p16INK4a Overexpression and Human Papillomavirus Infection in Small Cell Carcinoma of the Uterine Cervix NOBUO MASUMOTO, MD, TAKUMA FUJII, MD, MITSUYA ISHIKAWA, MIYUKI SAITO, CT, TAKASHI IWATA, MD, TAKESHI FUKUCHI, MD, NOBUYUKI SUSUMU, MD, MAKIO MUKAI, MD, KANEYUKI KUBUSHIRO, MD, KATSUMI TSUKAZAKI, MD, AND SHIRO NOZAWA, MD Carcinogenesis of cervical cancer has been investigated, and p16INK4a overexpression in squamous cell carcinoma of the cervix has been reported as a result of infection by human papillomavirus (HPV) (eg, HPV 16), and the consequence of the retinoblastoma (Rb) protein inactivation by HPV E7 protein. However, to our knowledge, there have been no studies on the relation between p16INK4a overexpression associated with HPV and small cell carcinoma of the cervix, which behaves more aggressively clinically than squamous cell carcinoma. The purpose of this study was to determine whether p16INK4a is overexpressed in small cell carcinoma, and if p16INK4a is overexpressed, the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined them for p16INK4a overexpression by immunohistochemistry. We also performed HPV typing with polymerase chain reaction (PCR)-sequencing analysis and in situ hybridization and found that p16INK4a was overexpressed in every case. PCR-sequencing analyses revealed that all cases were HPV-positive and that 9 cases were positive for HPV 18. Five of the 9 cases positive for HPV 18 were also positive by in situ hybridization and yielded a punctate signal, considered to represent the integrated form. In conclusion, p16INK4a was overexpressed and HPV 18 was frequently detected in an integrated form in small cell carcinoma. Therefore, inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogenesis of small cell carcinoma the same as inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogenesis of squamous cell carcinoma. HUM PATHOL 34:778-783. © 2003 Elsevier Inc. All rights reserved. Key words: small cell carcinoma, p16INK4a, human papillomavirus. Abbreviations: Cdks, cyclin-dependent kinase; CKIs, Cdk-inhibitory proteins; HPV, human papillomavirus; PCR, polymerase chain reaction; Rb, retinoblastoma; TBST, Tris buffered saline with Tween 20. Cancer of the uterine cervix is one of the most common cancers among women worldwide and is second only to breast cancer in incidence and mortality.1 Small cell carcinoma is one of the aggressive pathlogic types of cervical cancer that have a poor prognosis. The carcinogenesis of cervical cancer has been investigated, and an association between genital human papillomavirus (HPV) and carcinogenesis of this cancer has been reported.2,3 Some types of HPV (eg, HPV 16) are considered high risk for squamous cell carcinoma of the cervix, and p16INK4a, one of the cyclin-dependent kinase (Cdk) inhibitory proteins (CKIs) that regulates the cell cycle, has been reported to be overexpressed when retinoblastoma (Rb) protein is inactivated by HPV E7 protein or by mutation,4,5 suggesting that inactivation of Rb protein by HPV E7 protein results in abnormal cell cycle progression. The frequency of overexpression of p16INK4a has been reported to be 97% in cervical squamous cell carcinoma6 and 48% to 50% in cervical adenocarcinoma.7,8 However, there have been no studies on overexpression of p16INK4a in cervical small cell carcinoma, which is considered a very aggressive form of cervical cancer. This study was carried out to determine whether p16INK4a is overexpressed in small cell carcinoma, and if it is, to identify the types of HPV that are related to this cancer. We reviewed 10 cases of small cell carcinoma and examined tissue specimens from them for p16INK4a overexpression by immunohistochemistry. We also performed HPV typing by direct sequencing of polymerase chain reaction (PCR) products. We detected HPV in cytological specimens as described previously,9,10 and in the present study we applied this method to a PCR sequencing analysis using paraffin-embedded materials in this study. We also performed HPV typing with in situ hybridization to assess HPV status in tumor cells. To our knowledge, this is the first report of p16INK4a overexpression in small cell carcinoma. MATERIALS AND