1391 Endocrine Tumors of the Cervix Morphologic Assessment, Expression of Human Papillomavirus, and Evaluation for Loss of Heterozygosity on 1p, 3p, 11q, and 17p Ciaran Mannion, M.B., B.CH.1 Won-Sang Park, M.D., Ph.D.2 Yan Gao Man, M.D., Ph.D.1 Zhengping Zhuang, M.D., Ph.D.2 Jorge Albores-Saavedra, M.D.3 Fattaneh A. Tavassoli, M.D.1 1 Department and Laboratory of Gynecologic and Breast Pathology, Armed Forces Institute of Pathology, Washington, DC. 2 Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland. 3 Department of Anatomic Pathology, University of Texas Southwestern Medical Center, Dallas, Texas. BACKGROUND. Cervical endocrine tumors are rare lesions, with a varied diagnostic nomenclature. A recent consensus meeting proposed a standardized terminology. This study evaluated: 1) applicability of histopathologic guidelines; 2) evidence of loss of heterozygosity (LOH) at selected sites; and 3) the presence of human papillomavirus (HPV) detected by nonisotopic in situ hybridization (ISH). METHODS. Thirty-eight cases (patient age range, 19 – 88 years; mean, 48 years) were retrieved. Outcome data were available for 32 patients. Classification was based on architectural and cytologic features. Tissue was available from 15 cases for LOH analysis with D3S1234(3p14), D3S1289(3p21), THRB(3p24), TP53(17p13), D1S468(1p36), and INT-2(11q13). In ten cases, tissue was analyzed by nonisotopic ISH with HPV probes for types 6/11, 16/18, and 31/33. RESULTS. Tumors were divided into four groups: small cell carcinoma (SCC) (n 5 25); large cell neuroendocrine carcinoma (LCNC) (n 5 5); SCC with focal LCNC differentiation (n 5 3), and carcinoid tumor (n 5 5). Tumors defined as exclusively or predominantly SCC had a particularly poor prognosis, with 20 patients dead of disease (,6 years after diagnosis) and 6 alive with disease (after ,3 years of follow-up). LOH at various 3p loci (3p14, 3p21, and 3p24) was observed in eight cases. One patient demonstrated LOH on 17p(TP53). Eight of ten cases assessed by ISH showed nuclear staining using a combined HPV-16/18 probe. CONCLUSIONS. Cervical endocrine tumors are highly aggressive and can be subdivided into definable categories. LOH at 3p loci is a frequent finding, as is nuclear staining with a combined HPV-16/18 probe. LOH at 17p(TP53 locus) appears to be relatively uncommon, suggesting that p53 mutations may not be developmentally significant. Cancer 1998;83:1391– 400. © 1998 American Cancer Society. KEYWORDS: cervical carcinoma, endocrine carcinoma, small cell carcinoma, human papillomavirus, in situ hybridization, loss of heterozygosity, 3p, p53. Presented in part at the 86th Annual Meeting of the United States and Canadian Academy of Pathology, Orlando, Florida, March 1–7, 1997. Address for reprints: Jorge Albores-Saavedra, M.D., Department of Anatomic Pathology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75235-9072. The opinions and assertions contained herein are the private views of the authors and are not to be construed as official or reflecting the views of the Department of the Army or the Department of Defense. Received October 29, 1997; revision received March 19, 1998; accepted April 8, 1998. © 1998 American Cancer Society O n a global basis, carcinoma of the uterine cervix is one of the most frequent causes of death from malignant neoplasms among women. In developing countries, cervical carcinoma accounts for an estimated 340,000 new cases and 160,000 deaths every year.1 The introduction of cytologic screening programs has had some measurable success in developed countries, in which cervical carcinoma currently ranks as fifth in incidence and seventh in mortality for female cancer patients of all ages.2 Nonetheless, in 1997, it is estimated that there were 14,500 new cases of cervical carcinoma and 4800 deaths attributable to carcinoma of the cervix in the U.S.3 Four decades ago, criteria were introduced for the classification of cervical carcinoma.4 This system, which subsequently was adopted by the World Health Organization, served to divide cervical carcinomas into two broad categories: squamous cell carcinoma (large