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Molecular Abnormalities Associated with Endocrine Tumors of the Uterine Cervix
Gynecologic Oncology 72, 3–9 (1999) Article ID gyno.1998.5248, available online at http://www.idealibrary.com on
Molecular Abnormalities Associated with Endocrine Tumors of the Uterine Cervix Ignacio I. Wistuba, M.D.,* ,† Bilue Thomas,* Carmen Behrens, M.D.,* Naoyoshi Onuki, M.D.,* Guy Lindberg, M.D.,‡ Jorge Albores-Saavedra, M.D.,‡ and Adi F. Gazdar, M.D.* ,‡ ,1 *Hamon Center for Therapeutic Oncology Research and ‡Division of Anatomic Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; and †Department of Pathology, Pontificia Universidad Catolica de Chile, Santiago, Chile Received June 23, 1998
tive terms [3]. In 1997 a workshop sponsored by the Cancer Committee of the College of American Pathologists and the National Cancer Institute recommended a uniform terminology for these tumors [3]. As the morphologic features of the endocrine tumors of the uterine cervix are similar to those occurring in the lung, the same classification existent in the lung was proposed [3]. Thus, four categories of endocrine tumors of the uterine cervix are recognized: (1) typical carcinoid tumor; (2) atypical carcinoid tumor; (3) large cell neuroendocrine carcinoma (LCNC); and (4) small cell carcinoma. Several studies have described a spectrum of molecular changes involved in the pathogenesis of cervical squamous cell carcinomas [4 – 6]. While human papillomavirus (HPV) infection plays an important role in the pathogenesis of this neoplasm [7], other frequent molecular changes have also been detected [4 – 6]. Relatively high frequencies of loss of heterozygosity (LOH) at chromosomal regions 4p, 6q, and localized 3p deletions have been reported [4 – 6]. In contrast, low frequencies of TP53 and RB gene abnormalities are usually present, consistent with HPV-mediated inactivation of their protein products [6, 8]. On the other hand, many molecular abnormalities involved in the pathogenesis of the neuroendocrine tumors of the lung have been identified, especially those present in small cell carcinomas [9]. These tumors demonstrate a very high incidence of TP53 and RB gene mutations, as well as extensive deletions occurring at chromosome 3p, frequently involving losses of most or all of the arm [9]. Recently the gene for multiple endocrine neoplasia type 1 (MEN1) [10] localized at 11q13 has been implicated in the pathogenesis of some sporadic neuroendocrine tumors [11]. Mutations and deletions at the MEN-1 gene have been identified in a subset (36%) of sporadic lung carcinoids [12]. Although cervical endocrine neoplasms are not included in the spectrum of MEN1, no data about the role of the MEN1 gene in their pathogenesis have been reported. Other than the presence of HPV sequences, especially highrisk strain 18 [13, 14], no information about genetic changes involved in endocrine tumors of uterine cervix is available. We
Objective. We studied the molecular abnormalities involved in the pathogenesis of endocrine tumors of the uterine cervix. Methods. We obtained DNA from precisely microdissected archival tissue from 15 endocrine tumors of the uterine cervix, consisting of 5 carcinoids (1 typical, 4 atypical), 2 large cell neuroendocrine carcinomas, and 8 small cell carcinomas. We investigated the presence of high-risk (types 16 and 18) and intermediate-risk (types 31 and 33) human papilloma virus (HPV) sequences, TP53 and K-ras gene mutations, and loss of heterozygosity (LOH) at 9 genes/chromosomal regions, including 3p14.2/ FHIT, 3p14 –p21, 3p21, 3p22–p24, 5q21– q22/APC-MCC region, 9p21/CDKN2, 11q23/MEN1, 13q/RB, and 17p/TP53. Results. HPV sequences were detected in 8 (53%) tumors, HPV 16 in 2 cases, and HPV 18 in 2 cases. LOH at 9p21 (43%) and localized 3p deletions (47%) were the most frequent allelic losses found. Allelic losses at 5q21– q22/APC-MCC region, 11q23/MEN1, and 13q/RB were infrequent. TP53 gene mutations were detected in 7 (47%) tumors (1 atypical carcinoid and 6 carcinomas). HPV sequences were demonstrated in 4 of the 7 cases with TP53 gene mutations. No K-ras mutations were detected. Conclusion. The molecular changes present in endocrine tumors of the uterine cervix have distinct features. They incorporate those present in the neuroendocrine tumors of the lung (high frequency of TP53 gene abnormalities and 9p21 deletions) with those detected in squamous cell carcinomas of the cervix (high-risk HPV sequences and localized 3p deletions). © 1999 Academic Press Key Words: neuroendocrine tumors; tumors of the uterine cervix; loss of heterozygosity; chromosome 3p; TP53 gene mutations.
INTRODUCTION Although rare, malignant tumors with endocrine differentiation are a morphologically distinct group of cervical neoplasms having a highly aggressive clinical course [1–3]. Following their initial description, several reports emphasized their broad morphologic spectrum using a variety of descrip1
To whom correspondence and reprint requests should be addressed at Hamon Center for Therapeutic Oncology Research, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 752358593. Fax: (214) 590-6424. 3
0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Summary of Genetic Abnormalities in 15 Endocrine Tumors of the Uterine Cervix
Genetic abnormality
Carcinoid (n 5 1)
Atypical carcinoids (n 5 4)
Large cell neuroendocrine carcinomas (n 5 2)
Small cell carcinomas (n 5 8)
Total (%) (n 5 15)
HPV sequences 3p LOH (any region) 3p14.2 (FHIT gene) 3p14–p21 3p21 3p22–p24 5q22 (APC-MCC region) LOH 9p21 (CDKN2 gene) LOH 11q13 (MEN1 gene) LOH 13q (RB gene) LOH TP53 gene abnormalities 17q (TP53 gene) LOH TP53 gene mutations
0/1 0/1 0/1 — 0/1 0/1 0/1 0/1 — 0/1 0/1 0/1 0/1
3/4 2/4 0/2 1/4 2/4 0/4 1/2 3/4 2/3 0/4 2/4 1/3 1/4
1/2 2/2 0/2 0/1 2/2 0/2 0/2 0/2 0/1 0/1 1/2 0/1 1/2
4/8 3/8 0/5 1/3 0/8 0/8 2/5 3/17 0/5 1/5 6/18 2/5 5/8
8/15 (53) 7/15 (47) 0/10 3/8 (38) 4/15 (27) 1/15 (7) 3/10 (30) 6/14 (43) 2/9 (22) 1/11 (9) 9/15 (60) 3/10 (30) 7/15 (47)
investigated whether the molecular changes present in squamous cell carcinomas of the uterine cervix and endocrine tumors of the lung are detected in endocrine tumors of the uterine cervix. We investigated the presence of high-risk HPV sequences, TP53 and K-ras gene mutations, and LOH at 9 chromosomal regions, including 3p14.2/FHIT, 3p14 –p21, 3p21, 3p22–p24, 5q21– q22 at the APC-MCC region, 9p21/ CDKN2, 11q13/MEN1, 13q/RB, and 17p/TP53, in a series of 15 endocrine tumors of the uterine cervix. MATERIALS AND METHODS Neuroendocrine Tumor Specimens and Patient Information Neuroendocrine tumors of the cervix are relatively rare, and a search of our files including consultation materials yielded blocks from a total of 15 confirmed neuroendocrine tumors of the uterine cervix including 5 carcinoid tumors (1 typical and 4 atypical carcinoids), 2 LCNC, and 8 small cell carcinomas (Table 1). Histological diagnoses were made by one of us (J.A.-S.), a reference pathologist who helped establish the criteria recently published for the classification and terminology of endocrine tumors of the uterine cervix [3]. Immunohistochemical stains for general neuroendocrine markers including chromogranin and synaptophysin were performed in all tumors to confirm the neuroendocrine origin of the tumors. The patients ranged in age from 31 to 88 years (median 43). Microdissection and DNA Extraction Serial 5-mM sections were cut from archival, formalin-fixed, paraffin-embedded tissue. All slides were stained with hematoxylin– eosin, and one of the slides was coverslipped. The coverslipped slide was used as a guide to localize regions of interest for microdissection for the other slide. Microdissection