METHODS Sample Preparation Between 1994 and 2002, 449 patients with invasive cervical cancer were evaluated and treated at Keio University Hospital, Tokyo, Japan, and 10 cases (2.2%) of small cell carcinoma of the cervix were identified. The clinical records of these patients were examined, and their follow-up data are shown in Table 1. No patients were lost to follow-up. From the Department of Obstetrics and Gynecology and Department of Pathology, Keio University, School of Medicine, Tokyo, Japan. Accepted for publication April 2, 2003. Supported in part by a Grant-in-Aid for Scientific Research (A) for Japanese Society for the promotion of Science and Research Grants for Life Science and Medicine, the Keio University Medical Science Fund, and by a Grant-in-Aid for Scientific Research on Priority Area (C) in 2000-2003 from the Ministry of Education, Science, Sports, Culture, and Technology in Japan. Address correspondence and reprint requests to Dr. Takuma Fujii, Department of Obstetrics and Gynecology, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan. © 2003 Elsevier Inc. All rights reserved. 0046-8177/03/3408-0008$30.00/0 doi:10.1016/S0046-8177(03)00284-3 778Immunohistochemistry Sections for immunohistochemistry were stained with antibodies to p16INK4a, neuron-specific enolase (NSE), leu-7, chromogranin, synaptophysin, and somatostatin. The immunohistochemistry technique used for p16INK4a was as follows. Sections from a formalin-fixed, paraffin-embedded block were mounted on a saline-coated glass slide, dewaxed in xylene, and rinsed in phosphate-buffered saline (PBS). Endogeneous peroxidase activity was blocked by incubating in 1% H2O2-PBS for 5 minutes, and after rinsing in PBS and rehydration, the sections were microwaved in citrate buffer (pH 6.0) at 95°C for 20 minutes as an antigen-retrieval step. Sections were then incubated with normal serum for 15 minutes, followed by incubation with a 1:200 dilution of anti-p16INK4a monoclonal antibody (clone E6H4; MTM Laboratories, Heidelberg, Germany) for 3 hours. Sections were exposed to HISTOFINE Simple Stain PO(M) (Nichirei, Tokyo, Japan) for 30 minutes, and peroxidase was visualized with 0.02% 3, 3'-diaminobenzidine tetrahydrochloride. Finally, the sections were counterstained lightly with hematoxylin. Immunoreactivity for p16INK4a was classified as negative, weakly positive, moderately positive, or strongly positive based on both staining intensity and the percentage of positively stained tumor cells in each section (Table 2). DNA Extraction Formalin-fixed, paraffin-embedded sections of cervical histopathologic material were dewaxed in xylene, and genomic DNA was extracted by the proteinase K and phenolchloroform treatment. The quality and quantity of the genomic DNA extracted was monitored by agarose gel electrophoresis with ethidium bromide staining. PCR Analysis The presence of intracellular HPV DNA was determined by PCR analysis with the consensus primer pairs (L1C1 and L1C2).11 The consensus primer pairs target the HPV L1 open reading frame and detect a broad range of genital HPVs. A 50 L volume containing 20 mmol/L Tris HCl buffer (pH 8.0), 50 mmol/L KCl, 0.2 mmol/L dNTP mix, 2 mmol/L MgCl2, 0.5 mol/L of each forward and reverse primer, and 0.25 U of Taq polymerase (TaKaRa, Otsu, Japan) was used for each reaction. After an initial denaturation at 95°C for 10 minutes, 43 cycles of reactions were performed, each consisting of denaturation at 95°C for 1.5 minutes, annealing at 48°C for 1.5 minutes, and extension at 70°C for 2 minutes. PK114/K, a variant HPV16 clone (kindly provided by Dr. Mattias Durst) was used as the positive control. Direct Sequencing of PCR Products A partial L1 sequence was amplified by the L1C1 primer. The amplified PCR products were purified, and automated sequencing was performed with an ABI Prism 3100 Genetic Analyzer (Applied Biosystems, Foster City, CA). The HPV type was determined based on approximately 200 base pairs of the HPV DNA sequence and a search of the National Center for Biotechnology Information (NCBI) database (GenBank sequences; http://www.ncbi.nlm.nih.gov/blast/Blast.cgi) with Sequencing Analysis 3.3 software (Perkin-Elmer, Norwalk, CT). In