cell 1392 CANCER October 1, 1998 / Volume 83 / Number 7 keratinizing, nonkeratinizing, and small cell undifferentiated carcinoma) and adenocarcinoma of the cervix. Small cell neoplasms of the cervix have long been recognized as an uncommon, but highly aggressive, subset of uterine cervical neoplasms with a particularly poor prognosis compared with other phenotypes.5–10 A similarly aggressive behavior also has been documented with small cell carcinoma at other extrapulmonary sites.11 Over the past 25 years, a variety of terms have been used in the diagnosis of cervical lesions with neuroendocrine differentiation.12–39 This varied and inconsistent nomenclature has represented a major impediment to comparative studies of the true incidence, clinicopathologic characteristics, and natural history of this family of tumors. A recent consensus workshop was convened to address the issue of terminology.40,41 The participants acknowledged that neuroendocrine neoplasms of the cervix share many clinical and histopathologic features with those in other sites, particularly the lung.42 The proposed terminology was recognized as a first step toward a more uniform classification. In view of the limited experience to date with endocrine tumors of the cervix, the inherent prognostic and therapeutic implications of rendering such diagnoses, and the lack of understanding of the molecular changes underlying these phenotypes at this site, the current study resolved to examine cervical endocrine tumors at the light microscopic and molecular levels. Specifically, we proposed to: 1) evaluate the applicability of the recently introduced consensus terminology40 to cervical endocrine tumors from the case files of two large referral centers; 2) assess these cases for mutations, in the form of loss of heterozygosity (LOH) at multiple chromosomal loci, some of which have been associated with small cell tumors at other anatomic sites43– 48 or with cervical tumors with nonendocrine phenotypes;49 –55 and 3) document the presence of human papillomavirus (HPV) by in situ hybridization (ISH). MATERIALS AND METHODS Cases received between January 1970 and June 1996 were retrieved from the files of the Armed Forces Institute of Pathology (AFIP), the files of the University of Texas Southwestern Medical Center, and the personal consultation files of one of the authors (J.A-S.) under a variety of diagnostic codes: small cell carcinoma (SCC), carcinoid tumor (CT), neuroendocrine carcinoma, and poorly differentiated carcinoma. With respect to the latter two codes, only cases with positive staining for chromogranin and/or synaptophysin were accepted into the study group. In the course of initial review, cases with a predominantly squamous or glandular morphology with only isolated endocrine cells were excluded, as advocated by the recent consensus group.40 Hematoxylin and eosin stained sections were reviewed in 38 cases, the majority of which were biopsies. Using an Olympus BX40 microscope (Olympus America Inc., Lake Success, NY), each case was assessed for the presence of a variety of architectural and cytologic attributes: growth pattern; prominent areas of tumor necrosis at low power (310 wide field eyepiece, 34 objective 5 340 magnification 5 field area of 22.89 mm2); character of the tumor margin (circumscribed versus infiltrative); vascular invasion; cell shape; nuclear to cytoplasmic ratio; nuclear pleomorphism; nuclear chromatin; nucleoli; mitotic activity at high power (310 wide field eyepiece, 340 objective 5 3400 magnification 5 field area of 0.92 mm2); apoptotic bodies; cytoplasmic granules; rosette-like arrangements; metaplastic changes; squamous or glandular intraepithelial lesion/in situ component; and concomitant presence of other carcinoma and dysplasia patterns. The patients ranged in age from 19 – 88 years (mean, 48 years). In 26 patients, the major presenting symptom was a cervical mass protruding through the cervical os. In the colposcopic impressions and/or macroscopic descriptions accompanying these consultation cases, the tumors typically were described as “exophytic” or “polypoid” and showed “ulcerated” or “friable” surface features. Survival data were obtained for 32 patients. Of cases defined as SCC or SCC with focal large cell neuroendocrine carcinoma (LCNC) differentiation, follow-up indicated a very poor prognosis (20 patients were dead of disease , 6 years after diagnosis; 3 were alive with disease after ,3 years of follow-up; and 3 were alive with disease after ,1 year of follow-up). Four patients with CT were alive (after ,3 years of follow-up), 2 with evidence of disease. One of the 5 patients with a LCNC had died 42 months after the initial diagnosis, whereas another was alive with metastatic disease , 3 years after initial presentation. Additional material, in the form of formalin fixed, paraffin embedded tissue blocks, was available from 15 cases. After hematoxylin staining of sections from this tissue, microdissection was performed under direct light microscopic visualization, as has been reported previously.56 This involved the manual use of a disposable, sterile, 30-gauge needle. Tumor cells were selected to avoid contamination by surrounding elements; in cases with more than one morphologically distinct malignant cell population, separate areas of tumor were dissected individually so as to provide pure representative samples of the respective tumoral Endocrine Tumors of the Cervix/Mannion et al. 1393 TABLE 1 Analysis of Loss of Heterozygosity in Endocrine Tumors of the Cervix at Multiple Chromosomal Loci Case no. Diagnosis D1S468(1p36) D3S1234(3p14) D3S1289(3p21) THRB(3p24) INT-2(11q13) TP53(17p13) 1 2 3 4 5 6 7 8 9 10 SCC SCC SCC SCC SCC SCC SCC SCC SCC SCC LCNC SCC LCNC SCC LCNC SqCCa LCNC LCNC LCNC NW NW NI NW NI NI NI NI NI NI NI — — LOH LOH LOH NW NI NI LOH — LOH LOH NI NI — NI NI LOH — LOH — NI NI NI LOH — NI LOH NI LOH LOH NI LOH — NW — NI NI — NI NI NI NI NW — NW LOH NA NA NA NA NA LOH NA NI LOH — LOH LOH NA NA NA NA NI NI NW NW — — NW — NW LOH NI NI NI NI NI — — — NW LOH NI — — — — — — NI — — NI NI — — — — — LOH NI NI 11 12 13 14 15 SCC: small cell carcinoma; LCNC: large cell neuroendocrine carcinoma; SqCCa: squamous cell carcinoma; NW: test did not work; NI: not informative as a result of homozygosity; —: no loss of heterozygosity; LOH: loss of heterozygosity; NA: no available tissue remained to assess. populations. To assess reproducibility, two separate samples were taken from each area of interest and from normal internal controls (e.g., benign-appearing squamous and/or glandular epithelium, adjacent normal stroma, inflammatory cells, etc.). One sample was processed at the Gynecologic and Breast Pathology Department at the AFIP, whereas the other was processed in an identical manner at the Laboratory of Pathology at the National Cancer Institute to assure consistency of results. Procured cells were resuspended in a 20-mL solution containing 0.05 M Tris-hydrochloric acid, 0.001 M ethylenediamine tetraacetic acid (EDTA), 1% Tween 20, and 0.1 mg/mL proteinase K. After overnight incubation at 37 °C, the mixture was boiled for 10 minutes to inactivate proteinase K. Polymorphic DNA markers were selected for analysis of loci, based largely on reports of LOH in small cell neoplasms at other body sites or other tumor phenotypes in the cervix (Table 1).43–55 These included: D3S1234(3p14); D3S1289(3p21); THRB(3p24); TP53(17p13); D1S468(1p36); and INT-2(11q13). Polymerase chain reaction (PCR) was performed in 10-mL volumes and contained 1 mL 10X PCR buffer; 1.5 mL DNA extract; 50 pM of each primer; 20 nM each of dCTP, dGTP, dATP, and dTTP; 0.1 mLp32 dCTP (6000 Ci/mM); and 0.1 U Taq DNA polymerase. The reactions were performed in a Perkin-Elmer Cetus thermal cycler (Perkin-Elmer Cetus Instruments, Norwalk, CT) as follows: 30 cycles of amplification, each comprised of 94 °C for 30 seconds, followed by 55 °C for 30 seconds, and finally 72 °C for 30 seconds. The amplified product was mixed with an equal volume of formamide loading dye (95% formamide, 20 nM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol). Samples were denatured for 5 minutes at 94 °C and resolved on 6% acrylamide gel. After electrophoresis at 1800 volts for 2 hours, the