and DNA extraction were performed as previously described from noncoverslipped hematoxylin– eosin stained slides [15]. Precisely identified areas of endocrine tumor and stromal cells (as source of constitutional DNA) were microdissected under microscopic visualization (Fig. 1). Detection of HPV Sequences The presence of HPV sequences was tested by PCR using general and type-specific primers designed for paraffin-extracted DNA [16]. Specific primers were used to identify high (types 16 and 18) and intermediate (types 31 and 33) oncogenic risk HPV strains [16]. The HPV analysis was done using one round PCR. DNA extracted from human cell lines CaSki (HPV 16) and HeLa (HPV 18) (obtained from the American Type Culture Collection, Rockville, MD) were used as positive controls for HPV analyses. Polymorphic DNA Markers and PCR–LOH Analysis To evaluate LOH, we used primers flanking 18 dinucleotide and multinucleotide microsatellites repeat polymorphisms located at the following 9 genes or chromosomal regions: 3p14.2 at the FHIT gene (D3S4103), 3p14 –p21 (D3S1766), 3p21 (D3S1029, D3S1478, K1.CA, ITIH-1, D3S1447), 3p22–p24 (D3S1537, D3S1351, D3S2432), 5q21– q22 (L5.71 in the APC-MCC region), 9p21 near the CDKN2 gene (IFNA, D9S1748), 11q13 at the MEN1 gene region (D11S4907, PYGM), RB gene at 13q14 (dinucleotide repeat), and TP53 gene at 17p13 (dinucleotide and pentanucleotide repeats). Primer sequences can be obtained from the Genome Database, with three exceptions (dinucleotide repeat in the RB gene, pentanucleotide and dinucleotide repeats in the TP53 gene) previously published and referenced [15]. Nested PCR or tworound PCR (using the same set of primers in two consecutive
MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS
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FIG. 1. Representative example of microdissection of small cell carcinoma of the uterine cervix (a) before and (b) after microdissection. Hematoxylin and eosin 375.
amplifications) methods were used as previously described [15]. Multiplex PCR was performed during the first amplification, followed by uniplex PCR for individual markers. In the multiplex PCR, four markers were amplified during the same reaction. LOH was scored by visual detection of complete absence of one allele (Fig. 2). TP53 and ras Gene Mutation Analyses We examined for mutations in exons 5 to 8 of the TP53 gene using nested PCR methodology, followed by SSCP analysis and sequencing as previously described [6]. For detection of K-ras mutations, we used a designed RFLP method, utilizing nested PCR methodology, followed by sequencing, as described previously [17]. Using the designed RFLP method we screened for mutations in codons 12, 13, and 61 of K-ras gene [17]. RESULTS Detection of HPV Sequences Eight (53%) of the 15 endocrine tumors of the uterine cervix were positive for high-risk HPV sequences (types 16 and 18) using general and type-specific HPV primers (Tables 1 and 2). The HPV distribution was 2 cases (25%) of HPV-16 and 6 cases (75%) of HPV-18. No intermediate-risk HPV strains were detected. With the exception of the single typical carcinoid analyzed, oncogenic HPV sequences were detected in the entire spectrum of endocrine tumors of the uterine cervix.
43%) were the most frequent deletions detected in endocrine carcinomas of the uterine cervix (Table 1). The 3p deletions detected in endocrine tumors were in all cases localized, occurring at 3p14 –p21 (38%), 3p21 (27%), and 3p22–p24 (7%) regions (Tables 1 and 2). No deletion at FHIT gene region (3p14.2) was detected. In contrast, allelic losses at 5q21– q22 (APC-MCC region; 3 of 10 cases, 30%), 11q13 (MEN1 gene locus; 2 of 9 cases, 22%), and RB gene (13q; 1 of 11 cases, 9%) were relatively infrequent. Frequency of TP53 Gene Abnormalities TP53 gene abnormalities (either point mutation or LOH) were frequent events (60%) in endocrine tumors of the uterine cervix (Table 1). Seven (47%) of the 15 tumors demonstrated TP53 gene point mutations (Table 2 and Fig. 2C). Allelic loss at the TP53 loci was detected in 3 (30%) of 10 informative tumors, and occurred in 1 atypical carcinoid and 2 small cell carcinomas. TP53 gene mutations were detected in 1 (25%) of 4 atypical carcinoids and 6 (60%) of 10 endocrine carcinomas. Five mutations were detected in exon 5 of the TP53 gene and the remaining 2 in exon 6. The specific mutations detected in the TP53 gene are listed in Table 2. Two of the four HPV-negative endocrine tumors that were informative for the polymorphic markers analyzed at the TP53 (17p13) locus lacked both gene abnormalities (LOH and point mutation). In contrast, oncogenic HPV-16 strain was detected in the only case demonstrating both allelic loss and point mutation at the TP53 gene.
LOH Analysis With the exception of the single typical carcinoid analyzed, allelic losses were demonstrated in the entire spectrum of endocrine tumors of the uterine cervix. Allelic losses at one or more 3p regions (7 of 15 cases, 47%) and 9p21 (6 of 14 cases,
DISCUSSION Several studies have described a spectrum of molecular changes involved in the pathogenesis of squamous cell carci-
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FIG. 2. (A) Agarose gel showing HPV-18-positive sequences in atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNC), and two small cell carcinoma (SCC) cases. Hela, positive control. (B) Representative autoradiographs of loss of heterozygosity (LOH) analyses show allelic loss at P53 gene marker in an atypical carcinoid and in 3p22–p24 and P53 gene markers in a small cell carcinoma. N indicates normal stromal cells; T, tumor. Bars represent the position of the major allelic bands. (C) Representative gels of TP53 gene sequencing of two neuroendocrine tumors of the uterine cervix. The left panel (small cell carcinoma) demonstrates a point mutation in exon 5, codon 168. The right panel (large cell neuroendocrine carcinoma, LCNC) demonstrates a point mutation in exon 5, codon 177.
noma [4 – 6], the most frequent histologic type of cervical cancer. However, other than HPV sequences, no data about genetic changes present in endocrine tumors of the uterine cervix have been reported. The morphologic and clinical features of the endocrine tumors of the uterine cervix are similar to those occurring in the lung, and a similar histologic classification has been adopted [3]. In a series of 15 endocrine tumors of the cervix uteri we studied the spectrum of genetic abnormalities frequently detected in squamous cervical carcinoma and neuroendocrine tumors of the lung, including oncogenic HPV sequences, LOH at 9 chromosomal regions, and TP53 gene mutations.
