Marina Frimer

HPV16 methyl-haplotypes determined by a novel next-generation sequencing method are associated with cervical precancer

We have developed and evaluated a next‐generation bisulfite sequencing (NGS) assay to distinguish HPV16 cervical precancer (CIN2–3; N =59) from HPV16‐positive transient infections (N = 40). Cervical DNA was isolated and treated with bisulfite and HPV16 methylation was quantified by (i) amplification with barcoded primers and massively parallel single molecule sequencing and (ii) site‐specific pyrosequencing. Assays were evaluated for agreement using intraclass correlation coefficients (ICC). Odds ratios (OR) for high methylation vs. low methylation were calculated. Single site pyrosequencing and NGS data were correlated (ICC = 0.61) and both indicated hypermethylation was associated with precancer (ORs of 2–37). Concordant NGS and pyrosequencing results yieled ORs that were stronger when compared with using either assay separately. Within the L1 region, the ORs for CIN2–3 were 14.3 and 22.4 using pyrosequencing and NGS assays, respectively; when both methods agreed the OR was 153. NGS assays provide methylation haplotypes, termed methyl‐haplotypes from single molecule reads: cases had increased methyl‐haplotypes with ≥ 1 methylated CpG site(s) per fragment compared with controls, particularly in L1 (p = 3.0 × 10−8). The maximum discrimination of cases from controls for a L1 methyl‐haplotype had an AUC of 0.89 corresponding to a sensitivity of 92.5% and a specificity of 73.1%. The strengthening of the OR when the two assays were concordant suggests the true association of CpG methylation with precancer is stronger than with either assay. As cervical cancer prevention moves to DNA testing methods, DNA based biomarkers, such as HPV methylation could serve as a reflex strategy to identify women at high risk for cervix cancer.

Micrometastasis of endometrial cancer to sentinel lymph nodes: Is it an artifact of uterine manipulation?

OBJECTIVE To determine if micrometastasis (MM) and isolated tumor cells (ITCs) in sentinel lymph nodes (SLNs) of endometrial cancer patients are artifactual and related to uterine manipulation at the time of diagnosis and surgery. METHODS We reviewed a prospectively maintained database of all patients with endometrial cancer undergoing SLN mapping between 2005 and 2009. MM was defined as a focus of metastatic cancer ranging from 0.2 to 2mm. ITCs were defined as metastasis measuring ≤ 0.2mm, including the presence of single, non-cohesive cytokeratin-positive tumor cells. We reviewed the effect of diagnostic procedure such as dilatation and curettage (D&C) versus biopsy and type of hysterectomy performed on the presence of MM and ITCs in SLNs. RESULTS In all, 175 patients had successful SLN mapping. Of these, 145 (83%) had negative nodes, 11 (6%) had positive nodes, and 19 (11%) met the criteria for MM and ITC. The uterine procedure used to diagnose endometrial cancer, type of hysterectomy, tumor grade, histology, positive pelvic washings, and type of uterine manipulator utilized, did not appear to be associated with MM/ITC. However, the presence of lymphovascular invasion (P < 0.001) and the depth of myometrial invasion (P = 0.01) were significantly higher in the MM/ITC group. CONCLUSIONS These data demonstrate that the presence of MM and ITCs in SLNs of endometrial cancer patients is not an artifact of uterine manipulation or instrumentation. Rather, it is a real pathologic finding likely associated with lymphovascular invasion and depth of myoinvasion.

Characterization of HPV DNA methylation of contiguous CpG sites by bisulfite treatment and massively parallel sequencing—the FRAGMENT approach