gels were transferred to 3-mm Whatman paper (Daigger Scientific, Wheeling, IL) and dried. Autoradiography was performed with Kodak X-Omat film (Eastman Kodak, Rochester, NY). The case was considered informative for a polymorphic marker at a given locus if the DNA retrieved from the normal tissue showed two different alleles (heterozygosity). For informative cases, the intensities of the two alleles in the tumor samples were compared. In this study, LOH was defined as complete or near-complete ($75%) absence of one allele in the tumor DNA as assessed by direct visualization. Cases were classified as “not informative” at the polymorphic locus of interest on the basis of homozygosity in DNA extracted from histologically normal tissue in the specimen, thereby precluding evaluation for LOH. In the instances of test samples that “did not work”, the sample material was either of insufficient quantity or possessed degraded, uninterpretable DNA. In some cases, due to the scant quantity of lesional material in the biopsy, no tumor remained in deeper sections 1394 CANCER October 1, 1998 / Volume 83 / Number 7 FIGURE 1. (a) Representative example of a case of small cell carcinoma (SCC) of the cervix. Note the high nuclear:cytoplasmic ratio, numerous mitotic figures (arrow heads), and prominent apoptosis (H & E, original magnification 3240). (b) Large cell neuroendocrine carcinoma (LCNC). Although there are prominent mitotic figures (arrows), the tumor cells have more abundant cytoplasm than SCC and vesicular nuclei showing occasional prominent eosinophilic nucleoli (arrowhead) (H & E, original magnification 3300). (c) Carcinoid tumor. The perivascular, rosette-like architecture and uniform appearance of the cell nuclei are evident (H & E, original magnification 3200). (d) Case of SCC showing focal LCNC differentiation. Nests of tumor cells with more conspicuous eosinophilic cytoplasm (arrowheads) are evident in a background of hyperchromatic small cells (H & E, original magnification 3150). from the paraffin blocks and was “not available” for assessment. In the 10 cases in which tissue remained after LOH analysis, consecutive 5-mm sections were prepared on positively charged slides (Fisher Scientific, Pittsburgh, PA). Sections were deparaffinized and ISH was performed with HPV typing kits for types 6/11, 16/18, and 31/33 (Kreatech Diagnostics, Amsterdam, The Netherlands). This process involved incubation with pepsin digestion solution at 37 °C for 30 minutes, rinsing with distilled water, and dehydration in ethanol. The sections were incubated with selected HPV DNA probes at 95 °C for 5 minutes, followed by overnight incubation at 37 °C. The slides were treated with differentiation solution and washed with Tris-buffered saline (TBS) buffer. A HPV-infected cell line was used as a positive control. Several different negative controls were employed including control probe or hy- bridization buffer (in place of the HPV DNA probes), use of normal kidney and breast tissue (as a substitute for cervical tumor sections), and omission of the incubation phase with HRP-conjugated a-digoxigenin. Reactivity was classified, on the basis of signal pattern, as punctate and/or diffuse, as outlined in previous reports.57– 62 RESULTS Histopathologic Evaluation In accordance with the terminology guidelines recommended by the consensus workshop,40 three distinct phenotypic patterns were recognized: SCC (Fig. 1a); LCNC (Fig. 1b); and CT (Fig. 1c). Based on these profiles, the cases were subdivided into four groups: SCC (25 cases); LCNC (5 cases); SCC with focal LCNC differentiation (3 cases) (Fig. 1d); and CT (5 cases). Of the Endocrine Tumors of the Cervix/Mannion et al. 1395 FIGURE 1. (continued) five carcinoid lesions, four were atypical carcinoid tumors and one was a single typical (classic) carcinoid. Cases of SCC and/or LCNC could be distinguished from CT using a combination of architectural and cytologic features: infiltrative margin, extensive geographic areas of necrosis, relatively high mitotic rate (. 