ, Arbyn M, Kitchener H, Segnan N, Gilham C, Giorgi-Rossi P, Berkhof J. Efficacy of HPV-based screening for prevention of invasive cervical cancer: follow-up of four European randomized controlled trials. The lancet. 2014 Feb 14;383(9916):524-32.

World Health Organization (WHO). WHO guidance note: Comprehensive cervical cancer prevention and control – a healthier future for girls and women.[accessed on 15th August 2017]. Available from: http://www.who.int/reproductivehealth/publications/cancers/9789241505147/en/

Safaeian M1, Kiddugavu M, Gravitt PE, Ssekasanvu J, Murokora D, Sklar M, Serwadda D, Wawer MJ, Shah KV, Gray R. Comparability of self-collected vaginal swabs and physician-collected cervical swabs for detection of human papillomavirus infections in Rakai, Uganda. Sex Transm Dis. 2007 Jul;34(7):429-36.

Sankaranarayanan& Wesley. A Practical Manual on Visual Screening for Cervical Neoplasia IARC Technical Publication No. 41; Edited by Sankaranarayanan R, Wesley RS:2003

Sauvaget C, Fayette JM, Muwonge R, Wesley R, Sankaranarayanan R. Accuracy of visual inspection with acetic acid for cervical cancer screening. Int J Gynaecol Obstet. 2011 Apr;113(1):14-24. doi: 10.1016/j.ijgo.2010.10.012. Epub 2011 Jan 22.

McCredie MR, Sharples KJ, Paul C, Baranyai J, Medley G, Jones RW, et al. Natural history of cervical neoplasia and risk of invasive cancer in women with cervical intraepithelial neoplasia 3: a retrospective cohort study. Lancet Oncol. 2008 May 1;9(5):425–34.

Sankaranarayanan R, Esmy PO, Rajkumar R, Muwonge R, Swaminathan R, Shanthakumari S, Fayette JM, Cherian J. Effect of visual screening on cervical cancer incidence and mortality in Tamil Nadu, India: a cluster-randomized trial. Lancet. 2007 Aug 4;370(9585):398-406.

22. Joshi S, Sankaranarayanan R, Muwonge R, Kulkarni V, Somanathan T, Divate U. Screening of cervical neoplasia in HIV-infected women in India. AIDS. 2013 Feb 20;27(4):607-15. doi: 10.1097/QAD.0b013e32835b1041.

23. Vikrant V. Sahasrabuddhe, Ramesh A. Bhosale, Anita N. Kavatkar, Chandraprabha A. Nagwanshi, Smita N. Joshi, Cathy A. Jenkins, Bryan E. Shepherd, Rohini S. Kelkar, Seema Sahay, Arun R. Risbud, Sten H. Vermund, and Sanjay M. Mehendale. Comparison of visual inspection with acetic acid and cervical cytology to detect high-grade cervical neoplasia among HIV-infected women in India Int J Cancer. 2012 Jan 1; 130(1): 234–240.Published online 2011 Apr 20.

24. Sankaranarayanan R., Rajkumar R., Esmy P., Fayette J., Shanthakumary S., Frappart L., Thara S., Cherian J. Effectiveness, safety and acceptability of ‘see and treat’ with cryotherapy by nurses in a cervical screening study in India. Br. J. Cancer. 2007;96:738–743. doi: 10.1038/sj.bjc.6603633

25. Mwanahamuntu MH, Sahasrabuddhe VV, Kapambwe S, Pfaendler KS, Chibwesha C, Mkumba G, et al. (2011) Advancing Cervical Cancer Prevention Initiatives in Resource-Constrained Settings: Insights from the Cervical Cancer Prevention Program in Zambia. PLoS Med 8(5): e1001032. https://doi.org/10.1371/journal.pmed.1001032

26. Parham GP, Mwanahamuntu MH, Kapambwe S, Muwonge R, Bateman AC, Blevins M, et al. (2015) Population-Level Scale-Up of Cervical Cancer Prevention Services in a Low-Resource Setting: Development, Implementation, and Evaluation of the Cervical Cancer Prevention Program in Zambia. PLoS ONE 10(4): e0122169. https://doi.org/10.1371/journal.pone.0122169

