Characterizing TP53 mutations in ovarian carcinomas with and without concurrent BRCA1 or BRCA2 mutations.

OBJECTIVES: Mutations in the TP53 tumor suppressor gene are common in ovarian carcinoma (OC) but their impact on outcomes is controversial. We sought to define the relationship of TP53 mutations to cancer outcomes and their interactions with co-occurrent BRCA1 or BRCA2 (BRCA) mutations, comparing three different TP53 mutation classification schemes. METHODS: We performed next generation sequencing on 393 cases of OC prospectively followed for survival. TP53 mutations were classified according to three schemes termed Structural, Functional, and Hotspot. Mutation distribution was compared between cases with and without BRCA mutations. In a subset of 281 cases of high grade serous carcinoma (HGSC), overall survival was compared using Kaplan-Meier curves, logrank testing, and multivariate Cox regression analysis, both stratified and adjusted for BRCA mutation status. Multivariate logistic regression was used to analyze the effects of TP53 mutation type on platinum resistance. RESULTS: TP53 mutations were identified in 76.8% of the total cohort (n = 302/393) and 87.9% of HGSC (n = 247/281). Cases with BRCA mutations demonstrated significantly higher TP53 mutation frequency overall (n = 84/91, 92.3% vs. n = 218/302, 72.2%, p < 0.001). TP53 mutations were not associated with overall survival, even when stratified by BRCA mutation. TP53 mutations were associated with platinum sensitivity, even after adjusting for BRCA mutation status (OR 0.41, p = 0.048). The choice of TP53 mutation classification scheme was not found to alter any significant outcome. CONCLUSIONS: BRCA mutations significantly co-occur with TP53 mutations. After adjusting for BRCA mutations, TP53 mutations are associated with platinum sensitivity, and this effect is not dependent on TP53 mutation type.

Pathologic findings and clinical outcomes in women undergoing risk-reducing surgery to prevent ovarian and fallopian tube carcinoma: A large prospective single institution experience.

OBJECTIVES: Risk-reducing salpingo-oophorectomy (RRSO) is recommended for women at increased risk of ovarian, fallopian tube (FT), and peritoneal carcinoma (collectively OC). We describe rates of occult neoplasia in the largest single-institution prospective cohort of women undergoing RRSO, including those with mutations in non-BRCA homologous repair (HRR) genes. METHODS: Participants undergoing RRSO enrolled in a prospective tissue bank between 1999 and 2017. Ovaries and FTs were serially sectioned in all cases. Participants had OC susceptibility gene mutations or a family history suggesting OC risk. Analyses were completed in Stata IC 15.1. RESULTS: Of 644 women, 194 (30.1%) had mutations in BRCA1, 177 (27.5%) BRCA2, 27 (4.2%) other HRR genes, and 15 (2.3%) Lynch Syndrome-associated genes. Seventeen (2.6%) had occult neoplasms at RRSO, 15/17 (88.2%) in the FT. Of BRCA1 carriers, 14/194 (7.2%) had occult neoplasia, 8/194 (4.1%) invasive. One PALB2 and two BRCA2 carriers had intraepithelial FT neoplasms. Occult neoplasm occurred more frequently in BRCA1/2 carriers ≥45 years of age (6.5% vs 2.2%, chi square, p = .04), and 211/371 (56.9%) BRCA1/2 carriers had surgery after guideline-recommended ages. Four in 8 (50%) invasive and 2/9 (22%) intraepithelial neoplasms had positive pelvic washings. None with intraepithelial neoplasms developed recurrence or peritoneal carcinoma. CONCLUSIONS: BRCA1 carriers have the highest risk of occult neoplasia at RRSO, and the frequency increased with age. Women with BRCA1/2 mutations often have RRSO beyond recommended ages. One PALB2 carrier had FT intraepithelial neoplasia, a novel finding. Serial sectioning is critical to identifying occult neoplasia and should be performed for all risk-reducing surgeries.

Inherited mutations in fallopian tube, ovarian and primary peritoneal carcinoma: Changes in diagnoses and mutational frequency over 20 years.

