Published papers

Human papillomavirus (HPV) is a sexually transmitted virus with an approximately 8-kilo base DNA genome, which establishes long-term persistent infection in anogenital tissues. High levels of genetic variations, including viral genotypes and intra-type variants, have been described for HPV genomes, together with geographical differences in the distribution of genotypes and variants. Here, by employing a maximum likelihood method, we performed phylogenetic analyses of the complete genome sequences of HPV16, HPV18 and HPV58 available from GenBank (n = 627, 146 and 157, respectively). We found several characteristic clusters that exclusively contain HPV genomes from Japan: two for HPV16 (sublineages A4 and A5), one for HPV18 (sublineage A1) and two for HPV58 (sublineages A1 and A2). Bayesian phylogenetic analyses of concatenated viral gene sequences showed that divergence of the most recent common ancestor of these Japan-specific clades was estimated to have occurred ~98,000 years before present (YBP) for HPV16 A4, ~39,000 YBP for HPV16 A5, ~38,000 YBP for HPV18 A1, ~26,000 for HPV58 A1 and ~25,000 YBP for HPV58 A2. This estimated timeframe for the divergence of the Japan-specific clades suggests that the introduction of these HPV variants into the Japanese archipelago dates back to at least ~25,000 YBP and provides a scenario of virus co-migration with ancestral Japanese populations from continental Asia during the Upper Paleolithic period.

Immunodeficient mice engrafted with human peripheral blood cells are promising tools for in vivo analysis of human patient individual immune responses. However, when human peripheral blood mononuclear cells (PBMCs) are transferred into NOG (NOD/Shi-scid, IL-2rg(null)) mice, severe graft versus host disease (GVHD) hinders long term detailed analysis. Administration of human PBMCs into newly developed murine MHC class I- and class II-deficient NOG (NOG-dKO; NOG- Iab, B2m-double-knockout) mice showed sufficient engraftment of human immune cells with little sign of GVHD. Immunization with influenza vaccine resulted in an increase in influenza-specific human IgG Ab, indicating induction of antigen-specific B cells in the NOG-dKO mice. Immunization with human dendritic cells pulsed with HLA-A2 restricted cytomegalovirus peptide induced specific cytotoxic T cells, indicating the induction of antigen-specific T cells in the NOG-dKO mice. Adoptive cell therapies (ACTs) using melanoma antigen recognized by T cells (MART-1)-specific TCR-transduced activated T cells showed strong tumor growth inhibition in NOG-dKO mice without any sign of GVHD accompanied by preferential expansion of the transferred MART-1-specific T cells. ACTs using cultured human melanoma infiltrating T cells also showed anti-tumor effects against autologous melanoma cells in NOG-dKO mice, in which changes in human cancer phenotypes by immune intervention, such as increased CD271 expression, could be evaluated. Therefore, NOG-dKO mice are useful tools for more detailed analysis of both the induction and effector phases of T-cell and B-cell responses for a longer period than regular NOG mice.Cellular & Molecular Immunology advance online publication, 20 November 2017; doi:10.1038/cmi.2017.106.

© 2020 Elsevier Inc. Objectives: To (i) identify correlations between selected immunogenic factors and clinicopathological characteristics, (ii) determine whether intratumoral abundance of various specific tumor-infiltrating lymphocytes (TILs) is a prognostic indicator in women with Stage II and III cervical cancer who undergo treatment with cisplatin-based concurrent chemoradiotherapy (CCRT), and (iii) investigate subtypes of FOXP3+ T cells in 15 fresh samples of cervical cancer. Methods: In this retrospective study, intratumoral lesions in colposcopic biopsies from 55 women with advanced cervical cancer who subsequently underwent CCRT at our institution were subjected to automatic immunological staining using the following six mouse monoclonal antibodies: anti-CD3, anti-CD4, anti-CD8, anti-CD20, anti-CD206, and anti-FOXP3. Associations between the findings on automatic scoring of the number of each type of TIL in each specimen and various clinicopathological characteristics were analyzed, as were associations between the abundance of various specific types of TIL and survival. Subtypes of FOXP3+ TILs in 15 additional fresh tumor samples were also investigated using flow cytometry. Results: Infiltration with CD8+ TILs was associated with pelvic lymph node metastasis. Abundant infiltration by CD3+, CD4+, CD8+, CD206+, and FOXP3+ TILs were statistically significant indicators of better progression-free and overall survival. Regarding subtypes of FOXP3+ TILs, non-Tregs (Fr-III) were found in all samples tested for this. Conclusions: The abundance of various specific intratumoral TILs may be prognostic indicators in patients with advanced cervical cancer undergoing CCRT.