27. Sankaranarayanan R, Nessa A, Esmy PO, et al. Visual inspection methods for cervical cancer prevention Best Pract Res Clin Obstet Gynaecol. 2012. 26(2):221–232

The LAST Terminology: The Lower Anogenital Squamous Terminology

Carcinoma cervix is not the only cancer caused by the Human Papilloma Virus(HPV). High risk HPV is implicated in the causation of various other cancers like anal cancers, oropharyngeal cancers, vulval cancers, vaginal cancers, and penile cancers. Low risk HPV viruses similarly cause infections of the perianal and genital region in males and females. Currently, the histopathological terminology for describing the lesions caused by low and high risk HPV viruses does not reflect, either the HPV type or the risk prognostication. This is because the terminology has evolved before the understanding of the role of HPV and is a descriptive terminology for the lesion as seen grossly or under the microscopy. Thus we have terms such as Wart, Bowen's disease, Erythroplasia of Queret, which are used by dermatologists. The Bethesda classification incorporated an understanding of HPV biology and mechanism of pathogenesis and cytologically the intraepithelial lesions were divided into two tiers1; those of low and high squamous intraepithelial lesions. However histologically we still have three grades of dysplasia: mild, moderate and severe.

To address all these issues a project was undertaken by the College of American Pathologists and American Society for Colposcopy and Cervical Pathology. The report with the consensus of all working groups is The Lower Anogenital Squamous Terminology (LAST). The report hopefully has the LAST word considering numerous changes and classifications evolved so far. The recommendations encompass the HPV associated lesions of the squamous epithelium of Cervix, vagina, and Vulva in females, penis, and scrotum in males and anal region and perianal skin in both genders.

Biologically HPV infects squamous epithelium in two basic ways.2,3 In first, the virus, often of type 6 & 11, completes its life cycle in the squamous epithelium, produces virions and while growing inside the epithelium, it produces changes secondary to an infection, seen histologically as grade 1 squamous intraepithelial lesions. The condyloma of skin and genital organs is another manifestation of this type of infection.

In a second way, the virus integrates with the host genome with overexpression of viral oncogenes, leads to epithelial cell proliferation and expansion of relatively undifferentiated clone, expressing persistent viral antigens. These are caused by oncogenic viruses of type 16 & 18, mediated through two viral proteins E 6 and E7 which interfere with cell cycle regulation, unlock the regulatory mechanisms. These proliferating cells evade apoptosis, proliferate and gather further mutations to develop into cancer. A hallmark of these lesions is persistence and overexpression of viral antigens which potentially can differentiate the pre-cancer from other lesions, that mimic dysplasia.

The rationale for Unified Terminology for Lower Anogenital region is as follows.

1. Epithelial infections of HPV are biologically similar.

2. Each cytological or histological sample is a representation of the true biology of viral infection then and in the epithelium studied. Thus over some time and in multiple biopsies true biological picture of viral infection will emerge

3. The diagnostic variations due to these facts can be improved by the use of biomarkers and by assigning terminology that correlates with biologically relevant categories

The recommendations of the LAST project group are as follows:

Squamous Intraepithelial Lesion: All pre-invasive lesions of the lower anogenital tract which are HPV associated, to be called Intraepithelial Neoplasia with IN suffix and labeled as per site (Refer Table 1)

The Intraepithelial neoplasia is to be reported as a two-tier system: Low grade and High grade

The rationale for recommending a 2 tier nomenclature: The current understanding of HPV biology does not support a 3 tier nomenclature.The -IN2 is an admixture of Low and High grade of SIL that can not be separated by morphology in H & E sections. Biomarkers can help to classify this accurately. In young girls and adolescents –the IN2 category is managed conservatively and thus its use is maintained in this age group. Thus an HSIL in the cervix will be reported as HSIL-CIN3.

Low SIL is defined as a proliferation of squamous or metaplastic cells with abnormal nuclear features of increased nuclear size, irregular nuclear membrane, increased n: C ratio and little cytoplasmic maturation in the lower third. Mitosis is seen in the lower third.