With the exception of the single typical carcinoid tumor analyzed, oncogenic HPV sequences, especially HPV-18 (6 of 8 HPV-positive cases), were detected in all types of endocrine tumors of the uterine cervix (53%). HPV sequences are strongly associated with the development of cervical squamous cell carcinoma and its precursor lesions [7]. To date more than 70 types of HPV have been identified on the basis of DNA sequences, and over a dozen have been associated with genital tract neoplasms [7]. Despite this heterogeneity, oncogenic HPV types 16 and 18 in particular have been shown to predominate in cervical carcinomas [7]. However, HPV-16 is two to four times more frequently detected than HPV-18 in squamous cell carcinoma of the uterine cervix [18, 19]. In contrast, as shown earlier and confirmed in this study, endocrine tumors of the cervix uteri are more frequently associated with HPV type 18 [13, 14]. In a study of 20 small cell carcinomas of the uterine cervix, HPV-18 was five times more frequently detected than HPV type 16 [13]. The higher prevalence of HPV type 18 in the endocrine tumors of the uterine cervix supports the concept that even though particular lesions show no absolute viral specificity, there is a relative tropism of HPV type 18 for reserve or stem cells that are committed to differentiate along nonsquamous lines. This is analogous to the situation described for HPV type 18 tropism in cervical adenocarcinomas [20]. Deletions of the short arm of the chromosome 3 are common in many human cancers and at least four distinct 3p regions, including 3p12, 3p14, 3p21, and 3p24 –p25, are believed to each harbor tumors suppressor genes (TSG) [9]. Allelic losses involving restricted regions of the chromosome 3p were the most frequent deletions (47%) detected in our cases of endocrine tumors of the uterine cervix, and they were detected in both major tumor types (carcinoid and carcinoma). Of the four regions tested on 3p, those showing LOH were 3p14 –p21 (38%), 3p21 (33%), and 3p22–p24 (7%). We previously reported [6] that 85% of the squamous cell carcinomas of the uterine cervix demonstrated deletions involving restricted regions of 3p, in contrast to the more extensive losses present in lung tumors, including lung carcinomas with endocrine differentiation (small cell and large cell carcinomas) [9, 21]. While extensive deletions of chromosome 3p are detected in most large cell neuroendocrine and small cell carcinomas of the lung, a high frequency of more restricted deletions have been detected in carcinoid tumors of the lung [21]. Of interest, cytogenetic and allelotyping analyses performed in five extrapulmonary small cell carcinomas (including a tumor from the cervix uteri) demonstrated retention of chromosome 3p [22]. Attention has been focused on FHIT, a candidate TSG at 3p14.2 that spans FRA3B, the most common of the aphidilcolin-inducible fragile sites [23]. Whereas allelic losses involving the FHIT gene locus have been frequently detected in squamous cell carcinoma of the uterine cervix (56%) [6] and small cell lung carcinomas (100%) [23], no deletions at this region were detected in our cases of endocrine tumors of the uterine
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MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS
TABLE 2 HPV Sequences, 3p Deletions, and TP53 Gene Abnomralities in 15 Endocrine Tumors of the Uterine Cervix
Case No.
Tumor histology
HPV strain
3p LOH
TP53 gene LOH
TP53 gene mutation
TP53 gene exon/codon/ base substitution
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Typical carcinoid Atypical carcinoid Atypical carcinoid Atypical carcinoid Atypical carcinoid LCNC LCNC Small cell Small cell Small cell Small cell Small cell Small cell Small cell Small cell
No 18 16 18 No No 18 No No 18 18 16 No No 18
No Yes, 3p12–p21/3p21 No No Yes, 3p21 Yes, 3p21 Yes, 3p21 Yes, 3p14–p21 Yes, 3p14–p21 Yes 3p22–p24 No No No No No
No No Yes No NI NI No No NI Yes NI Yes No No NI
No No No No Yes No Yes Yes No No Yes Yes Yes No Yes
— — — — Exon 6/199/GGA 3 AGA — Exon 5 /177/CCC 3 TCC Exon 5 /181/CGC 3 CAC —
— — — — Glycine 3 arginine — Proline 3 serine Arginine 3 histidine —
Exon 5 /177/CCC 3 TCC Exon 5 /168/CAC 3 TAC Exon 6/209/AGA 3 AAA — Exon 5 /140/ACC 3 ATC
Proline 3 serine Histidine 3 tyrosine Arginine 3 lysine — Threonine 3 isoleucine
Amino acid change
Note. NI, not informative for both polymorphic markers analyzed at TP53 (17p13) locus.
cervix. Integration of HPV sequences in the FRA3B site has been described in only a very limited number of cervical tumors, and probably represents only one of several possible integration sites [24]. In any case, only HPV type 16 has been described to integrate into FRA3B, while neuroendocrine tumors of the cervix are predominantly associated with HPV type 18 (six of eight HPV-positive cases in our current series). Allelic losses of the short arm of the chromosome 9 have been identified in several cancer types, and a putative oncogene has been identified at 9p21 region [25]. The gene, known by several names including CDKN2, encodes the p16 protein. Although CDKN2 gene mutations are uncommon in small cell lung carcinomas, allelic losses at 9p21 locus have been frequently detected in this tumor type, indicating that other TSGs in this region may be involved [9]. However, we previously reported [6] a low incidence (11%) of LOH at this region in squamous cell carcinomas of the uterine cervix. Allelic loss at the 9p21 region was the second most frequent (43%) deletion detected in our cases of endocrine cervical tumors, indicating that deletions at this region may play an important role in the pathogenesis in a subset of these neoplasms. Inactivation of p53 and Rb proteins by formation of complexes with E6 and E7 proteins, respectively, of highrisk oncogenic HPV strains 16 and 18 is the major mechanism of inactivation of these genes in squamous cell cervical carcinomas of the cervix [26, 27]. However, we detected a high incidence of TP53 gene abnormalities (60%) (either mutations, 47%, and deletions, 30%) in our cases of endocrine tumors of the uterine cervix, especially in endocrine carcinomas. While genetic abnormalities involving TP53 gene are a relatively rare event in squamous cell carcinomas of the uterine cervix [6], they are one of the most frequent
changes detected in endocrine tumors of the lung [9], including atypical carcinoid tumors [21]. Although the presence of oncogenic HPV strains is usually associated with lack of TP53 changes, there are many exceptions reported in the literature showing tumors with both oncogenic HPV sequences and TP53 gene mutations [28]. Thus it is not surprising that oncogenic HPV 16 sequences were detected in the single case demonstrating both allelic loss and point mutation at the TP53 gene. Allelic losses at 5q21– q22 APC-MCC region (30%), 11q23 MEN1 (22%), and 13qRB (9%) were infrequent, and no K-ras mutations were detected in our cases of endocrine tumors of the cervix uteri, indicating that these genes and chromosomal regions do not play an important role in the pathogenesis of these neoplasms. While deletions at 5q21– q22 APC-MCC region and 13q RB gene are frequent changes in endocrine carcinomas of the lung [9, 21], they are rare abnormalities in squamous cervical carcinoma [4, 29]. The recently cloned MEN1 gene localized at 11q13 [10], has been implicated in the pathogenesis of a fraction of the sporadic neuroendocrine tumors [11, 12]. Deletions at 11q13 region are the most frequent allelic loss detected in sporadic carcinoids [30, 31], and both mutations and deletions at the MEN-1 gene have been identified in sporadic lung carcinoids [12]. Whereas abnormalities of the MEN-1 gene have been described in some sporadic carcinoids [11, 12], to date no data exist for endocrine tumors of the uterine cervix. Our findings indicate that deletion at the MEN-1 gene locus is a rare event in the pathogenesis of carcinoids and endocrine carcinomas of the uterine cervix. Our previous studies of genetic abnormalities in endocrine tumors of the lung supported the concept that they represent a clinicopathological spectrum ranging from the