Invasive cervix cancer (ICC) is the third most common malignant tumor in women and human papillomavirus 16 (HPV16) causes more than 50% of ICC. DNA methylation is a covalent modification predominantly occurring at CpG dinucleotides and increased methylation across the HPV16 genome is strongly associated with ICC development. Next generation (Next Gen) sequencing has been proposed as a novel approach to determine DNA methylation. However, utilization of this method to survey CpG methylation in the HPV16 genome is not well described. Moreover, it provides additional information on methylation “haplotypes.” In the current study, we chose 12 random samples, amplified multiple segments in the HPV16 bisulfite treated genome with specific barcodes, inspected the methylation ratio at 31 CpG sites for all samples using Illumina sequencing, and compared the results with quantitative pyrosequencing. Most of the CpG sites were highly consistent between the two approaches (overall correlation, r = 0.92), thus verifying that Next Gen sequencing is an accurate and convenient method to survey HPV16 methylation and thus can be used in clinical samples for risk assessment. Moreover, the CpG methylation patterns (methylation haplotypes) in single molecules identified an excess of complete-and non-methylated molecules and a substantial amount of partial-methylated ones, thus indicating a complex dynamic for the mechanisms of HPV16 CpG methylation. In summary, the advantages of Next Gen sequencing compared to pyrosequencing for HPV genome methylation analyses include higher throughput, increased resolution, and improved efficiency of time and resources.

Mullerian adenosarcoma of the cervix: Report of two large tumors with sarcomatous overgrowth or heterologous elements

Highlights • Two cases of large cervical mullerian adenosarcoma with sarcomatous overgrowth or heterologous elements and contrasting survival outcomes are reported.• When the diagnosis of mullerian adenosarcoma is uncertain or suspected, review of pathology by a national expert may be considered.• Rhabdomyoblastic differentiation of mullerian adenosarcoma may be a more aggressive histologic type.

The Clinical Relevance of Rising CA-125 Levels Within the Normal Range in Patients With Uterine Papillary Serous Cancer

The utility of cancer antigen 125 (CA-125) levels as an adjunct method of monitoring patients with uterine papillary serous carcinoma (UPSC) or endometrial serous carcinoma after surgery and adjuvant treatment has been reported. Our goal was to determine the significance of rising CA-125 levels within the normal range in these patients in the posttreatment surveillance setting. All patients with UPSC who underwent surgical staging and had preoperative CA-125 measurement from 1999 to 2008 were included in this analysis. Information was extracted from records to assess the changes in CA-125 values with clinical and/or radiographic detection of recurrence. Of the 56 evaluable patients, 23 (41%) recurred. Of the 23 patients that recurred, 11 had serial CA-125 levels measured in remission. Elevated CA-125 levels at diagnosis were significantly associated with disease recurrence and advanced stage (P = .01, P = .001, respectively). The rise in CA-125 by 10 U/mL in the normal range and ≥15 U/mL were associated with disease recurrence (P < .001, P < .001, respectively). In multivariate analysis, only CA-125 level ≥15 U/mL was significantly associated with worse progression-free survival. In this small cohort of patients with recurrent UPSC after remission, surveillance of CA-125 levels may have a role in disease surveillance and management.

Pseudoepitheliomatous hyperplasia mimicking vulvar cancer in a patient with AIDS

Background. Pseudoepitheliomatous hyperplasia (PEH) clinically and histologically mimics squamous cell carcinoma (SCC), specifically in patients with HIV and AIDS. Case. A 51-year-old G3P2 with AIDS and history of vulvar cancer presented with large bilateral exophytic lesions on the vulva, grossly appearing neoplastic. Initial biopsies of the lesions were interpreted as vulvar SCC. After resolution with empiric treatment with acyclovir for possible herpes simplex virus type 2 outbreak, additional slides were reviewed, and cells with viral inclusions were identified, making the final diagnosis PEH in association with herpes simplex virus type 2 infection. Conclusions. Although PEH is infrequently encountered, PEH should be considered in the differential diagnosis of vulvar lesions. A multidisciplinary approach including the gynecologist, pathologist, and infectious disease specialists can optimize patient outcome.

Vesical clear cell adenocarcinoma arising from endometriosis: A mullerian tumor, indistinguishable from ovarian clear cell adenocarcinoma

Endometriosis is associated with increased rates of ovarian, particularly clear cell, adenocarcinomas. Malignant transformation of ovarian endometriosis is most common but rare cases have been reported in the bladder, abdominal wall, diaphragm, and rectum. We present the case of a 44-year-old female with vesical clear cell adenocarcinoma arising in a background of endometriosis in the absence of other pelvic endometriosis. The malignancy was diagnosed on transurethral resection of bladder tumor and managed with radical surgery. Histology and immunohistochemical findings were consistent mullerian origin and indistinguishable from similar tumors arising in the female genital tract. Extrapolating from the gynecologic literature, the recommendation was made for adjuvant chemotherapy. Further studies are needed to clarify the optimal treatment paradigm for ovarian and bladder clear cell adenocarcinomas.