Koilocytic cells that show nuclear pleomorphism, multinucleation, perinuclear halo are seen along with. (Figure 1)

High SIL is defined as a proliferation of squamous or metaplastic cells with abnormal nuclear features of increased nuclear size, irregular nuclear membrane, increased n: C ratio and little cytoplasmic maturation in middle third\superficial layers. Mitosis is not confined to the lower third. (Figure 2)

Special circumstances: where p16 staining is recommended

1. Marked atypia in a Low SIL

In thin ( less than 10 layers thick) epithelium(Figure 3)

Atypia in the keratinising epithelium is considered high grade

Full-thickness Dysplasia extending into endocervical glands

Superficially Invasive Squamous Cell Carcinoma: This term is reserved for minimally invasive squamous cancers which are excised and do not show positive resection margins

1.Superficially Invasive Squamous Cell Carcinoma of Cervix (SISCCA of Cervix): It is defined as an invasive squamous cell carcinoma that is not a grossly visible lesion AND has an invasive depth of 3mm from the basement membrane of the point of origin, AND Has a horizontal spread of 7mm to the maximal extent, AND Has been completely excised. (Figure 4)

A report of Superficially Invasive Squamous Cell Carcinoma Of Cervix should include

a)Presence or absence of lymphovascular invasion

b)Presence of number and size of independent multifocal carcinoma.

In a biopsy, if the invasive carcinoma exceeds dimensions of SISCCA, it should be reported as such.

In a biopsy showing SISCCA and the dimensions of SISCCA within the parameters, if the margins are

positive then it is reported as at least a SISCCA

In carcinoma cervix, multiple biopsies reveal a true picture of the biological behavior of squamous

intraepithelial lesion. These biopsies should be properly numbered as per location and colposcopic

findings and documented

The criteria for Anal intraepithelial lesion and PERIAnal SISCCA are the same as those for carcinoma Cervix5,8

The morphological hallmark of squamous cell carcinoma in these lesions is abnormal cellular

proliferation with nuclear atypia, enlargement, pleomorphism, changes in nuclear chromatin

texture and irregular nuclear borders.

No recommendation of the use of term SISCCA is made for early invasive vaginal carcinoma due to rarity

and lack of separate treatment modality for early lesions

Vulvar SISCCA is defined as an AJCC T1a (FIGO 1A) vulvar cancer i e Tumor 2 cm or less size, confined

to the vulva or perineum AND Stromal invasion of 1mm or less.14,15

No change in the current definition of T1a vulvar cancer is recommended4,7,9,10

Anal and Perianal SISCCA has definition same as cervical SISCCA8

SISCCA of Penis is defined as T1a of AJCC i.e.tumor invading only the subepithelial connective tissue9

. There is no lymphovascular invasion and morphologically it is not poorly differentiated.

Table 1: The Lower Anogenital Squamous Terminology

The calculation of the depth of invasion is defined as the distance from the epidermal stromal junction of the adjacent most dermal papilla to the deepest point of tumor invasion. (Figure 5).When HSIL colonizes an endocervical gland, the depth should be measured from the basement membrane of the gland.

Figure 5: Measurement of Depth of Invasion: done from the junction of the adjacent dermal papilla to the deepest point of invasion

Biomarker Recommendations: Biomarker recommended is p16INK4a. No benefit of the addition of other biomarkers like p63 or ki67 is found in problem-solving in the differentiation of HSIL from mimics or LSIL. The positivity of p16 should be strong and block-like(Figure 6)

Routine use of biomarkers is not advocated.

Use of p16 is recommended in the following situations12,13,17:

To differentiate HSIL from morphological mimics like squamous metaplasia, atrophy, reparative changes.

When the lesion is diagnosed as CIN 2, to better classify it as LSIL or HSIL for further management, p 16 can be useful

To resolve professional disagreement due to inter-observer variation

In High risk colposcopy referral situations where biopsy shows a lesion LSIL or lower but colposcopy, cytology or clinical features indicate a higher lesion

There is no role in the use of p16 to differentiate between LSIL and its mimics

There has been a more accurate classification of HSIL with use of biomarkers in these situations

The proliferation marker ki67 which trends with p16 is not recommended unless p16 is inconclusive 19,20 (Figure 8)

References:

Stoler MH. Human Papillomavirus and cervical neoplasia: a model for carcinogenesis. Int J GynecolPathol2000;19:16-28

Soloman D, Davey D, Kurman R et al 2001 Bethesda System: terminology for reporting cervical cytology; JAMA 2002;287:2114-9