Situ Hybridization HPV in situ hybridization was carried out with the Gen-Point kit catalyzed signal amplification system (Dako Cytomation, Kyoto, Japan), following the manufacturer’s instruction manual. Briefly, formalin-fixed, paraffin-embedded tissue sections were deparaffinized, pretreated with Target retrieval solution at 95°C for 40 minutes, and then exposed to proteinase K diluted 1:6000 to 1:10,000 at room temperature for 10 minutes. The tissues were then soaked in 0.3% hydrogen peroxide in methanol at room temperature for 20 minutes, dehydrated through a graded alcohol series, and air-dried. Biotin-labeled HPV-wide spectrum, HPV 16, HPV 18, and HPV 31/33 probes were applied to the tissues. After denaturing at 95°C for 5 minutes and hybridization at 37°C overnight, the slides were washed in Stringent wash solution at 45°C or 55°C for 20 minutes and rinsed in Trisbuffered saline with Tween 20 (TBST) (S3306; Dako Cytoma-TABLE 1. Clincal and Histological Features of 10 Cases Case Age (yr) Stage pTNM Histology DI (mm) LVSI Treatment Follow-up (mo) Site of Recurrence 1 27 I b pT2bN1M0 SCC 16 Op C R DoD, 5 Bone 2 28 I b pT1bN0M0 SCCAC 10 Op C NED, 24 NA 3 31 I b pT2bN1M0 SCCAC 3 Op C SwR, 24 Lung 4 33 I b pT1bN1M0 SCC 4 Op C SwR, 28 Bone 5 38 I b pT1bN1M0 SCC 12 Op C DoD, 10 Lung 6 53 I b pT1bN0M0 SCC 12 Op R NED, 3 NA 7 49 II a ypT1bN0M0 SCCSqCC 12 NAC Op R DoD, 12 Multiple organ 8 48 II b pT2bN1M0 SCC to serosa Op R C DoD, 14 Pelvis 9 69 II b pT4N1M1 SCC to serosa Op C DoD, 1 Pleural effusion 10 51 IV pT4N1M1 SCC 15 Op C DoD, 8 LN Abbreviations: DI, depth of invasion; LVSI, lymph-vascular space involvement; SCC, small cell carcinoma; SqCC, squamous cell carcinoma; AC, adenocarcinoma; , positive; , negative; Op, operation; C, adjuvant chemotherapy; R, radiation therapy; NAC, neoadjuvant chemotherapy; DoD, Dead of disease; SwR, survival with recurrence; NED, no evidence of disease; NA, not applicable. TABLE 2. Evaluation of p16INK4a Immunostaining Intensity Positive cells (%) 0% 5% 50% 50%N N N N N W W W N W M M N W M S Abbreviations: N, negative; W, weakly positive; M, moderately positive; S, strongly positive. p16INK4a OVEREXPRESSION AND HPV INFECTION IN CERVICAL CARCINOMA (Masumoto et al) 779tion). The slides were then incubated with 1:100 diluted horseradish peroxidase-conjugated streptavidin at room temperature for 15 minutes. After the slides were rinsed with TBST 3 times, they were incubated with biotinyl tyramine at room temperature for 15 minutes; after the slide glasses were washed with TBST 3 times, they were incubated with horseradish peroxidase-conjugated streptavidin at room temperature for 15 minutes and again washed 3 times in TBST. For the signal development, 3,3'-diaminobenzidine tetrahydrochloride was used as the substrate, and a positive signal was detected as a brown color under a light microscope. The sections were weakly counterstained with hematoxylin. The signal type was assessed as described previously.12 A type 1 signal is defined as the presence of diffuse positivity throughout the nuclei; a type 2 signal, as a punctate, localized signal in the nuclei; and a type 3, signal as a combination of type 1 and type 2. RESULTS Patients The age of the patients ranged from 27 to 69 (42.7 12.8) years old. Six cases were clinical stage Ib, 1 case was clinical stage IIa, 2 cases were clinical stage IIb, and 1 case was clinical stage IV (Table 1). At surgery, complete resection of tumor was found to be impossible in 3 patients (cases 8, 9, and 10). In 5 patients (cases 1, 3, 4, 5, and 7), the disease recurred between 1 and 22 (9.1 8.4) months after complete resection. Lymph node metastases were present in 4 (67%) of the 6 stage Ib patients. Six of the 10 patients have died (60%), and only 2 patients were free of disease (20%) at the last follow-up examination. The mean survival period of the 6 patients who died was 8.2 4.1 months. Histological Findings In all 10 cases, the tumor cells contained scanty cytoplasm and round, hyperchromatic nuclei (Figs 1A and 2A). Mitoses were seen, and the nucleus to cytoplasm ratio was high. Vessel permeation was observed in 8 cases (80%). More than 1 of the neuroendocrine