This study examined the association between peritoneal cytology and survival in early-stage cervical cancer. This is a nationwide multicenter retrospective study, examining consecutive women with clinical stage IB1-IIB cervical cancer who underwent radical hysterectomy with available peritoneal cytology results from 2004–2008. Propensity score inverse probability of treatment weighting was used to assess the impact of malignant peritoneal cytology on survival. Among 1409 analyzed cases, 88 (6.2%) had malignant peritoneal cytology. On weighted models, malignant peritoneal cytology was associated with decreased disease-free survival (hazard ratio (HR) 1.78, 95% confidence interval (CI) 1.36–2.32) and overall survival (OS, HR 1.93, 95% CI 1.44–2.59). On sensitivity analyses, malignant peritoneal cytology was associated with decreased OS in adenocarcinoma/adenosquamous carcinoma, high-risk early-stage disease and those who received concurrent chemo-radiotherapy. However, among women who received postoperative systemic chemotherapy, malignant peritoneal cytology was not associated with OS (HR 1.21, 95% CI 0.72–2.04). A systematic review, including our results, showed that malignant peritoneal cytology was associated with decreased OS (HR 4.03, 95% CI 1.81–8.99) and increased recurrence in squamous carcinoma (odds ratio 1.89, 95% CI 1.05–3.39) and adenocarcinoma (odds ratio 4.30, 95% CI 2.30–8.02). In conclusion, the presence of malignant cells in peritoneal cytology is associated with decreased survival in early-stage cervical cancer. The possible benefit of systemic chemotherapy in this subgroup merits further investigation.

Objective: To evaluate timing and a prediction model for pregnancy in early-stage cervical cancer patients who underwent fertility-sparing trachelectomy. Design: Retrospective cohort. Setting: Academic multicenter. Patient(s): Women ages <45 years with clinical stage I–II cervical cancer were enrolled between 2009 and 2013 (n = 393). Intervention(s): Planned fertility-sparing trachelectomy. Main Outcome Measure(s): Cumulative incidences and clinicopathological characteristics of those who developed subsequent pregnancy were examined. Result(s): There were 77 (21.6%) women who had subsequent pregnancies after fertility-sparing trachelectomy with 1-, 2-, and 5-year cumulative pregnancy rates of 2.8%, 6.2%, and 17.4%, respectively. The median time to develop subsequent pregnancy was 3.2 years. In a competing risk analysis, women had a higher risk of recurrent cancer than conception during the first 11 months postsurgery. On multivariable analysis, younger age, being married, and postoperative reproductive treatment were independently associated with an increased chance of developing a subsequent pregnancy. The subsequent pregnancy index (SPI) score to predict the likelihood of having pregnancy was proposed, and it was calculated based on age, marital status, and reproductive treatment (2, 2, and 4 points, respectively). Women with a higher SPI score had significantly higher subsequent pregnancy rates (5-year pregnancy rate; the score was 3 in 4.7% of cases; 4 to 5 in 11.3%; 6 to 7 in 27.4%; and 8 in 50.8%), but they had similar recurrence rates (5.0%). Conclusion(s): The SPI score proposed in our study is useful in predicting subsequent pregnancy in women with early-stage cervical cancer undergoing fertility-sparing trachelectomy.