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low-grade typical carcinoid and intermediate-grade atypical carcinoid to the highly malignant large cell neuroendocrine and small cell carcinomas [21]. Although a small number of cases have been analyzed, our findings in endocrine tumors of the uterine cervix support the concept of a clinicopathological spectrum for this type of neoplasms. While no molecular abnormality in our single case of typical carcinoid of the uterine cervix was detected, atypical carcinoids demonstrated an intermediate level of genetic changes compared to highly malignant carcinoma types. In fact, a higher incidence of TP53 gene mutations was detected in our cases of cervical large cell neuroendocrine and small cell carcinomas than in the carcinoid tumors. Our findings indicate that the molecular changes present in endocrine tumors of the uterine cervix have distinct features. They demonstrate a high incidence of oncogenic HPV sequences (especially HPV type 18) and TP53 gene abnormalities, relatively small deletions of chromosome 3p regions, and occasional 9p21 deletions. Thus, they share some changes present in the neuroendocrine tumors of the lung (TP53 gene abnormalities and 9p21 allelic loss) as well those in squamous carcinomas of the cervix (oncogenic HPV sequences and localized 3p deletions). However, their overall genetic profile is distinct and different from that of the other two tumor types. REFERENCES 1. Albores-Saavedra J, Rodriguez-Martinez HA, Larraza-Hernandez O: Carcinoid tumor of the cervix. Pathol Annu 14:273–291, 1979 2. Albores-Saavedra J, Ashfaq R, Henson DE: Carcinoid and small cell carcinoma of the uterine cervix. Lab Invest 74:87A, 1996 3. Albores-Saavedra J, Gersell F, Gilks CB, Henson DE, Lindberg G, Santiago H, Scully RE, Silva E, Sobin LH, Tavassoli FJ, Travis WD, Woodruff JM: Terminology of endocrine tumors of the uterine cervix. Arch Pathol Lab Med 121:34 –39, 1997
Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM, Burns AL, Marx SJ: Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276:404 – 407, 1997 11. Zhuang Z, Vortmeyer AO, Pack S, Huang S, Pham TA, Wang C, Park WAS, Agarwal SK, Debelenko LV, Kester M, Guru SC, Manickam P, Olufemi SE, Yu F, Heppner C, Crabtree JS, Skarulis MC, Venzon DJ, Emmert-Buck MR, Spiegel AM, Chandrasekharappa SC, Collins FS, Burns AL, Marx SJ, Lubensky IA, et al.: Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res 57:4682– 4686, 1997 12. Debelenko LV, Brambilla E, Agarwal SK, Swalwell JI, Kester MB, Lubensky IA, Zhuang Z, Guru SC, Manickam P, Olufemi SE, Chandrasekharappa SC, Crabtree JS, Kim YS, Heppner C, Burns AL, Spiegel AM, Marx SJ, Liotta LA, Collins FS, Travis WD, Emmert-Buck MR: Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung. Hum Mol Genet 6:2285–2290, 1997 13. Stoler MH, Stacey EM, Gersell DJ, Walker AN: Small-cell neuroendocrine carcinoma of the cervix. A human papillomavirus type-18 associated cancer. Am J Surg Pathol 15:28 –32, 1991 14. Abeler VM, Holm R, Nesland JM, Kjorstad KE: Small cell carcinoma of the cervix. A clinicopathologic study of 26 patients. Cancer 73:672– 677, 1994 15. Wistuba II, Behrens C, Milchgrub S, Virmani AK, Jagirdar J, Thomas B, Ioachim HL, Litzky LA, Brambilla EM, Minna JD, Gazdar AF: Comparison of molecular changes in lung cancers in HIV-positive and HIVindeterminate subjects. JAMA 279:1554 –1559, 1998 16. Baay MF, Quint WG, Kuodstaal J, Hollema H, Duk JM, Burger MP, Stolz E, Herbrink P: Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 34:745–747, 1996 17. Sugio K, Molberg K, Albores-Saavedra J, Virmani AK, Kishimoto Y, Gazdar AF: K-ras mutations and allelic loss at 5q and 18q in the development of human pancreatic cancers. Int J Pancreatol 21:205– 217, 1997 18. Fuchs PG, Girardi F, HP: Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri. Int J Cancer 41:41– 45, 1988
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21. Onuki N, Wistuba II, Travis WD, Virmani AK, Yashima K, Brambilla E, Hasleton P, Gazdar AF: Genetic changes in the spectrum of neuroendocrine lung tumors. Cancer, in press. 22. Johnson BE, Whang-Peng J, Naylor SL, Zbar B, Brauch H, Lee E, Simmons A, Russell E, Nam MH, Gazdar AF: Retention of chromosome 3 in extrapulmonary small cell cancer shown by molecular and cytogenetic studies. J Natl Cancer Inst 81:1223–1228, 1989
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23. Fong KM, Biesterveld EJ, Virmani A, Wistuba I, Sekido Y, Bader SA, Ahmadian M, Tiong Ong S, Rassool FV, Zimmerman PV, Giaccone G, Gazdar AF, Minna JD: FHIT and FRA3B allele loss are common in lung cancer and preneoplastic bronchial lesions and are associated with cancer-related FHIT cDNA splicing aberrations. Cancer Res 57:2256 – 2267, 1997
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MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites. Hum Mol Genet 5:187–195, 1996 25. Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP, Forrester K, Gerwin B, Serrano M, Beach DH, Harris CC: Mutations and altered expression of p16 INK4 in human cancer. Proc Natl Acad Sci USA 91:11045–11049, 1994 26. Dyson N, Howley PM, Munger K, Harlow E: The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934 –937, 1989 27. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76 –79, 1990
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28. Kim JW, Cho YH, Lee CG, Kim JH, Kim HK, Kim EJ, Han KT, Namkoong SE: Human papillomavirus infection and TP53 gene mutation in primary cervical carcinoma. Acta Oncol 36:295–300, 1997 29. Mullokandov MR, Kholodilov NG, Atkin NB, Burk RD, Johnson AB, Klinger HP: Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. Cancer Res 56:197–205, 1996 30. Friedman E, De Marco L, Gejman PV, Norton JA, Bale ARE, Aurbach GD, Spiegel AM, Marx SJ: Allelic loss from chromosome 11 in parathyroid tumors. Cancer Res 52:6804 – 6809, 1992 31. Sawicki MP, Wan YJ, Johnson CL, Berenson J, Gatti R, Passaro EJ: Loss of heterozygosity on chromosome 11 in sporadic gastrinomas. Hum Genet 89:445– 449, 1992
Molecular Abnormalities Associated with Endocrine Tumors of the Uterine Cervix Ignacio I. Wistuba, M.D.,* ,† Bilue Thomas,* Carmen Behrens, M.D.,* Naoyoshi Onuki, M.D.,* Guy Lindberg, M.D.,‡ Jorge Albores-Saavedra, M.D.,‡ and Adi F. Gazdar, M.D.* ,‡ ,1 *Hamon Center for Therapeutic Oncology Research and ‡Division of Anatomic Pathology, Department of Pathology, University of Texas Southwestern Medical Center, Dallas, Texas; and †Department of Pathology, Pontificia Universidad Catolica de Chile, Santiago, Chile Received June 23, 1998