Nayar R, Wilbur DC: The PAP test and Bethesda 2014: Acta Cytologica 2015, 59: 121-32

Jones RW.Vulval Intraepithelial Neoplasia: Current perspectives. Eur. J Gynecol Oncol; 22: 393-402

Machlek DA, Poyten m, Jin F, et al; Anal Human Papillomavirus infection and associated neoplastic lesions in men who have sex with men: A systematic review and meta-analysis: Lancel Oncol.2012; May, 13(5):487-500

Yifei Hu, Han zui Quin, Jianping Sun, et al.Anal human papillomavirus infections among HIV infected and uninfected men who have sex with men in Beijing China, J Acquir Immune Defic Syndr 2013, Sep1: 64(1): 103-114

IARC working group on the evaluation of carcinogenic risks to humans: Human Papillomaviruss, Lyon, France: Internation Agency for Research on Cancer:1995

Billimoria KY, Bentrem DJ, Rock CE, et al, Outcomes and prognostic factors for squamous cell carcinoma of the anal canal: Analysis of patients from National Cancer DataBase: Dis Col Rectum 2009; 52:624-631

Edge SB, Byrd Dr, Compton CC, et al; eds: AJCC Cancer Staging Manual. 7th Ed New York; Springer 2010

Sideri M, JonesRW, Wilkinson EJ et al; Squamous vulvar Intraepithelial Neoplasia: 2004; Modified terminology, ISSVD vulvar oncology subcommittee: J Reprod Med 2005; 50: 807-10

Kurman R J, Ronett J, Sherman ME, et al, eds: Atlas of Tumor Pathology: Tumors of cervix, vagina, and vulva; Washington DC, Armed Forces Institute of Pathology, American Registry of Pathology, 2010

Dijkstra Mg, Heidema D A, De Roy Sc, et al, p16INK4a immunostaining as an alternative to histology review for reliable grading of cervical Intraepithelial Lesions. J Clin Pathol 2010,63: 972-7

Del Pino M, Garcia S, Fuste V, et al, Value of p 16(INK4a) as a marker of progression\regression in cervical intraepithelial neoplasia grade 1: Am J Obstet Gynecol 2009; 201:488 e1-7

Reyes M C, Cooper K; An update on vulvar intraepithelial neoplasia: terminology and practical approach to diagnosis; J Clin Pathol 2014, Apr 67(4) 290-4

Hoang LN, Park J, Soslow RA, et al, Squamous Precursor lesions of the vulva: Current classification and diagnostic challenges. Pathology.2016 Jun48(4) 291-302

Darragh T M, Colgan T J, Cox JT, et al The Lower Anogenital Squamous Terminology standardization project for HPV associated lesions: background and consensus recommendations from the College of American Pathologists and American Society for Colposcopy and Cervical Pathology. Arch Pathol Lab Med:2012 oct 136(10)1266-97

Clinton Lk, Miyazaki K, Ayabe A, et al, The LAST guidelines in clinical practice, Implementing recommendations for p16 use Am J Clin Pathol 2015 Dec, 144(6)844-9

Gustinucci D, Passamonti B, Casarini E, et al, Role of p16(INK4a) cytology testing as an adjunct to enhance the diagnostic specificity and accuracy in human papillomavirus positive women within an organized cervical cancer program Acta Cytol 2012: 56(5)506-14

19Darragh TM, Colgan TJ, Thomas Cox J, Heller DS, Henry MR, Luff RD, McCalmont T, Nayar R, Palefsky JM, Stoler MH, Wilkinson EJ, Zaino RJ, Wilbur DC; Members of the LAST Project Work Groups. The Lower Anogenital Squamous Terminology Standardization Project for HPV-Associated Lesions: background and consensus recommendations from the College of American Pathologists and the American Society for Colposcopy and Cervical Pathology.Int J Gynecol Pathol. 2013 Jan;32(1):76-115. doi: 10.1097/PGP.0b013e31826916c7. Erratum in: Int J Gynecol Pathol. 2013 Jul;32(4):432. Int J Gynecol Pathol. 2013 Mar;32(2):241

20.Hebbar A1, Murthy VS1Role of p16/INK4a and Ki-67 as specific biomarkers for cervical intraepithelial neoplasia: An institutional study. J Lab Physicians. 2017 Apr-Jun;9(2):104-110.

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