markers, such as NSE, chromogranin, and synaptophysin, were positive in 9 cases (Table 3). One case (case 7) had a squamous cell carcinoma component, and 2 other cases (cases 2 and 3) had an adenocarcinoma component adjacent to the small cell carcinoma. p16INK4a Analysis p16INK4a was overexpressed in all 10 cases (100%). The tumor cells were strongly positive in 9 cases, especially within the nuclei (Figs 1B and 2B). Only 1 case (case 10) was weakly positive. A component of the squamous cell carcinoma in case 7 and components of the adenocarcinoma in cases 2 and 3 were also positive for p16INK4a (results not shown). No positive staining was seen in the normal mucosa and stroma. HPV Analysis All 10 cases were HPV positive by PCR; 9 cases (90%) were positive for HPV 18 by PCR sequencing analysis, and 1 case (10%) was positive for HPV 16. Six cases were also positive by in situ hybridization; 5 HPV 18–positive cases yielded punctate signals and were type 2 (Fig 1C), and the single HPV 16–positive case yielded a combination of diffuse and punctate signals and was type 3 (Fig 2C). No type 1 signals were detected in any of the cases. Therefore, in all HPV 18 cases in FIGURE 1. Case 2, HPV 18 positive. (A) Hematoxylin and eosin–stained section. The tumor cells were small and hyperchromatic with scant cytoplasm. (B) Immunohistochemical staining for p16INK4a. Widespread positivity within tumor cells was seen. (C) In situ hybridization by the HPV 18 probe. Punctate signals (arrow) within tumor cell nuclei were detected. HUMAN PATHOLOGY Volume 34, No. 8 (August 2003) 780our study, HPV 18 was detected in the pure integrated form, and HPV16 was detected as a combination of the episomal and integrated form. The results of HPV typing by PCR and in situ hybridization were concordant in all of the cases that were HPV-positive by in situ hybridization. The results are summarized in Table 4. DISCUSSION Our study found that p16INK4a is overexpressed in small cell carcinoma of the cervix. p16INK4a was overexpressed in all 10 cases we examined, and 9 cases were strongly positive for p16INK4a. Previous studies have reported that p16INK4a is overexpressed in 97% of squamous cell carcinomas of the cervix6 and 48% to 50% of adenocarcinomas.7,8 However, many reports have showed that functional p16INK4a is deficient in a variety of human cancers, including melanoma, lung cancer, and leukemia.13,14 Therefore, the association between p16INK4a and carcinogenesis appears to be different in these cancers and cervical cancer. p16INK4a plays important roles in the regulation of the mammalian cell cycle. Cdk4/6 form complexes with cyclin D, which regulates the progression from the G1 to S phase of the cell cycle. Rb protein binds to the transcriptional factors, E2F, and suppresses their roles in transcription during the G1 phase. Rb protein is inactivated and dissociated from the Rb protein E2F after phosphorylation by cyclin D–Cdk4/6 in the G1 phase.15-17 The activity of cyclin D–Cdk4/6 is negatively regulated by CKIs, such as p16INK4a, which bind directly FIGURE 2. Case 7, HPV 16 positive. (A) Hematoxylin and eosin–stained section. The tumor cells were small and hyperchromatic with scant cytoplasm. (B) Immunohistochemical staining for p16INK4a. Widespread positivity within tumor cells was seen. (C) In situ hybridization by the HPV 16 probe. Diffuse intranuclear staining and punctate signals (arrow) within tumor cell nuclei were detected. TABLE 3. Histochemical and Immunohistochemical Marker for Small Cell Carcinoma Case Grimelius NSE Leu-7 CGR SYN SMT 1 ND ND ND ND ND 2 ND ND 3 ND 4 ND ND 5 ND ND ND 6 ND ND ND 7 ND ND ND ND ND 8 ND ND ND ND ND 9 ND ND 10 ND ND Abbreviations: NSE, neuron-specific enolase; CGR, chromogranin; SYN, synaptophysin; SMT, somatostatin; , positive; , focally positive; , negative; ND, not determined. TABLE 4. Summary of HPV Typing and p16INK4a Overexpression Case L1 PCR ISH Type Signal Type Dot Number IHC p16 1 18 18 P 0-2 2 18 18 P 1 3 18 18 P 1-2 4 18 ND – – 5 18 18 P 0-1 6 18 18 P 0-1 7 16 16 PD 1-2 8 18 ND – – 9 18 ND – – 10 18 ND – – Abbreviations: ISH, in situ hybridization; IHC, immunohistochemistry; ND, not detected; P, punctate signal; D, diffuse