Objective: To assess the efficacy and safety of dose-dense weekly paclitaxel plus carboplatin (ddTC) with or without bevacizumab compared to conventional, tri-weekly paclitaxel plus carboplatin (cTC) with or without bevacizumab, in metastatic or recurrent cervical carcinoma not amenable to curative local therapy. Methods: Patients were randomly assigned to either the cTC or ddTC arm. The cTC regimen was paclitaxel 175 mg/m2 and carboplatin at an area under the curve (AUC) of 5 on day 1. The ddTC regimen was paclitaxel 80 mg/m2 on day 1, 8, 15 and carboplatin at AUC of 5 on day 1. Both cTC and ddTC treatments were repeated every 3 weeks for up to 9 cycles. After bevacizumab was approved in Japan, patients in both arms received bevacizumab 15 mg/kg if not contraindicated. The primary endpoint of phase II part was response rate (RR). If the RR of ddTC+bevacizumab was found to be at least 5% better than to cTC + bevacizumab, the study would proceed to phase III part, which had overall survival as its primary endpoint. Clinical trial information: jRCTs031180007. Results: In total, 122 patients were randomly assigned to either the cTC arm (cTC + bevacizumab: 32; cTC:29) or the ddTC arm (ddTC+bevacizumab: 30; ddTC:31). The RR for patients on cTC + bevacizumab was 67.9%, and for patients on ddTC+bevacizumab 60.7%, cTC: 55.2%, and ddTC: 50.0%. Conclusions: The study did not meet the primary endpoint of phase II portion. Dose-dense, weekly paclitaxel plus carboplatin is not promising for metastatic or recurrent cervical carcinoma.

To evaluate the feasibility of adoptive cell therapy (ACT) using ex vivo-expanded tumor-infiltrating lymphocytes (TILs) in Japanese patients with melanoma who failed immune-checkpoint inhibitor therapy, an open-label, single-arm, pilot study was conducted. We investigated the immunological and genetic factors of the pretreatment tumor and expanded TILs that may be associated with the clinical response. The treatment protocol comprised preparation of TIL culture, lympho-depleting non-myeloablative preconditioning with cyclophosphamide and fludarabine, TIL infusion, and intravenous administration of low-dose IL-2. Three patients of clinical subtypes mucosal, superficial spreading, and acral melanoma underwent TIL-ACT. Most severe adverse events, including fever and leukopenia, were manageable with the supportive regimen specified in the protocol, suggesting that the TIL-ACT regimen is suitable for Japanese patients with melanoma. One patient showed a short-term partial response, one relatively long-stable disease, and one experienced disease progression. Whole-exome and transcriptional sequencing of isolated tumor cells and immunohistochemical analyses before TIL-ACT revealed various immunostimulatory factors, including a high tumor mutation burden and immune cell-recruiting chemokines, as well as various immunosuppressive factors including TGF-β, VEGF, Wnt/β-catenin, and MAPK signaling and epithelial-to-mesenchymal transition, which might influence the efficacy of TIL-ACT. Our results imply mechanisms for the antitumor effect of and resistance to TIL-ACT. Further studies of immune-resistant mechanisms of TIL-ACT are warranted. This study is registered with the UMIN Clinical Trial Registry (UMIN 000011431).

Objective: Liquid-based cytology (LBC) and conventional cytology (CS) are routine diagnostic techniques in cervical cytology, but few studies have compared their diagnostic performances with each other and with histologic diagnosis. This study aimed to compare the diagnostic performances of these techniques in subjects with abnormal cervical cytology of atypical cells of undetermined significance (ASC-US) or worse. Methods: A total of 312 patients diagnosed with ASC-US or worse were enrolled in this prospective study in Japan from 2013 to 2014. LBC and CS samples were prepared by a split-sampling technique and evaluated blindly. The results were classified using the Bethesda System 2001. Colposcopy and biopsy were conducted simultaneously or within 4 weeks of cytology-specimen collection in all cases. Diagnostic performance was calculated in terms of sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) for detection of CIN2 or worse, with a cut-off ASC-US or worse. Results: There was one unsatisfactory CS sample and the remaining 311 cases were evaluated. The sensitivities of LBC and CS were 100.0% and 98.8%, specificities were 17.2% and 23.8%, PPVs were 56.1% and 57.9% and NPVs were 100.0% and 94.7%, respectively. LBC had slightly higher sensitivity and NPV for detection of CIN2, but there was no significant difference between the two methods. Conclusions: There was no significant difference in the diagnostic performances of LBC and CS in patients with ASC-US or worse. LBC may therefore be an alternative approach to CS for cervical cancer screening.

