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ORIGINAL CONTRIBUTIONS

Received: February 17^th 2015

Accepted: March 4^th 2015

History of the development of screening tests for cervical cancer

Yelda A. Herrera,^a Patricia Piña-Sánchez^b

^aRegistro de Cáncer, Unidad de Investigación Médica Yucatán, Instituto Mexicano del Seguro Social, Mérida, Yucatán

^bLaboratorio de Oncología Molecular, Unidad de Investigación Médica en Enfermedades Oncológicas, Centro Médico Nacional Siglo XXI, Distrito Federal

México

Communication with: Patricia Piña-Sánchez

Telephone: 55 5627 6900, extensión 22710

Email: 1307patricia@gmail.com

Cervical cancer (CC) is one of the best known malignancies. Currently, it is accepted that the etiological factor is persistent infection with high-risk human papillomavirus (HPV). Even before the identification of its etiological factors, methods such as Pap cytology and colposcopy were developed as tools for early diagnosis on CC and its precursor lesions. At the time when such tests were being developed, they were not fully accepted by the scientific community of the time; however, as time went by, the dissemination of knowledge, and more extensive application, these tests were finally included within the international guidelines. The implementation of programs with adequate coverage and quality allowed a significant reduction in the incidence and mortality of CC. However this did not occur widely, and CC is still a public health problem in developing countries. From the epidemiological and molecular viewpoint, knowledge on HPVs laid the foundations for the development of new prevention strategies based on vaccination and molecular detection of the causal agent, currently accepted as strategies for primary and secondary prevention. It is expected that the implementation of these strategies will have a greater impact on the control on CC and other malignancies associated with HPV infection.

Keywords: Uterine cervical neoplasms, Human papillomavirus, Diagnosis, History.

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From Papanicolaou to colposcopy

In 1914 Dr. George Nicolas Papanicolaou (1883-1962) entered the department of anatomy at Weill Cornell University Medical College in New York to analyze the menstrual cycle of guinea pigs by cytology. He published his first article in 1915.¹ In 1920 he began cytological studies in humans, and his wife was his first long-term test subject; he then focused on physiological studies of the ovary and uterus by cytology, and in 1925 published his study on the early diagnosis of pregnancy by vaginal cytology, in which he described navicular cells. In parallel he studied cytology of cancer patients, a work presented at the third Race Betterment Conference in Battle Creek, Michigan in 1928, published in Proceedings of the Conference in the same year, and reissued in 1973.² Unfortunately, this work was not well received by the scientific community of the time, which caused frustration for Dr. Papanicolaou.³ Simultaneously, in 1927 Dr. Aurel Babes (1886-1962) presented his work on cervical cancer diagnosis by smears to the Bucharest Society of Gynecology, published in 1928.⁴    

Moreover, in 1930 Hans Peter Hinselmann (1884-1959), professor of gynecology at the University of Bonn, Germany, published a chapter on the etiology, symptoms, and diagnosis of cervical cancer, in the third edition of the book Handbook on Gynecology; however, he was not satisfied with the methods of visual inspection and palpation to diagnose cervical cancer, and he wanted to analyze large images of the cervix with three-dimensional binocular vision. This led to the construction of the colposcope, which was first used in December 1924; the development and use of the colposcope itself were published the following year. His efforts continued to improve the images obtained; he proposed to remove the cervical mucus to get a better visual field, so he experimented with various chemicals, until he described the effect of acetic acid on abnormal areas of the cervix due to the effect on cellular proteins, which he called the "acetic acid test." In 1933 he published Introduction to colposcopy, where he described the vascular patterns and early cervical cancer called superficial carcinoma or intraepithelial carcinoma, later called in situ carcinoma. All observations were documented rigorously; however, the lack of correlation between colposcopic and histological images caused confusion, which was one of the causes of the lack of acceptance of colposcopy at that time. In 1935 Hinselmann said that the transition from preinvasive to invasive lesions took approximately 10 to 15 years. While Hinselmann’s contributions were critical to the development of diagnostic methods, he is also remembered for his participation in the concentration camps of Auschwitz, where he participated in the sterilization of Gypsy women.^5,6

In parallel, Walter Schiller (1887-1960) developed Lugol’s test in 1928, later known as Schiller's test, which was included as part of the cervical exam starting in 1933 and was also incorporated by Hinselmann. Moreover, Schiller improved the scraping technique using sharpened curettes to obtain samples from suspicious areas for colposcopy, allowing histological analysis, which may be considered the precursor to the PAP test.⁷