OBJECTIVES: Women with fallopian tube carcinoma (FTC) are reported to have a higher frequency of inherited BRCA mutations than those with ovarian carcinoma (OC) or primary peritoneal carcinoma (PPC). We hypothesized that routine serial sectioning of fallopian tubes would increase the proportion of cases designated as FTC and change the frequency of inherited mutations between carcinoma types. METHODS: Eight hundred and sixty-seven women diagnosed from 1998 to 2018 were enrolled at diagnosis into an institutional tissue bank. Germline DNA, available from 700 women with FTC (N = 124), OC (N = 511) and PPC (N = 65), was assessed using targeted capture and massively parallel sequencing for mutations in ovarian carcinoma susceptibility genes. Cases were divided between those prior to routine serial sectioning (1998-2008) and after (2009-2019), and the frequency of FTC and inherited mutations was assessed. RESULTS: The proportion of carcinomas attributed as FTC after 2009 was 28% (128/465), significantly higher than before 2009 [5% (21/402), p < .0001, OR 6.9, 95% CI 4.3-11.2], with subsequent decreases in OC and PPC. In the sequenced group, overall inherited mutation frequency in FTC (24/124, 19%), OC (106/511, 21%, p = .42), and PPC (16/65, 25%, p = .25) were similar. Germline mutation rates in FTC were lower after 2009,16/107 cases (15%), compared to 8/17 cases (47.1%) before 2009 (p = .005, OR 0.20, 95% CI 0.06-0.64). CONCLUSIONS: The prevalence of inherited mutations is similar in FTC compared to OC or PPC when using modern pathological assignment. Complete serial sectioning of fallopian tubes has significantly increased the diagnosis of FTC, and subsequently decreased the frequency of inherited mutations within this group.

An Alternate Diagnostic Algorithm for the Diagnosis of Intraepithelial Fallopian Tube Lesions.

Intraepithelial fallopian tube neoplasia is thought to be a precursor lesion to high-grade serous carcinoma of the Müllerian adnexae, particularly in women with BRCA1 or BRCA2 mutations. This association has led to recommendations to assess fallopian tubes for intraepithelial atypia. However, the diagnostic reproducibility of a diagnosis of intraepithelial neoplasia is unclear. In this study, 2 gynecologic pathologists independently evaluated sections of fallopian tubes from a sample of women (N=198, 623 slides) undergoing salpingectomy. A total of 101 (54%) women were undergoing risk-reducing salpingo-oophorectomy. Pathologists were blinded to patient histories and prior diagnoses. Pathologists rendered one of three diagnoses for each slide: "negative for fallopian tube intraepithelial neoplasia (FTIN)," "indeterminate for FTIN," or "definite for FTIN." Cases that were considered by histology definite for FTIN or suspicious for FTIN were stained with p53 and Ki67. Pathologists agreed on the diagnosis of "definite for FTIN" 61.5% of the time. There was no agreement on any cases for the diagnosis of "indeterminate for FTIN." Fifteen "indeterminate for FTIN" and 12 "definite for FTIN" cases were stained with p53 and Ki67. Two of the "indeterminate" cases (13%) had p53-positive foci. Five of the "definite" cases had p53-positive foci. In 3 of the other 8 "definite" cases, there was obvious carcinoma present, but the carcinoma did not stain with p53, suggesting a possible null phenotype. We propose that immunostains should only be used to aid in the diagnosis of FTIN in cases with indeterminate histology. The use of p53 immunohistochemistry in cases that were considered "definite for FTIN" by histology was minimally helpful, and in fact often served to further confuse the diagnosis.

Tumor infiltrating lymphocytes and homologous recombination deficiency are independently associated with improved survival in ovarian carcinoma.

OBJECTIVE: The presence of tumor infiltrating lymphocytes (TIL) and defects in homologous recombination (HR) are each important prognostic factors in ovarian carcinoma (OC). We characterized the association between HR deficiency (HRD) and the presence of TILs in a cohort of OC patients and the relative contribution to overall survival. METHODS: Patients with carcinoma of the ovary, fallopian tube, or peritoneum were prospectively enrolled. Malignant neoplasm and serum samples were collected. Immunohistochemistry for CD3+ T cells and CD68+ tumor associated macrophages (TAMs) was performed on specimens collected at primary surgery. Damaging germline and somatic mutations in genes in the HR-mediated repair (HRR) pathway were identified using BROCA sequencing. HRD was defined as a damaging mutation in one of 12 genes in the HRR pathway or promoter hypermethylation in BRCA1 or RAD51C. RESULTS: Ninety-eight of 250 patients included in the analysis had HRD OC (39.2%). HRD OC were enriched for CD3+ TILs and CD68+ TAMs. High CD3+ TIL was present in 65.3% of HRD OC compared to 43.4% of non-HRD OC (P = 0.001). High CD68+ TAM was present in 66.3% of HRD OC compared to 50.7% of non-HRD OC (P = 0.015). Patients with HRD OC and high CD3+ TILs had the longest median overall survival compared to non-HRD OC with low CD3+ TILs (70.9 vs. 35.8 months, adjusted HR 0.38, 95% CI (0.25-0.59)). CONCLUSIONS: Patients that have both CD3+ TILs and HRD OC are afforded the greatest improvement in overall survival. This finding may have therapeutic implications for OC patients treated with emerging immunotherapies.