This study seeks to identify risk factors associated with ovarian metastasis and to characterize a population with minimum risk of ovarian metastasis in young women with stage IB-IIB cervical cancer. This was a nation-wide multicenter retrospective study in Japan examining consecutive cases of surgically-treated women with clinical stage IB-IIB cervical cancer who had oophorectomy at radical hysterectomy (n = 5,697). Multivariable analysis was performed to identify independent risk factors for ovarian metastasis. Ovarian metastasis was seen in 70 (1.2%, 95% confidence interval 0.9-1.5) cases. In the entire cohort, adenocarcinoma, lympho-vascular space invasion, uterine corpus tumor invasion, and pelvic/para-aortic nodal metastases remained independent risk factors for ovarian metastasis (all, adjusted-p < 0.05). In a sensitivity analysis of 3,165 women aged <50 years (ovarian metastasis, 1.0%), adenocarcinoma, parametrial tumor involvement, uterine corpus tumor involvement, and pelvic/para-aortic nodal metastases remained independent risk factors for ovarian metastasis (all, adjusted-P < 0.05). In the absence of these five risk factors (representing 46.1% of women aged <50 years), the incidence of ovarian metastasis was 0.14%. With the presence of adenocarcinoma alone (representing 18.9% of women aged <50 years), the incidence of ovarian metastasis was 0.17% and was not associated with increased risk of ovarian metastasis compared to the subgroup without any risk factors (p = 0.87). In conclusion, nearly two thirds of women aged <50 years with clinical stage IB-IIB cervical cancer had no risk factor for ovarian metastasis or had adenocarcinoma alone: these subgroups had ovarian metastasis rates of around 0.1% and may be a candidate population for ovarian conservation at surgical treatment.

PURPOSE: We aimed to determine appropriate treatment guidelines for patients with stages I-II high-grade neuroendocrine carcinomas (HGNEC) of the uterine cervix in a multicenter retrospective study. PATIENTS AND METHODS: We reviewed the clinicopathological features and prognoses of 93 patients with HGNEC of International Federation of Gynecology and Obstetrics (FIGO) stages I and II. All patients were diagnosed with HGNEC by central pathological review. RESULTS: The median overall survival (OS) and disease-free survival (DFS) were 111.3months and 47.4months, respectively. Eighty-eight patients underwent radical surgery, and five had definitive radiotherapy. The hazard ratio (HR) for death after definitive radiotherapy to death after radical surgery was 4.74 (95% confidence interval [CI], 1.01-15.90). Of the surgery group, 18 received neoadjuvant chemotherapy. Pathological prognostic factors and optimal adjuvant therapies were evaluated for the 70 patients. Forty-one patients received adjuvant chemotherapy with etoposide-platinum (EP) or irinotecan-platinum (CPT-P). Multivariate analyses identified the invasion of lymphovascular spaces as a significant prognostic factor for both OS and DFS. Pelvic lymph node metastasis was also a prognostic factor for DFS. Adjuvant chemotherapy with an EP or CPT-P regimen appeared to improve DFS (HR=0.27, 95% CI, 0.10-0.69). A trend toward improved OS was also observed, but was not statistically significant (HR=0.39, 95% CI, 0.15-1.01). CONCLUSION: Radical surgery followed by adjuvant chemotherapy with an EP or CPT-P regimen was optimal treatment for stages I and II HGNEC of the uterine cervix.