In 1931 colposcopy was introduced to the United States by the gynecologist Frederick V. Emmert of the University of Saint Louis School of Medicine, who described colposcopic patterns associated with early cervical cancer. However, it was not well received, because his findings were considered somewhat cumbersome, because of the terminology in German among other things. Colposcopy as such began to be accepted in the United States only in the fifties, when the German Karl August Bolten (1912-1972) arrived in the country as a student in 1953 and developed a school of colposcopy at Jefferson Medical College in Philadelphia, and later (1954) at the University of Louisiana School of Medicine in New Orleans, where he trained a group of gynecologists in the development of colposcopy in the United States. Later he published the first American Atlas of Colposcopy in 1959 and a textbook in 1960.⁷

Despite the limited impact of George Papanicolaou’s work until then, he continued to work, and, in 1940, in collaboration with Herbert Traut, published the identification cancer cells of the cervix and endometrium in vaginal cytology with suspected cancer in the American Journal of Obstetrics and Gynecology. Subsequently in 1943 he published the monograph Diagnosis of uterine cancer by vaginal smear.⁸ His work began to gain importance in the gynecological community, and numerous studies have confirmed the usefulness of the test and some hospitals began to use it for the detection of cervical cancer; it was then called "Pap", as it is known to this day. Another merit of George Papanicolaou was the systematization of diagnostic technique for sampling, fixation, staining, and nomenclature, which is considered the beginning of modern clinical cytology.

In 1946 the American Cancer Society was founded, which promoted education about the Pap test, and in the same year the article "Diagnostic value of exfoliative cells from cancerous tissues"⁹ was published. In 1947 the first course of cytology was established, and in 1948 the first National Conference of Cytology was conducted in Boston. George Papanicolaou is considered the father of exfoliative cytology, as his technique was not applied solely to the detection of cervical cancer, but also in the detection of vaginal, endometrial, and fallopian tube cancer, as well as in fluids such as urine, sputum, pleural and peritoneal fluid, and so on. This was reflected in his classic book The Atlas of Exfoliative Cytology, published in 1954.¹⁰

Human papilloma virus as the causative agent of cancer

In Latin America in 1971 (Maracaibo, Venezuela) and 1973 (Yucatán, Mexico), works were presented that demonstrated the presence of viral particles in cervical lesions by electron microscopy. This was at the VIII and IX Latin American Congress on Pathology. At that time, it was thought that the perinuclear halos typical of koilocytes were due to glycogen overload, which was discarded when special stains were performed.¹¹

For his part, Dr. Harald zur Hausen proposed in 1975, based on diverse evidence, that human papillomavirus (HPV) was the etiologic agent of cervical cancer.

In 1907 Giuseppe Ciuffo established the viral etiology of human warts, demonstrating that they can be transmitted from cell-free filtrate. In 1911 Peyton Rous (1879-1970) discovered the avian sarcoma virus (from RNA), known as Rous sarcoma virus (RSV), which was the first tumor virus described. In 1935 Rous and Beard showed that the papilloma virus of the cottontail rabbit (CRPV) causes carcinomas in domestic rabbits. This was the first virus with tumor-inducing DNA to be described. In the following decades CRPV became a study model for viral tumorigenesis, and in 1949 the existence of viral particles in skin tags was demonstrated.

In 1965 the first tumor virus was discovered in humans, when the association of Burkitt lymphoma (described by Denis Parsons Burkitt, 1911-1993) with viral DNA was established, called Epstein-Barr virus in honor of its discoverers: Michael Anthony Epstein (1921-) and Yvonne Barr (1932-), who visualized virus-like particles in cell lines using electron microscopy, established based on Burkitt lymphoma.¹² In 1965 the first reports were released describing circular double-stranded DNA from HPV. In the early seventies the diversity of papilloma viruses became apparent, because antigenic differences were described between viral particles isolated from skin tags and genitals, both by electron microscopy studies and agglutination assays. In 1972, studies began to try to establish the link between HPV and cervical cancer. The key works on the heterogeneity of HPV originated from hybridization and in vitro transcription studies of HPV isolated from skin and genital warts, published by Dr. Harald zur Hausen in 1974. In 1976 Meisels and Fortin proposed that koilocytes represented the pathognomonic figure of HPV infection, which was subsequently demonstrated by Della Torre (1978), and Hills and Laberty (1978).¹³