tive terms [3]. In 1997 a workshop sponsored by the Cancer Committee of the College of American Pathologists and the National Cancer Institute recommended a uniform terminology for these tumors [3]. As the morphologic features of the endocrine tumors of the uterine cervix are similar to those occurring in the lung, the same classification existent in the lung was proposed [3]. Thus, four categories of endocrine tumors of the uterine cervix are recognized: (1) typical carcinoid tumor; (2) atypical carcinoid tumor; (3) large cell neuroendocrine carcinoma (LCNC); and (4) small cell carcinoma. Several studies have described a spectrum of molecular changes involved in the pathogenesis of cervical squamous cell carcinomas [4 – 6]. While human papillomavirus (HPV) infection plays an important role in the pathogenesis of this neoplasm [7], other frequent molecular changes have also been detected [4 – 6]. Relatively high frequencies of loss of heterozygosity (LOH) at chromosomal regions 4p, 6q, and localized 3p deletions have been reported [4 – 6]. In contrast, low frequencies of TP53 and RB gene abnormalities are usually present, consistent with HPV-mediated inactivation of their protein products [6, 8]. On the other hand, many molecular abnormalities involved in the pathogenesis of the neuroendocrine tumors of the lung have been identified, especially those present in small cell carcinomas [9]. These tumors demonstrate a very high incidence of TP53 and RB gene mutations, as well as extensive deletions occurring at chromosome 3p, frequently involving losses of most or all of the arm [9]. Recently the gene for multiple endocrine neoplasia type 1 (MEN1) [10] localized at 11q13 has been implicated in the pathogenesis of some sporadic neuroendocrine tumors [11]. Mutations and deletions at the MEN-1 gene have been identified in a subset (36%) of sporadic lung carcinoids [12]. Although cervical endocrine neoplasms are not included in the spectrum of MEN1, no data about the role of the MEN1 gene in their pathogenesis have been reported. Other than the presence of HPV sequences, especially highrisk strain 18 [13, 14], no information about genetic changes involved in endocrine tumors of uterine cervix is available. We
Objective. We studied the molecular abnormalities involved in the pathogenesis of endocrine tumors of the uterine cervix. Methods. We obtained DNA from precisely microdissected archival tissue from 15 endocrine tumors of the uterine cervix, consisting of 5 carcinoids (1 typical, 4 atypical), 2 large cell neuroendocrine carcinomas, and 8 small cell carcinomas. We investigated the presence of high-risk (types 16 and 18) and intermediate-risk (types 31 and 33) human papilloma virus (HPV) sequences, TP53 and K-ras gene mutations, and loss of heterozygosity (LOH) at 9 genes/chromosomal regions, including 3p14.2/ FHIT, 3p14 –p21, 3p21, 3p22–p24, 5q21– q22/APC-MCC region, 9p21/CDKN2, 11q23/MEN1, 13q/RB, and 17p/TP53. Results. HPV sequences were detected in 8 (53%) tumors, HPV 16 in 2 cases, and HPV 18 in 2 cases. LOH at 9p21 (43%) and localized 3p deletions (47%) were the most frequent allelic losses found. Allelic losses at 5q21– q22/APC-MCC region, 11q23/MEN1, and 13q/RB were infrequent. TP53 gene mutations were detected in 7 (47%) tumors (1 atypical carcinoid and 6 carcinomas). HPV sequences were demonstrated in 4 of the 7 cases with TP53 gene mutations. No K-ras mutations were detected. Conclusion. The molecular changes present in endocrine tumors of the uterine cervix have distinct features. They incorporate those present in the neuroendocrine tumors of the lung (high frequency of TP53 gene abnormalities and 9p21 deletions) with those detected in squamous cell carcinomas of the cervix (high-risk HPV sequences and localized 3p deletions). © 1999 Academic Press Key Words: neuroendocrine tumors; tumors of the uterine cervix; loss of heterozygosity; chromosome 3p; TP53 gene mutations.
INTRODUCTION Although rare, malignant tumors with endocrine differentiation are a morphologically distinct group of cervical neoplasms having a highly aggressive clinical course [1–3]. Following their initial description, several reports emphasized their broad morphologic spectrum using a variety of descrip1
To whom correspondence and reprint requests should be addressed at Hamon Center for Therapeutic Oncology Research, University of Texas, Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 752358593. Fax: (214) 590-6424. 3
0090-8258/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved.
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TABLE 1 Summary of Genetic Abnormalities in 15 Endocrine Tumors of the Uterine Cervix
Genetic abnormality
Carcinoid (n 5 1)
Atypical carcinoids (n 5 4)
Large cell neuroendocrine carcinomas (n 5 2)
Small cell carcinomas (n 5 8)
Total (%) (n 5 15)
HPV sequences 3p LOH (any region) 3p14.2 (FHIT gene) 3p14–p21 3p21 3p22–p24 5q22 (APC-MCC region) LOH 9p21 (CDKN2 gene) LOH 11q13 (MEN1 gene) LOH 13q (RB gene) LOH TP53 gene abnormalities 17q (TP53 gene) LOH TP53 gene mutations
0/1 0/1 0/1 — 0/1 0/1 0/1 0/1 — 0/1 0/1 0/1 0/1
3/4 2/4 0/2 1/4 2/4 0/4 1/2 3/4 2/3 0/4 2/4 1/3 1/4
1/2 2/2 0/2 0/1 2/2 0/2 0/2 0/2 0/1 0/1 1/2 0/1 1/2
4/8 3/8 0/5 1/3 0/8 0/8 2/5 3/17 0/5 1/5 6/18 2/5 5/8
8/15 (53) 7/15 (47) 0/10 3/8 (38) 4/15 (27) 1/15 (7) 3/10 (30) 6/14 (43) 2/9 (22) 1/11 (9) 9/15 (60) 3/10 (30) 7/15 (47)
investigated whether the molecular changes present in squamous cell carcinomas of the uterine cervix and endocrine tumors of the lung are detected in endocrine tumors of the uterine cervix. We investigated the presence of high-risk HPV sequences, TP53 and K-ras gene mutations, and LOH at 9 chromosomal regions, including 3p14.2/FHIT, 3p14 –p21, 3p21, 3p22–p24, 5q21– q22 at the APC-MCC region, 9p21/ CDKN2, 11q13/MEN1, 13q/RB, and 17p/TP53, in a series of 15 endocrine tumors of the uterine cervix. MATERIALS AND METHODS Neuroendocrine Tumor Specimens and Patient Information Neuroendocrine tumors of the cervix are relatively rare, and a search of our files including consultation materials yielded blocks from a total of 15 confirmed neuroendocrine tumors of the uterine cervix including 5 carcinoid tumors (1 typical and 4 atypical carcinoids), 2 LCNC, and 8 small cell carcinomas (Table 1). Histological diagnoses were made by one of us (J.A.-S.), a reference pathologist who helped establish the criteria recently published for the classification and terminology of endocrine tumors of the uterine cervix [3]. Immunohistochemical stains for general neuroendocrine markers including chromogranin and synaptophysin were performed in all tumors to confirm the neuroendocrine origin of the tumors. The patients ranged in age from 31 to 88 years (median 43). Microdissection and DNA Extraction Serial 5-mM sections were cut from archival, formalin-fixed, paraffin-embedded tissue. All slides were stained with hematoxylin– eosin, and one of the slides was coverslipped. The coverslipped slide was used as a guide to localize regions of interest for microdissection for the other slide. Microdissection
and DNA extraction were performed as previously described from noncoverslipped hematoxylin– eosin stained slides [15]. Precisely identified areas of endocrine tumor and stromal cells (as source of constitutional DNA) were microdissected under microscopic visualization (Fig. 1). Detection of HPV Sequences The presence of HPV sequences was tested by PCR using general and type-specific primers designed for paraffin-extracted DNA [16]. Specific primers were used to identify high (types 16 and 18) and intermediate (types 31 and 33) oncogenic risk HPV strains [16]. The HPV analysis was done using one round PCR. DNA extracted from human cell lines CaSki (HPV 16) and HeLa (HPV 18) (obtained from the American Type Culture Collection, Rockville, MD) were used as positive controls for HPV analyses. Polymorphic DNA Markers and PCR–LOH Analysis To evaluate LOH, we used primers flanking 18 dinucleotide and multinucleotide microsatellites repeat polymorphisms located at the following 9 genes or chromosomal regions: 3p14.2 at the FHIT gene (D3S4103), 3p14 –p21 (D3S1766), 3p21 (D3S1029, D3S1478, K1.CA, ITIH-1, D3S1447), 3p22–p24 (D3S1537, D3S1351, D3S2432), 5q21– q22 (L5.71 in the APC-MCC region), 9p21 near the CDKN2 gene (IFNA, D9S1748), 11q13 at the MEN1 gene region (D11S4907, PYGM), RB gene at 13q14 (dinucleotide repeat), and TP53 gene at 17p13 (dinucleotide and pentanucleotide repeats). Primer sequences can be obtained from the Genome Database, with three exceptions (dinucleotide repeat in the RB gene, pentanucleotide and dinucleotide repeats in the TP53 gene) previously published and referenced [15]. Nested PCR or tworound PCR (using the same set of primers in two consecutive
MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS
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FIG. 1. Representative example of microdissection of small cell carcinoma of the uterine cervix (a) before and (b) after microdissection. Hematoxylin and eosin 375.