© 2019 Elsevier B.V. Objectives: Radical hysterectomy is performed for invasive cervical cancer. In this surgery, separation of the anterior leaf and posterior leaves of the vesicouterine ligament (VUL) is important. We studied the local anatomy of the anterior leaf of the VUL, especially the branches of the umbilical artery from the view point of surgery and cadaver dissection. Study Design: We observed the cervicovesical blood vessels and the connective tissue layer continued from umbilical artery and searched for the origin of the cervicovesical blood vessels in radical hysterectomy. We also dissected a formalin-fixed female cadaver, and observed the same points. Results: After separation of the connective tissue of urinary bladder from the cervical fascia, we could discern the outline of the distal ureter near the ureterovesical junction. We separate the connective tissue of the so-called anterior leaf of the VUL enwrapping the ureter gently, and then the ureter with the connective tissue is completely rolled out laterally. We identified a cervicovesical vessel crossing over the ureter. We looked for the central side of the cervicovesical vessel and found that cervicovesical vessel was a branch of the superior vesical artery. And, during cadaver dissection, we found that the connective tissue and the branches of the superior vesical artery were similarly observed. Conclusions: We found the precise anatomy of the connective tissue layer from umbilical artery to urinary bladder and the superior vesical artery. Our procedure based on the precise anatomy obtained in this study is reasonable anatomically as a method for separation of the vesicouterine ligament during radical hysterectomy.

© 2019 Elsevier Inc. Objective: To determine the involvement of homeobox D9 (HOXD9) in the survival, proliferation, and metastasis of cervical cancer cells through regulating the expression of human papillomavirus (HPV) 16 E6/E7 genes using the P97 promoter. Methods: One hundred cases of cervical cancer (CC), CC cell lines SKG-I, SKG-II, SKG-IIIa, SKG-IIIb, HeLa, and SiHa, and a human tumor xenograft mouse model were used to examine the roles of HOXD9 in CC. Knockdown experiments employed RNA interference of HOXD9. qPCR, functional assays, western blotting, DNA microarray, and luciferase and ChIP assays were applied for assessments. Results: All CC cell lines expressed HOXD9 mRNA and protein. In uterine CC, HOXD9 gene expression was significantly higher than in normal cervical tissues. A positive correlation of lymphovascular space invasion and lymph node metastasis with high levels of HOXD9 expression was found in patient samples. HOXD9-knockdown cells in the mouse xenograft model only formed small or no tumors. Knockdown of HOXD9 markedly reduced CC cell proliferation, migration and invasion, induced apoptosis, increased P53 protein expression, and suppressed HPV E6/E7 expression by directly binding to the P97 promoter of HPV16 E6/E7 genes. A positive correlation between HOXD9 and HPV16 E6 expression was found in CC patients. Conclusions: HOXD9 promotes HPV16 E6 and E7 expression by direct binding to the P97 promoter, which enhances proliferation, migration, and metastasis of CCr cells. Our results suggest that HOXD9 could be a prognostic biomarker and potential therapeutic target in CC.

© 2019 by the authors. Licensee MDPI, Basel, Switzerland. Recent large-scale genomics studies of human papillomaviruses (HPVs) have shown a high level of genomic variability of HPV16, the most prevalent genotype in HPV-associated malignancies, and provided new insights into the biological and clinical relevance of its genetic variations in cervical cancer development. Here, we performed deep sequencing analyses of the viral genome to explore genetic variations of HPV16 that are prevalent in Japan. A total of 100 complete genome sequences of HPV16 were determined from cervical specimens collected from Japanese women with cervical intraepithelial neoplasia and invasive cervical cancer, or without cervical malignancies. Phylogenetic analyses revealed the variant distribution in the Japanese HPV16 isolates; overall, lineage A was the most prevalent (94.0%), in which sublineage A4 was dominant (52.0%), followed by sublineage A1 (21.0%). The relative risk of sublineage A4 for cervical cancer development was significantly higher compared to sublineages A1/A2/A3 (odds ratio = 6.72, 95% confidence interval = 1.78–28.9). Interestingly, a novel cluster of variants that branched from A1/A2/A3 was observed for the Japanese HPV16 isolates, indicating that unique HPV16 variants are prevalent among Japanese women.