10/10 high-power fields (hpf) 5 .10/9.2 mm2), and prominent apoptotic bodies in the former tumors versus a predominantly pushing border, absence of or only focal necrosis, lower mitotic rate (# 10/10 hpf), and fewer apoptotic bodies in CT. Of note, vascular invasion was not a reliable discriminator. Distinction between SCC and LCNC was achieved on the basis of cytologic attributes: sparse cytoplasm, finely granular chromatin, and absence of nucleoli in SCC versus relatively abundant cytoplasm, vesicular chromatin, and prominent nucleoli in LCNC. LOH Analysis A total of 15 lesions were microdissected and evaluated for LOH with microsatellite markers at 5 chromosomal loci (Table 1): 9 SCCs, 3 cases with SCC and focal LCNC, and 3 LCNCs. In the remaining cases, which included the cases diagnosed as CT, analysis for LOH was not possible either because no archival tissue was available or the only tissue present was tumor without adjacent uninvolved tissue for comparison. Several markers for loci in the short arm of chromosome 3 (Fig. 2) showed a high frequency of LOH, with .50% of the informative cases showing at least 1 loss (Table 1). Of note, two of the three cases with SCC and focal LCNC revealed LOH for D3S1234 in the SCC component, but not in the LCNC elements (Fig. 2; a1, Case 11). Although only one case proved informative for LOH with D1S468, the findings are intriguing in that three different tumor phenotypes (SCC, LCNC, and squamous cell carcinoma) were present in the lesion and all showed LOH under direct visualization with this marker (Fig. 3a; Case 12). This case was unique with respect to the demonstration of LOH in association with both LCNC and squamous cell carcinoma phenotypes. Remarkably, the LOH in the squamous cell samples involved a different allele than that observed with the endocrine elements. LOH at TP53 was uncommon, being present in only 1 of 11 informative cases (Fig. 3b; Case 13). With INT-2, LOH was detected in two of six informative cases (Fig. 3c; Case 8). 1396 CANCER October 1, 1998 / Volume 83 / Number 7 FIGURE 3. (a) Sequencing gel demonstrates loss of heterozgosity (LOH) for FIGURE 2. (a) Sequencing gels demonstrate loss of heterozygosity (LOH) for D3S1234 (3p14) in the small cell carcinoma (SCC) component, but not in the coexistent large cell neuroendocrine carcinoma (LCNC) (a1; Case 11) or squamous carcinoma (SqCCa) (a2; Case 1). (b) Sequencing gel shows LOH for D3S1289 (3p21) in a SCC (Case 4). Sq Epi: squamous cell epithelium tissue; N: normal tissue. HPV In Situ Hybridization In ten cases, sufficient lesional material remained for HPV analysis (Table 2). Eight of the ten cases demonstrated positive staining with the combined HPV type 16/18 probe (Fig. 4a and 4b), which exclusively was a punctate nuclear staining pattern. Of note, this staining was confined to viable tumor cells and was not present in the background stroma or adjacent necrotic areas. Three of these eight cases showed similar, but rare, punctate staining for the HPV type 31/33 probe in a few tumor cells. None of the cases were found to be positive for the HPV type 6/11 probe. DISCUSSION Little is known regarding the genetic alterations underlying cervical carcinoma. This is particularly true of D1S468 (1p36) in three coexistent phenotypically distinct tumors (large cell neuroendocrine carcinoma [LCNC], small cell carcinoma [SCC], and squamous cell carcinoma [SqCCa]) from the same patient (Case 12); although the same allele is involved in the LCNC and SCC lesions, the other allele is lost in the SqCCa. (b) LOH for TP53 (17p13) in Case 13. (c) LOH for INT-2 (11q13) in Case 8. endocrine tumors of the cervix, which are rare, but highly aggressive, malignancies. In addition to reaffirming the particularly poor prognosis associated with these lesions,5–10,13,31,33,37,39 the current study also provided an uncommon opportunity to evaluate a large number of these tumors at both the light microscopic and molecular levels. By adhering to the guidelines of the recent consensus workgroup,40 it was possible to define distinct light microscopic phenotypes within the endocrine category. SCC represented the most common cervical endocrine tumor by far, confirming the observations