signal; , diffusely positive; , focally positive. p16INK4a OVEREXPRESSION AND HPV INFECTION IN CERVICAL CARCINOMA (Masumoto et al) 781to Cdk4/6 or the cyclin D–Cdk4/6 complexes. Therefore, lack of functional p16INK4a results in abnormal cell cycle progression, which is associated with carcinogenesis of cancers that show deficiency of p16INK4a. Although inactivation of functional p16INK4a has been demonstrated to be due to homozygous deletion, mutation with loss of heterozygosity, and inactivation of the promoter with methylation,18 overexpression of p16INK4a is reportedly common in cervical neoplasia.6-8 The overexpression of p16INK4a in cervical neoplasia has been reported to be associated with HPV infection,19,20 and an inverse correlation has been found between Rb protein function and overexpression of p16INK4a.4,21 Inactivation of Rb protein results in overexpression of p16INK4a,4 and transcriptional activity of p16INK4a appeared to be repressed by Rb protein.16 Khleif et al22 reported that HPV16 E7 protein is responsible for the increase in p16INK4a level. They found that p16INK4a mRNA was strongly induced by E2F1 and concluded that accumulation of E2F might induce the overexpression of p16INK4a. According to these hypotheses, dysfunction of Rb protein through HPV E7 protein should increase free E2F and result in both abnormal cell cycle progression and p16INK4a overexpression.4,5,17 We therefore hypothesized that overexpression of p16INK4a in small cell carcinoma is associated with HPV infection just as it is in other types of cervical cancer. To test this hypothesis, we performed HPV typing with PCR sequencing analysis and in situ hybridization. The results showed the presence of HPV in all 10 cases. Small cell carcinoma was reported to be associated with HPV 18 infection in previous studies,23-26 and we detected HPV 18 in 9 of the 10 cases in our study. Although HPV 16 has been the most common HPV type in squamous cell carcinoma of the cervix, HPV 16 was detected in only 1 of our 10 cases of small cell carcinoma. Interestingly, the HPV-16 positive case had a squamous cell carcinoma component adjacent to the small cell carcinoma region. It has also been reported that HPV 18 integration into genomic DNA is common in HPV 18–positive cervical cancer.27 In our study, in situ hybridization showed the pure integrated form in all 5 cases in which HPV 18 infection was present, and a mixed type consisting of both episomal and integrated forms in the case in which HPV 16 was present. Therefore, we suspected that inactivation of Rb protein by HPV E7 protein occurred after HPV infection (especially HPV 18 in an integrated form), and inactivation of Rb protein resulted in overexpression of p16INK4a. Although HPV 18 integration into the human genome has been considered an important event in the carcinogenesis of small cell carcinoma, exactly how neuroendrocrine differentiation occurs is unknown. Small cell carcinoma of the cervix has a poorer prognosis than squamous cell carcinoma. Six of the 10 patients with small cell carcionoma died, and the mean survival time of those 6 patients was only 8.2 4.1 months. Lymph node metastasis in stage Ib was detected in 67% of these patients. These data indicate that small cell carcinoma is a very aggressive form of cervical carcinoma. In conclusion, the results of this study showed that p16INK4a is overexpressed in small cell carcinoma of the cervix and that this tumor, which behaves very aggressively clinically, is associated with HPV infection, especially HPV 18 in an integrated form. The overexpression of p16INK4a and HPV 18 infection in small cell carcinoma cases suggests that inactivation of Rb protein by HPV 18 E7 protein may be associated with carcinogensis of small cell carcionoma in the same way that inactivation of Rb protein by HPV 16 E7 protein is associated with carcinogesis of squamous cell carcinoma. These events may contribute to the aggressive clinical behavior of small cell carcinoma. Acknowledgment. The authors thank Mr. Yoichi Tani and Ms. Nahoko Okitsu, Dako Cytomation Co, Ltd. for the cooperation and stimulating discussion. 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