© 2020 Elsevier B.V. Among the oncogenic genotypes of human papillomavirus (HPV), HPV18 is the second most common type detected in cervical cancer worldwide and is primarily involved in the generation of cervical adenocarcinoma. Although HPV intra-type variants confer different risks of cervical carcinogenesis, there is little information on the genetic diversity of HPV18 compared to the most prevalent type, HPV16. In this study, we investigated the genetic variation of HPV18 in cervical specimens obtained from Japanese women with normal cervices or cervical cancers and precancers. Of the 101 HPV18-positive samples analyzed, viral whole genome amplification followed by next-generation sequencing led to the determination of viral complete genome sequences of 18 samples. Phylogenetic analysis of these HPV18 whole genome sequences identified a distinct variant cluster consisting of only Japanese samples (n = 7) belonging to sublineage A1. Viral genome sequences were also analyzed for the E6/E7 (n = 66) and E2 (n = 27) genes by Sanger sequencing. Phylogenetic analyses of these regions showed that the variant distribution among Japanese women was strongly biased toward sublineage A1 (72 of 87; 82.8%). No significant differences were observed in the prevalence of specific sublineages between cervical cancer/precancer cases and controls, and between squamous cell carcinoma and adenocarcinoma cases. These data contribute to our understanding of the genetic diversity of HPV18 in Japanese women.

© 2019 S. Karger AG, Basel. All rights reserved. Objective: The exfoliative cell analyzer, LC-1000 (Sysmex Corporation, Japan), is a medical device that presents the cell proliferation index and 23 research parameters as indicators of cellular proliferative potential. The objective was to evaluate the clinical usability of qualitative assessment by LC-1000 compared with cytology, the human papillomavirus (HPV) test, and histology as gold standard. Study Design: Women that visited 3 sites between July 2015 and March 2017 were registered. The primary endpoint in this study was the comparison between LC-1000 measurement and HPV test for sensitivity and specificity for cervical intraepithelial neoplasia 2+ (CIN2+). A tree model algorithm was newly constructed by a statistical method and its relationship with histological results was evaluated. Results: The sensitivity and specificity of LC-1000 were 78.3 and 74.1%, while those of the HPV test were 94.7 and 85.4%, respectively. A tree model comprising five categories was constructed. The proportion of advanced lesions was higher with the change in the rank classification results from 1 to 5. The positive predictive values of CIN2+ in the categories 4 and 5 were high. Despite the small number of subjects, cancer was undetected in categories 1 and 2. In addition, the comparison with follow-up results in 19 women assessed as CIN1 showed that the rate of progression in the categories 3-5 was 50% (7/14); progression in the categories 1 and 2 was 0% (0/5). Conclusions: LC-1000 may be useful for cervical cancer screening as an index to qualitatively evaluate CIN and cancer based on the changes in characteristics of cells.