These facts led Dr. zur Hausen to propose the hypothesis that cervical cancer could develop from viral infections found in genital warts. In the early eighties Lutz Guissmann published the first HPV sequences isolated from genital warts: HPV-6 and HPV-11.¹⁴ With these sequences, used as hybridization probes, analysis of other HPVs isolated from carcinomas continued, thanks to which other viral types were identified, such as HPV-16 isolated from a cervical carcinoma in 1983,¹⁵ and the sequence of HPV-18 in the following year.¹⁶ Due to advances in the techniques of molecular biology and specifically the development of polymerase chain reaction, the study of HPV expanded, and in the early nineties the first epidemiological studies were conducted. At the same time the role of viral proteins E6 and E7 was determined in the inactivation of tumor suppressor genes p53 and pRb, so the principles of immortalization and transformation of HPV were established.^17,18 In 1997 the compilation of HPV sequences was established, and in 1999 it was defined that HPV is found in almost 100% of cervical carcinomas.¹⁹ In 2003 a worldwide epidemiological study was published, which was fundamental to the classification of HPV associated with cancer.²⁰ In 2004, 118 papillomaviruses had been reported, and terms were established for the taxonomic classification of HPV.²¹ Many articles have shown the fundamental role of HPV in cervical cancer development, which in addition generated the interest of scientists and the pharmaceutical industry for the development of vaccines. Ian Frazer and Jian Zhou began studies for the development of prophylactic vaccines in the nineties; after several clinical phase trials showing the efficacy and safety of the vaccine, it was approved by the Food and Drug Administration (FDA) of the United States in 2006, and put on the market. In 2008 Dr. zur Hausen was awarded the Nobel Prize for Medicine and Physiology because of his contributions to the discovery of HPV as an etiological factor for cervical cancer, which has represented one of the culminating events in the investigation of viruses as causative agents of cancer.

To date nearly 200 viral types of HPV have been described.²² According to the International Agency for Research on Cancer (IARC), 13 of them (HPV-16, -18, -31, -33, -35, -39, -45, -51, -52, -56, -58, -59, and -66) are classified as type I carcinogens.²³ Globally the most prevalent HPV types in cervical cancer are HPV-16 (57%) and -18 (16%), followed by HPV -33, -45, -58, -31, -52, and -35.²⁴

HPV in cervical cancer

Currently HPV is considered a necessary cause for cervical cancer;²⁵ however, HPV infection is not enough for the development of this cancer, and in most cases (about 80%) the infection will be temporary, with morphological manifestations with minimal intraepithelial abnormalities that may resolve spontaneously, while a small percentage of patients will have persistent infection with one or more types of oncogenic HPV, which can allow the development of precancerous lesions that may progress to cervical cancer itself over a period of approximately 10 to 20 years.²⁶

According to the World Health Organization, it has been reported that about 291 million women worldwide are infected with HPV, which corresponds to a prevalence of 10.4%. The prevalence is highest in young women aged 25 or more.²⁷

Furthermore, a substantial fraction of other anogenital cancers (vulvar, vaginal, penile, anal, and oropharyngeal carcinoma) have been strongly related to HPV.²⁴

Cervical cancer is the third most common neoplasia in women in the world, in terms of incidence and mortality, with 528,000 new cases and 266,000 deaths annually; about 85% of these cases occur in developing countries, and the leading cause is HPV infection. A marked difference has been described in the rates of incidence and mortality in developing countries versus developed countries. Latin America and the Caribbean have registered an incidence (I) of 12.2 and a mortality (M) of 9.9 per 100,000 inhabitants; in Asia I = 9.3 and M = 80.0 per 100,000 inhabitants; and in Africa I = 20.4 and M = 19.2 per 100,000 inhabitants; while in Europe I = 3.6 and M = 3.1 per 100,000 inhabitants; in the United States I = 1.7 and M = 2.0 per 100,000 inhabitants; and in New Zealand I = 5.3 and M = 1.3 per 100,000 inhabitants (Figure 1).²⁷

Against this background, the women of Latin America as well as Asia and Africa have a higher risk of developing cervical cancer; however, it is in these countries where detection and screening programs are generally not as timely, mainly due to limited health care infrastructure. Colposcopy is a viable alternative as a first step for screening, as it is a useful method for the diagnosis and evaluation of intraepithelial neoplasia and preclinical invasive cancer, plus it allows detailed observation of the site where carcinogenesis happens; also, biopsies can be obtained from the specific site to delimit the extent of cervical lesions on one side. 