amplifications) methods were used as previously described [15]. Multiplex PCR was performed during the first amplification, followed by uniplex PCR for individual markers. In the multiplex PCR, four markers were amplified during the same reaction. LOH was scored by visual detection of complete absence of one allele (Fig. 2). TP53 and ras Gene Mutation Analyses We examined for mutations in exons 5 to 8 of the TP53 gene using nested PCR methodology, followed by SSCP analysis and sequencing as previously described [6]. For detection of K-ras mutations, we used a designed RFLP method, utilizing nested PCR methodology, followed by sequencing, as described previously [17]. Using the designed RFLP method we screened for mutations in codons 12, 13, and 61 of K-ras gene [17]. RESULTS Detection of HPV Sequences Eight (53%) of the 15 endocrine tumors of the uterine cervix were positive for high-risk HPV sequences (types 16 and 18) using general and type-specific HPV primers (Tables 1 and 2). The HPV distribution was 2 cases (25%) of HPV-16 and 6 cases (75%) of HPV-18. No intermediate-risk HPV strains were detected. With the exception of the single typical carcinoid analyzed, oncogenic HPV sequences were detected in the entire spectrum of endocrine tumors of the uterine cervix.
43%) were the most frequent deletions detected in endocrine carcinomas of the uterine cervix (Table 1). The 3p deletions detected in endocrine tumors were in all cases localized, occurring at 3p14 –p21 (38%), 3p21 (27%), and 3p22–p24 (7%) regions (Tables 1 and 2). No deletion at FHIT gene region (3p14.2) was detected. In contrast, allelic losses at 5q21– q22 (APC-MCC region; 3 of 10 cases, 30%), 11q13 (MEN1 gene locus; 2 of 9 cases, 22%), and RB gene (13q; 1 of 11 cases, 9%) were relatively infrequent. Frequency of TP53 Gene Abnormalities TP53 gene abnormalities (either point mutation or LOH) were frequent events (60%) in endocrine tumors of the uterine cervix (Table 1). Seven (47%) of the 15 tumors demonstrated TP53 gene point mutations (Table 2 and Fig. 2C). Allelic loss at the TP53 loci was detected in 3 (30%) of 10 informative tumors, and occurred in 1 atypical carcinoid and 2 small cell carcinomas. TP53 gene mutations were detected in 1 (25%) of 4 atypical carcinoids and 6 (60%) of 10 endocrine carcinomas. Five mutations were detected in exon 5 of the TP53 gene and the remaining 2 in exon 6. The specific mutations detected in the TP53 gene are listed in Table 2. Two of the four HPV-negative endocrine tumors that were informative for the polymorphic markers analyzed at the TP53 (17p13) locus lacked both gene abnormalities (LOH and point mutation). In contrast, oncogenic HPV-16 strain was detected in the only case demonstrating both allelic loss and point mutation at the TP53 gene.
LOH Analysis With the exception of the single typical carcinoid analyzed, allelic losses were demonstrated in the entire spectrum of endocrine tumors of the uterine cervix. Allelic losses at one or more 3p regions (7 of 15 cases, 47%) and 9p21 (6 of 14 cases,
DISCUSSION Several studies have described a spectrum of molecular changes involved in the pathogenesis of squamous cell carci-
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FIG. 2. (A) Agarose gel showing HPV-18-positive sequences in atypical carcinoid (AC), large cell neuroendocrine carcinoma (LCNC), and two small cell carcinoma (SCC) cases. Hela, positive control. (B) Representative autoradiographs of loss of heterozygosity (LOH) analyses show allelic loss at P53 gene marker in an atypical carcinoid and in 3p22–p24 and P53 gene markers in a small cell carcinoma. N indicates normal stromal cells; T, tumor. Bars represent the position of the major allelic bands. (C) Representative gels of TP53 gene sequencing of two neuroendocrine tumors of the uterine cervix. The left panel (small cell carcinoma) demonstrates a point mutation in exon 5, codon 168. The right panel (large cell neuroendocrine carcinoma, LCNC) demonstrates a point mutation in exon 5, codon 177.
noma [4 – 6], the most frequent histologic type of cervical cancer. However, other than HPV sequences, no data about genetic changes present in endocrine tumors of the uterine cervix have been reported. The morphologic and clinical features of the endocrine tumors of the uterine cervix are similar to those occurring in the lung, and a similar histologic classification has been adopted [3]. In a series of 15 endocrine tumors of the cervix uteri we studied the spectrum of genetic abnormalities frequently detected in squamous cervical carcinoma and neuroendocrine tumors of the lung, including oncogenic HPV sequences, LOH at 9 chromosomal regions, and TP53 gene mutations.
With the exception of the single typical carcinoid tumor analyzed, oncogenic HPV sequences, especially HPV-18 (6 of 8 HPV-positive cases), were detected in all types of endocrine tumors of the uterine cervix (53%). HPV sequences are strongly associated with the development of cervical squamous cell carcinoma and its precursor lesions [7]. To date more than 70 types of HPV have been identified on the basis of DNA sequences, and over a dozen have been associated with genital tract neoplasms [7]. Despite this heterogeneity, oncogenic HPV types 16 and 18 in particular have been shown to predominate in cervical carcinomas [7]. However, HPV-16 is two to four times more frequently detected than HPV-18 in squamous cell carcinoma of the uterine cervix [18, 19]. In contrast, as shown earlier and confirmed in this study, endocrine tumors of the cervix uteri are more frequently associated with HPV type 18 [13, 14]. In a study of 20 small cell carcinomas of the uterine cervix, HPV-18 was five times more frequently detected than HPV type 16 [13]. The higher prevalence of HPV type 18 in the endocrine tumors of the uterine cervix supports the concept that even though particular lesions show no absolute viral specificity, there is a relative tropism of HPV type 18 for reserve or stem cells that are committed to differentiate along nonsquamous lines. This is analogous to the situation described for HPV type 18 tropism in cervical adenocarcinomas [20]. Deletions of the short arm of the chromosome 3 are common in many human cancers and at least four distinct 3p regions, including 3p12, 3p14, 3p21, and 3p24 –p25, are believed to each harbor tumors suppressor genes (TSG) [9]. Allelic losses involving restricted regions of the chromosome 3p were the most frequent deletions (47%) detected in our cases of endocrine tumors of the uterine cervix, and they were detected in both major tumor types (carcinoid and carcinoma). Of the four regions tested on 3p, those showing LOH were 3p14 –p21 (38%), 3p21 (33%), and 3p22–p24 (7%). We previously reported [6] that 85% of the squamous cell carcinomas of the uterine cervix demonstrated deletions involving restricted regions of 3p, in contrast to the more extensive losses present in lung tumors, including lung carcinomas with endocrine differentiation (small cell and large cell carcinomas) [9, 21]. While extensive deletions of chromosome 3p are detected in most large cell neuroendocrine and small cell carcinomas of the lung, a high frequency of more restricted deletions have been detected in carcinoid tumors of the lung [21]. Of interest, cytogenetic and allelotyping analyses performed in five extrapulmonary small cell carcinomas (including a tumor from the cervix uteri) demonstrated retention of chromosome 3p [22]. Attention has been focused on FHIT, a candidate TSG at 3p14.2 that spans FRA3B, the most common of the aphidilcolin-inducible fragile sites [23]. Whereas allelic losses involving the FHIT gene locus have been frequently detected in squamous cell carcinoma of the uterine cervix (56%) [6] and small cell lung carcinomas (100%) [23], no deletions at this region were detected in our cases of endocrine tumors of the uterine
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MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS
TABLE 2 HPV Sequences, 3p Deletions, and TP53 Gene Abnomralities in 15 Endocrine Tumors of the Uterine Cervix
Case No.