of the workgroup.40 The presence of foci of LCNC differentiation in three cases of otherwise typical SCCs points to a close relation between these two phenotypes and is in keeping with the hypothesis of a common cell of origin,63 possibly a reserve or stem cell in the cervix. However, the finding of different LOH profiles on the short arm of chromosome 3 in respective Endocrine Tumors of the Cervix/Mannion et al. 1397 FIGURE 4. (a) Section of small cell carcinoma showing punctate positive (brown) staining with combined kit for human papillomavirus type 16/18, (HRP-conjugated a-digoxigenin, original magnification 3400). (b) Corresponding area on hematoxylin and eosin stained section confirms that the positive staining with the a-digoxigenin is confined to the viable tumor cells (arrowhead), but was not observed in the adjacent degenerated cells (arrow) (H & E, original magnification 3400). SCC and LCNC elements from the same lesion in two patients provides support for the practice of distinguishing between these definable phenotypic patterns. Numerous studies have reported a high frequency of LOH on 3p in malignancies with a variety of phenotypes at different body sites, most commonly in the lung43– 48,64 – 66 and kidney.67,68 Reports of LOH on 3p in tumors with SCC at noncervical sites43– 46,48 and in nonendocrine cervical carcinoma49 are particularly noteworthy. The high frequency of LOH on 3p in the current study cases provides further evidence that 3p deletions represent an important step in cervical tumor pathogenesis and also are a characteristic finding in endocrine tumors at this site. In view of the emerging concept of carcinogenesis as a multistep process involving more than one genetic change, the association of 3p loss with various tumor phenotypes suggests that this finding may, in some instances, represent an early change in such a process. Indeed, the observation of deletions of 3p in the early stages of lung carcinogenesis66,69,70 and the presence of LOH on multiple chromosomal loci45,48 in small cell lung carcinoma are consistent with such a model. Although a similar archetype may well apply in the evolution of cervical endocrine tumors, a number of fundamental differences exist between neuroendocrine tumors in the lung and those in the cervix, most notably in the response to therapeutic modalities. The biologic basis for these differences remain unclear. In contrast to SCLC,47 our cases of cervical SCC showed a low frequency of LOH of chromosome region 17p13 (p53 locus). This finding is provocative when viewed in the context of the rather high rate of detection of high risk HPV types in these lesions and supports the evidence from other studies of the inverse relation between the presence of HPV and p53 mutations in cervical carcinoma.51–55,71,72 Previous investigators have demonstrated in vitro that E6 oncoprotein from high risk HPV types has the capacity to 1398 CANCER October 1, 1998 / Volume 83 / Number 7 TABLE 2 Detection of Human Papillomavirus by Nonisotopic In Situ Hybridization Using HRP-Conjugated a-Digoxigenin Case no. HPV 6/11 HPV 16/18 HPV 31/33 1 2 3 7 9 10 11 12 13 15 (2) (2) (2) (2) (2) (2) (2) (2) (2) (2) (1) (1) (1) (1) (1) (1) (1) (2) (1) (2) (2) (2) (2) (2) (1) (1) (1) (2) (2) (2) 9. 10. HPV: human papillomavirus; 1: positive staining; 2: no staining. 11. 12. 13. 14. 15. bind and degrade wtp53 protein,73,74 whereas other studies have provided evidence that such E6-wtp53 interaction may be functionally equivalent to p53 mutations.51,75 Such a model would be consistent with our results. The punctate pattern of HPV expression, observed with the digoxigenin probes in this study, has been shown to be indicative of physical integration of HPV DNA.60 – 62 Other investigators have shown an association between SCC of the cervix and high risk HPV types, particularly HPV type 18.63,76 It is conceivable that certain tumors, including endocrine tumors of the cervix, require both 3p loss and HPV integration to transform an epithelial precursor stem cell into a cell with malignant potential. 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