The first program of population screening for cervical cancer was conducted in middle and high income countries, and was released between 1960 and 1980. This led to the implementation of more such programs that were based on the Pap smear or conventional cytology, and which allowed a 50 to 80% reduction in cervical cancer mortality.²⁸

Although a global trend of decreasing incidence of cervical cancer is currently seen, as shown in Figure 2, one can distinguish rates exceeding 15.0 per 100,000 population in developing countries such as Colombia or Costa Rica, in contrast with countries like the United States, Canada, New Zealand, and Australia, where the incidence is less than 15.0 per 100,000 inhabitants; this trend is observed in Figure 3. Given this, the implementation of more specific screening tests is necessary, especially in countries with a higher risk for developing cervical cancer.

Vaccines and molecular testing: current strategies to control cervical cancer

Thanks to the knowledge of HPV, efforts have been made to generate prophylactic vaccines for this virus. There are currently vaccines that protect against the main HPV infections in cervical cancer (HPV-16 and HPV-18), which were adopted starting in 2006 once their effectiveness was demonstrated. However, these are not the only HPV types responsible for the development of cervical cancer, so a nine-valent vaccine is currently in clinical phase, which seeks to provide approximately 90% protection. However, the impact of such vaccines will only be reflected decades after application.

Today, within the scope of technological development, the area of ​​diagnosis and detection of pathogens has evolved and is in a process of continuous development, thus changing the perspective of medicine and other areas. In this context, molecular tests have been one of the most significant advances resulting from genetic engineering, whose foundation involves the in vitro manipulation of nucleic acid (DNA/RNA); what is attempted with these techniques is to detect whether the virus is present through the identification of DNA or viral mRNA in samples from women even before the presence of premalignant or malignant cervical lesions.²⁹ Today there is a large repertoire of techniques such as polymerase chain reaction (PCR), in situ hybridization, sequencing, and more (Figure 4).^30-32

For this reason one of the strategies currently used to improve cervical cancer screening is the molecular detection of HPV, which has been reported highly effective, with a negative predictive value close to 100%, and a predictive value for the development of cervical lesions greater than that of cytology.³⁰ Based on this, several tests have been validated for diagnostic use in multicenter trials and have been approved for in vitro diagnostic (IVD) (Table I).

These molecular methods offer fast, accurate, and specific detection, and their processes are automated with quality controls to ensure their effectiveness, so they do not depend on a subjective observation as in cytology or colposcopy, whose outcome depends largely on the capacity of the observer. The impact of screening based on molecular evidence has been such that even in the United States it has been approved by the FDA as a primary screening test and cytology as a part of screening. While this has generated controversy, it is clear that the incorporation of molecular testing is a tool that provides information in screening, so the international consensus for several years has been for the incorporation of molecular evidence.^33,34 Another argument against the molecular test is its high cost compared to the conventional Pap test. In this regard there have been several cost-benefit studies supporting the use of molecular evidence.³⁵ Moreover, the costs of these tests has been reduced over time due to the diversity of available tests.

Conclusion

The discovery of HPV as an etiologic agent of cervical cancer has been the result of several contributions from various areas of study, such as cytology, pathology, colposcopy, epidemiology, and molecular biology. Cervical cancer is one of the most widely known malignancies today. Since 1950 there has been cytology for the detection of precursor lesions; in the decades of the eighties and nineties the mechanisms of carcinogenesis began to be described, and HPV was identified as the causative agent of cervical cancer. This knowledge laid the foundation for the development of vaccines and molecular tests for HPV, currently considered the strategies for primary and secondary prevention of cervical cancer.

The knowledge generated over the course of these years has had a major impact on disease control, from the implementation of national screening programs based on the Pap smear, to the development of molecular tests for the detection of the causative agent, and the development of prophylactic and therapeutic vaccines.

Despite this, cervical cancer remains a public health problem in developing countries because it ranks third in incidence, so the decrease in the short and medium term, especially in countries with low and middle income, will have greater impact by implementing more effective and timely screening programs. The development of molecular testing is a valuable tool, as its contribution to primary screening has been proven; therefore, its incorporation is part of current international guidelines. It is expected that the effectiveness of screening will improve considerably with the addition of molecular tests, and together with vaccination it will help control cervical cancer.

Acknowledgments

To the Red de Investigación del Virus del Papiloma Humano of the Instituto Mexicano del Seguro Social, in particular its coordinators, Doctors María Elena Furuya Meguro, Eduardo Almeida, María Elena Galván Plata, and Gabriela Borrayo.

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