Tumor histology
HPV strain
3p LOH
TP53 gene LOH
TP53 gene mutation
TP53 gene exon/codon/ base substitution
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15
Typical carcinoid Atypical carcinoid Atypical carcinoid Atypical carcinoid Atypical carcinoid LCNC LCNC Small cell Small cell Small cell Small cell Small cell Small cell Small cell Small cell
No 18 16 18 No No 18 No No 18 18 16 No No 18
No Yes, 3p12–p21/3p21 No No Yes, 3p21 Yes, 3p21 Yes, 3p21 Yes, 3p14–p21 Yes, 3p14–p21 Yes 3p22–p24 No No No No No
No No Yes No NI NI No No NI Yes NI Yes No No NI
No No No No Yes No Yes Yes No No Yes Yes Yes No Yes
— — — — Exon 6/199/GGA 3 AGA — Exon 5 /177/CCC 3 TCC Exon 5 /181/CGC 3 CAC —
— — — — Glycine 3 arginine — Proline 3 serine Arginine 3 histidine —
Exon 5 /177/CCC 3 TCC Exon 5 /168/CAC 3 TAC Exon 6/209/AGA 3 AAA — Exon 5 /140/ACC 3 ATC
Proline 3 serine Histidine 3 tyrosine Arginine 3 lysine — Threonine 3 isoleucine
Amino acid change
Note. NI, not informative for both polymorphic markers analyzed at TP53 (17p13) locus.
cervix. Integration of HPV sequences in the FRA3B site has been described in only a very limited number of cervical tumors, and probably represents only one of several possible integration sites [24]. In any case, only HPV type 16 has been described to integrate into FRA3B, while neuroendocrine tumors of the cervix are predominantly associated with HPV type 18 (six of eight HPV-positive cases in our current series). Allelic losses of the short arm of the chromosome 9 have been identified in several cancer types, and a putative oncogene has been identified at 9p21 region [25]. The gene, known by several names including CDKN2, encodes the p16 protein. Although CDKN2 gene mutations are uncommon in small cell lung carcinomas, allelic losses at 9p21 locus have been frequently detected in this tumor type, indicating that other TSGs in this region may be involved [9]. However, we previously reported [6] a low incidence (11%) of LOH at this region in squamous cell carcinomas of the uterine cervix. Allelic loss at the 9p21 region was the second most frequent (43%) deletion detected in our cases of endocrine cervical tumors, indicating that deletions at this region may play an important role in the pathogenesis in a subset of these neoplasms. Inactivation of p53 and Rb proteins by formation of complexes with E6 and E7 proteins, respectively, of highrisk oncogenic HPV strains 16 and 18 is the major mechanism of inactivation of these genes in squamous cell cervical carcinomas of the cervix [26, 27]. However, we detected a high incidence of TP53 gene abnormalities (60%) (either mutations, 47%, and deletions, 30%) in our cases of endocrine tumors of the uterine cervix, especially in endocrine carcinomas. While genetic abnormalities involving TP53 gene are a relatively rare event in squamous cell carcinomas of the uterine cervix [6], they are one of the most frequent
changes detected in endocrine tumors of the lung [9], including atypical carcinoid tumors [21]. Although the presence of oncogenic HPV strains is usually associated with lack of TP53 changes, there are many exceptions reported in the literature showing tumors with both oncogenic HPV sequences and TP53 gene mutations [28]. Thus it is not surprising that oncogenic HPV 16 sequences were detected in the single case demonstrating both allelic loss and point mutation at the TP53 gene. Allelic losses at 5q21– q22 APC-MCC region (30%), 11q23 MEN1 (22%), and 13qRB (9%) were infrequent, and no K-ras mutations were detected in our cases of endocrine tumors of the cervix uteri, indicating that these genes and chromosomal regions do not play an important role in the pathogenesis of these neoplasms. While deletions at 5q21– q22 APC-MCC region and 13q RB gene are frequent changes in endocrine carcinomas of the lung [9, 21], they are rare abnormalities in squamous cervical carcinoma [4, 29]. The recently cloned MEN1 gene localized at 11q13 [10], has been implicated in the pathogenesis of a fraction of the sporadic neuroendocrine tumors [11, 12]. Deletions at 11q13 region are the most frequent allelic loss detected in sporadic carcinoids [30, 31], and both mutations and deletions at the MEN-1 gene have been identified in sporadic lung carcinoids [12]. Whereas abnormalities of the MEN-1 gene have been described in some sporadic carcinoids [11, 12], to date no data exist for endocrine tumors of the uterine cervix. Our findings indicate that deletion at the MEN-1 gene locus is a rare event in the pathogenesis of carcinoids and endocrine carcinomas of the uterine cervix. Our previous studies of genetic abnormalities in endocrine tumors of the lung supported the concept that they represent a clinicopathological spectrum ranging from the
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low-grade typical carcinoid and intermediate-grade atypical carcinoid to the highly malignant large cell neuroendocrine and small cell carcinomas [21]. Although a small number of cases have been analyzed, our findings in endocrine tumors of the uterine cervix support the concept of a clinicopathological spectrum for this type of neoplasms. While no molecular abnormality in our single case of typical carcinoid of the uterine cervix was detected, atypical carcinoids demonstrated an intermediate level of genetic changes compared to highly malignant carcinoma types. In fact, a higher incidence of TP53 gene mutations was detected in our cases of cervical large cell neuroendocrine and small cell carcinomas than in the carcinoid tumors. Our findings indicate that the molecular changes present in endocrine tumors of the uterine cervix have distinct features. They demonstrate a high incidence of oncogenic HPV sequences (especially HPV type 18) and TP53 gene abnormalities, relatively small deletions of chromosome 3p regions, and occasional 9p21 deletions. Thus, they share some changes present in the neuroendocrine tumors of the lung (TP53 gene abnormalities and 9p21 allelic loss) as well those in squamous carcinomas of the cervix (oncogenic HPV sequences and localized 3p deletions). However, their overall genetic profile is distinct and different from that of the other two tumor types. REFERENCES 1. Albores-Saavedra J, Rodriguez-Martinez HA, Larraza-Hernandez O: Carcinoid tumor of the cervix. Pathol Annu 14:273–291, 1979 2. Albores-Saavedra J, Ashfaq R, Henson DE: Carcinoid and small cell carcinoma of the uterine cervix. Lab Invest 74:87A, 1996 3. Albores-Saavedra J, Gersell F, Gilks CB, Henson DE, Lindberg G, Santiago H, Scully RE, Silva E, Sobin LH, Tavassoli FJ, Travis WD, Woodruff JM: Terminology of endocrine tumors of the uterine cervix. Arch Pathol Lab Med 121:34 –39, 1997