Germline mutations of the DNA repair pathways in uterine serous carcinoma

OBJECTIVE Treatment options are limited for patients with uterine serous carcinoma (USC). Knowledge of USCs somatic mutation landscape is rapidly increasing, but its role in hereditary cancers remains unclear. We aim to evaluate the frequency and characteristics of germline mutations in genes commonly implicated in carcinogenesis, including those within homologous recombination (HR) and mismatch repair (MMR) pathways in patients with pure USC. METHODS By using targeted capture exome sequencing, 43 genes were analyzed in a cohort of 7 consecutive patients with paired tumor and non-tumor USC samples in our institutional tumor repository. Mutations predicted to have damaging effects on protein function are validated by Sanger Sequencing. RESULTS We found 21 germline mutations in 11 genes in our USC cohort. Five patients harbored 7 germline mutations (33.3%) within genes involved in the HR pathway, RAD51D being the most common. Four patients had 9 (42.8%) germline mutations in hereditary colon cancer genes, most commonly MLH. All patients (42.7%) who are platinum-sensitive had HR germline mutations (RAD50, NBN, ATM). Patients with HER2 overexpression (2/7, 28.6%) had germline HR mutations and were platinum-sensitive. Three patients in our cohort reported a personal history of breast cancer, one with HR germline mutation, and 2 in patients with germline mutations in HCC genes. In addition, 5 out of 7 patients had germline mutations in genes associated with growth factor signaling pathway. CONCLUSIONS A significant proportion of our cohort harbor germline mutations in DNA repair genes. This may be associated with the high rate of breast cancer in our patients and their family, and suggests a targeted cohort for genetic counseling. If validated in a larger cohort, our findings may allow clinicians to expand therapeutic options to include targeted therapies and inclusion of USC patient in preventative and genetic counseling.

Role of elevated cancer antigen 19-9 in women with mature cystic teratoma.

The objective of this study was to determine how often an elevated cancer antigen (CA) 19-9 (≥ 37 U/mL) was present during the preoperative evaluation of women with a mature cystic teratoma (MCT). This was a retrospective, consecutive case series (N = 139) of histologically proven MCT treated at Montefiore Medical Center from 1997 to 2008. Data were analyzed for patient and tumor characteristics, tumor markers (CA 19-9, CA 125, and carcinoembryonic antigen [CEA]), preoperative imaging, and procedure. CA 19-9 was elevated in 37.4% of patients. Elevated CA 19-9 was not significantly associated with age, race, CA 125 (≥35 U/mL), CEA (≥5 ng/mL), MCT size, or the presence of bilateral MCTs. Of the patients, 59% were ≥40 years old. Age <40 years was associated with cystectomy rather than oophorectomy (P < .001), regardless of CA 19-9 (P = .09). Elevated preoperative CA 19-9 in patients with MCT was associated with increased preoperative computed tomography (P = .04).

Human papillomavirus- associated cancers as acquired immunodeficiency syndrome defining illnesses

Abstract The Centers for Disease Control currently report cervical, vulvar, vaginal, anal and some head and neck cancers as human papillomavirus (HPV)-associated cancers. Only cervical cancer is listed amongst acquired immunodeficiency syndrome (AIDS) defining illnesses. All of these cancers may represent progression of the immunocompromised state with the inability to eradicate viral infection. This study reports the case of a 27-year old HIV positive female presenting with a persistent right vulvar exophytic lesion. High-risk HPV analysis and immunostaining for P16 were both positive. A biopsy of the lesion revealed invasive squamous cell carcinoma. The patient underwent neoadjuvant radiation and chemotherapy followed by a radical vulvectomy. During treatment, her CD4 T-lymphocyte count decreased to 120 advancing her condition from HIV to AIDS. This case suggests that all HPV-associated cancers should be included as AIDS defining illnesses.