© 2019 Elsevier Inc. Objective: To propose an ideal patient candidate with early-stage cervical cancer for undergoing fertility-sparing trachelectomy. Methods: This nationwide, multicenter, retrospective study was conducted by the Japan Society of Obstetrics and Gynecology involving women aged <45 years with clinical stage I-II cervical cancer who had planned fertility-sparing trachelectomy and pelvic lymphadenectomy between 2009 and 2013 (n = 393). Ideal candidates were defined to have a tumor size of ≤2 cm, no lymph node metastasis, no deep stromal invasion, and no high-risk histology (n = 284, 69.6%). Less-ideal candidates were defined to have any one of these four characteristics (n = 109, 30.4%). Propensity score inverse probability of treatment weighting was used to assess survival outcomes. Results: Less-ideal candidates were more likely to undergo hysterectomy conversion (22.9% versus 3.2%), receive postoperative radiotherapy (11.9% versus 0.4%), or chemotherapy (32.1% versus 3.2%) compared with ideal candidates (all, P < 0.05). The weighted model revealed that among those who underwent trachelectomy (ideal candidates, n = 275 and less-ideal candidates, n = 84), less-ideal candidates had significantly decreased disease-free survival (5-year rates: 85.5% versus 95.5%; HR 3.93, 95% CI 1.99–7.74; P < 0.001) and cause-specific survival (92.5% versus 98.6%; HR 5.47, 95% CI 1.68–17.8, P = 0.001) compared with ideal candidates. Similarly, less-ideal candidates were significantly associated with decreased disease-free survival compared with ideal candidates among those who were young age, had small tumors or squamous histology, and underwent surgery alone (all, P < 0.05). Conclusion: Less-ideal candidates had approximately four-fold higher recurrence risk and cancer mortality compared with ideal candidates. Ideal candidates for fertility-sparing trachelectomy for early-stage cervical cancer proposed in our study may be useful as the future framework for developing guidelines for fertility-sparing trachelectomy in Japan.

© 2020 The Author(s). Background: Human papillomavirus (HPV) infection is a primary cause of cervical cancer. Although epidemiologic study revealed that carcinogenic risk differs according to HPV genotypes, the expression patterns of HPV-derived transcripts and their dependence on HPV genotypes have not yet been fully elucidated. Methods: In this study, 382 patients with abnormal cervical cytology were enrolled to assess the associations between HPV-derived transcripts and cervical intraepithelial neoplasia (CIN) grades and/or HPV genotypes. Specifically, four HPV-derived transcripts, namely, oncogenes E6 and E6*, E1^E4, and viral capsid protein L1 in four major HPV genotypes-HPV 16, 18, 52, and 58-were investigated. Results: The detection rate of E6/E6*increased with CIN progression, whereas there was no significant change in the detection rate of E1^E4 or L1 among CIN grades. In addition, we found that L1 gene expression was HPV type-dependent. Almost all HPV 52-positive specimens, approximately 50% of HPV 58-positive specimens, around 33% of HPV 16-positive specimens, and only one HPV18-positive specimen expressed L1. Conclusions: We demonstrated that HPV-derived transcripts are HPV genotype-dependent. Especially, expression patterns of L1 gene expression might reflect HPV genotype-dependent patterns of carcinogenesis.

© Copyright © 2020 Hirose, Yamaguchi-Naka, Onuki, Tenjimbayashi, Tasaka, Satoh, Tanaka, Iwata, Sekizawa, Matsumoto and Kukimoto. Human papillomavirus type 16 (HPV16) is the most common HPV genotype found in invasive cervical cancer (ICC). Recent comprehensive genomics studies of HPV16 have revealed that a large number of minor nucleotide variations in the viral genome are present in each infected woman; however, it remains unclear whether such within-host variations of HPV16 are linked to cervical carcinogenesis. Here, by employing next-generation sequencing approaches, we explored the mutational profiles of the HPV16 genome within individual clinical specimens from ICC (n = 31) and normal cervix (n = 21) in greater detail. A total of 367 minor nucleotide variations (167 from ICC and 200 from the normal cervix) were detected throughout the viral genome in both groups, while nucleotide variations at high frequencies (>10% abundance in relative read counts in a single sample) were more prevalent in ICC (10 in ICC versus 1 in normal). Among the high-level variations found in ICC, six were located in the E1/E2 genes, and all of them were non-synonymous substitutions (Q142K, M207I, and L262V for E1; D153Y, R302T, and T357A for E2). In vitro functional analyses of these E1/E2 variants revealed that E1/M207I, E2/D153Y, and E2/R302T had reduced abilities to support viral replication, and that E2/D153Y and E2/R302T failed to suppress the viral early promoter. These results imply that some within-host variations of E1/E2 present at high levels in ICC may be positively selected for and contribute to cervical cancer development through dysfunction or de-stabilization of viral replication/transcription proteins.