Emmert-Buck MR, Debelenko LV, Zhuang Z, Lubensky IA, Liotta LA, Crabtree JS, Wang Y, Roe BA, Weisemann J, Boguski MS, Agarwal SK, Kester MB, Kim YS, Heppner C, Dong Q, Spiegel AM, Burns AL, Marx SJ: Positional cloning of the gene for multiple endocrine neoplasia-type 1. Science 276:404 – 407, 1997 11. Zhuang Z, Vortmeyer AO, Pack S, Huang S, Pham TA, Wang C, Park WAS, Agarwal SK, Debelenko LV, Kester M, Guru SC, Manickam P, Olufemi SE, Yu F, Heppner C, Crabtree JS, Skarulis MC, Venzon DJ, Emmert-Buck MR, Spiegel AM, Chandrasekharappa SC, Collins FS, Burns AL, Marx SJ, Lubensky IA, et al.: Somatic mutations of the MEN1 tumor suppressor gene in sporadic gastrinomas and insulinomas. Cancer Res 57:4682– 4686, 1997 12. Debelenko LV, Brambilla E, Agarwal SK, Swalwell JI, Kester MB, Lubensky IA, Zhuang Z, Guru SC, Manickam P, Olufemi SE, Chandrasekharappa SC, Crabtree JS, Kim YS, Heppner C, Burns AL, Spiegel AM, Marx SJ, Liotta LA, Collins FS, Travis WD, Emmert-Buck MR: Identification of MEN1 gene mutations in sporadic carcinoid tumors of the lung. Hum Mol Genet 6:2285–2290, 1997 13. Stoler MH, Stacey EM, Gersell DJ, Walker AN: Small-cell neuroendocrine carcinoma of the cervix. A human papillomavirus type-18 associated cancer. Am J Surg Pathol 15:28 –32, 1991 14. Abeler VM, Holm R, Nesland JM, Kjorstad KE: Small cell carcinoma of the cervix. A clinicopathologic study of 26 patients. Cancer 73:672– 677, 1994 15. Wistuba II, Behrens C, Milchgrub S, Virmani AK, Jagirdar J, Thomas B, Ioachim HL, Litzky LA, Brambilla EM, Minna JD, Gazdar AF: Comparison of molecular changes in lung cancers in HIV-positive and HIVindeterminate subjects. JAMA 279:1554 –1559, 1998 16. Baay MF, Quint WG, Kuodstaal J, Hollema H, Duk JM, Burger MP, Stolz E, Herbrink P: Comprehensive study of several general and type-specific primer pairs for detection of human papillomavirus DNA by PCR in paraffin-embedded cervical carcinomas. J Clin Microbiol 34:745–747, 1996 17. Sugio K, Molberg K, Albores-Saavedra J, Virmani AK, Kishimoto Y, Gazdar AF: K-ras mutations and allelic loss at 5q and 18q in the development of human pancreatic cancers. Int J Pancreatol 21:205– 217, 1997 18. Fuchs PG, Girardi F, HP: Human papillomavirus DNA in normal, metaplastic, preneoplastic and neoplastic epithelia of the cervix uteri. Int J Cancer 41:41– 45, 1988
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20. Teshima H, Beaudenon S, Koi S, Katase K, Hasumi K, Masabuchi K, Orth G: Human papillomavirus type 18 DNA sequences in adenocarcinoma and adenosquamous carcinoma of the uterine cervix. Arch Gynecol Obstet 259:169 –177, 1997
6. Wistuba II, Montellano FD, Milchgrub S, Virmani AK, Behrens C, Chen H, Ahmadian M, Nowak JA, Muller C, Minna JD, Gazdar AF: Deletions of chromosome 3p are frequent and early events in the pathogenesis of uterine cervical carcinoma. Cancer Res 57:3154 –3158, 1997 7. zur Hausen H: Human papillomavirus in the pathogenesis of anogenital cancer. Virology 184:9 –13, 1991 8. Pao CC, Kao SM, Chen JH, Tang GC, Chang PY, Tan TT: State of mutational alterations of p53 and retinoblastoma susceptibility genes in papillomavirus-negative small cell cervical carcinomas. J Surg Oncol 57:87–93, 1994
21. Onuki N, Wistuba II, Travis WD, Virmani AK, Yashima K, Brambilla E, Hasleton P, Gazdar AF: Genetic changes in the spectrum of neuroendocrine lung tumors. Cancer, in press. 22. Johnson BE, Whang-Peng J, Naylor SL, Zbar B, Brauch H, Lee E, Simmons A, Russell E, Nam MH, Gazdar AF: Retention of chromosome 3 in extrapulmonary small cell cancer shown by molecular and cytogenetic studies. J Natl Cancer Inst 81:1223–1228, 1989
9. Minna JD, Sekido Y, Fong K, Gazdar AF: Molecular Biology of Lung Cancer. in DeVita VT,Jr, Hellman S, Rosenberg SA (eds): Cancer: Principles and Practice of Oncology, 5th ed, Philadelphia, Lippincott, 1997, pp 849 – 857.
23. Fong KM, Biesterveld EJ, Virmani A, Wistuba I, Sekido Y, Bader SA, Ahmadian M, Tiong Ong S, Rassool FV, Zimmerman PV, Giaccone G, Gazdar AF, Minna JD: FHIT and FRA3B allele loss are common in lung cancer and preneoplastic bronchial lesions and are associated with cancer-related FHIT cDNA splicing aberrations. Cancer Res 57:2256 – 2267, 1997
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24. Wilke CM, Hall BK, Hoge A, Paradee W, Smith DI, Glover TW: FRA3B
MOLECULAR CHANGES IN CERVICAL ENDOCRINE TUMORS extends over a broad region and contains a spontaneous HPV16 integration site: direct evidence for the coincidence of viral integration sites and fragile sites. Hum Mol Genet 5:187–195, 1996 25. Okamoto A, Demetrick DJ, Spillare EA, Hagiwara K, Hussain SP, Bennett WP, Forrester K, Gerwin B, Serrano M, Beach DH, Harris CC: Mutations and altered expression of p16 INK4 in human cancer. Proc Natl Acad Sci USA 91:11045–11049, 1994 26. Dyson N, Howley PM, Munger K, Harlow E: The human papilloma virus-16 E7 oncoprotein is able to bind to the retinoblastoma gene product. Science 243:934 –937, 1989 27. Werness BA, Levine AJ, Howley PM: Association of human papillomavirus types 16 and 18 E6 proteins with p53. Science 248:76 –79, 1990
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28. Kim JW, Cho YH, Lee CG, Kim JH, Kim HK, Kim EJ, Han KT, Namkoong SE: Human papillomavirus infection and TP53 gene mutation in primary cervical carcinoma. Acta Oncol 36:295–300, 1997 29. Mullokandov MR, Kholodilov NG, Atkin NB, Burk RD, Johnson AB, Klinger HP: Genomic alterations in cervical carcinoma: losses of chromosome heterozygosity and human papilloma virus tumor status. Cancer Res 56:197–205, 1996 30. Friedman E, De Marco L, Gejman PV, Norton JA, Bale ARE, Aurbach GD, Spiegel AM, Marx SJ: Allelic loss from chromosome 11 in parathyroid tumors. Cancer Res 52:6804 – 6809, 1992 31. Sawicki MP, Wan YJ, Johnson CL, Berenson J, Gatti R, Passaro EJ: Loss of heterozygosity on chromosome 11 in sporadic gastrinomas. Hum Genet 89:445– 449, 1992