Clinical characteristics and outcomes of patients with BRCA1 or RAD51C methylated versus mutated ovarian carcinoma.

OBJECTIVE: In ovarian carcinoma, mutations in homologous recombination DNA repair (HRR) genes, including BRCA1 and RAD51C, are associated with increased survival and specific clinical features. Promoter hypermethylation is another mechanism of reducing gene expression. We assessed whether BRCA1 and RAD51C promoter hypermethylation is associated with similar survival and clinical characteristics. METHODS: Promoter methylation of BRCA1 and RAD51C was evaluated using methylation-sensitive PCR in 332 primary ovarian carcinomas. Damaging germline and somatic mutations in 16 HRR genes were identified using BROCA sequencing. RESULTS: BRCA1 methylation was detected in 22 carcinomas (6.6%) and RAD51C methylation in 9 carcinomas (2.7%). These small numbers limited the power to detect differences in survival and platinum sensitivity. Mutations in one or more HRR genes were found in 95 carcinomas (29%). Methylation of BRCA1 or RAD51C was mutually exclusive with mutations in these genes (P=0.001). Patients whose carcinomas had BRCA1 methylation (57.7years±2.5) or BRCA1 mutations (54.1years±1.4) were younger than those without (63.3years±0.8; P=0.029, P<0.0001). BRCA1 methylation and germline BRCA1 mutation were associated with high grade serous (HGS) histology (P=0.045, P=0.001). BRCA1 mutations were associated with increased sensitivity to platinum chemotherapy while BRCA1 methylation was not (P=0.034, P=0.803). Unlike HRR mutations, methylation was not associated with improved overall survival compared to cases without methylation or mutation. CONCLUSIONS: Patients with BRCA1-methylated carcinomas share clinical characteristics with patients with BRCA1-mutated carcinomas including younger age and predominantly HGS histology. However, unlike mutation, RAD51C and BRCA1 methylation were not associated with improved survival or greater sensitivity to platinum chemotherapy.

Evaluation of Intraoperative Assessments in the Management of Endometrial Carcinoma: A Retrospective Quality Assurance Review.

Intraoperative assessment (IA) of uteri is often used to help determine whether to perform lymphadenectomy in patients with endometrial carcinoma. We sought to perform a quality assurance review of the practice of IA at our institution. In a 1-yr period, 107 hysterectomies had an IA performed. Grade of neoplasm in preoperative endometrial biopsy, neoplasm size, depth of myometrial invasion at IA, operative management, and final histologic features were recorded. Operative reports were reviewed to assess the surgeon's interpretation of the IA and the effect on surgical management. The sensitivity and specificity for IA of deep myometrial invasion (>50% myometrial thickness) compared with final histology was 76.9% and 91.1%. The positive predictive value was 71.4%, negative predictive value 93.2% and accuracy 88%. Neoplasm size was provided in 47% of cases. In 10% of patients lymphadenectomy was performed despite low-risk features. IA results were included in the operative report in 87% of cases. There were differences in 8.4% of cases between the IA diagnosis and the surgeon's operative report. IA of deep myometrial invasion is reliable at our institution. Several metrics for quality improvement have been identified as a result of this retrospective review. These include but are not limited to more reliable reporting of neoplasm size, documentation, and communication with gynecologic oncologists.

Neoplastic cellularity is associated with clinical and molecular features of high-grade serous ovarian carcinoma.

OBJECTIVE: Most molecular analyses of high-grade serous ovarian, peritoneal and fallopian tube carcinomas (HGSC) require ≥70% tumor (neoplastic) cell nuclei. We characterized the distribution of the percentage of neoplastic nuclei (PNN) in a large cohort of HGSC and correlated PNN with clinical outcomes to determine the fraction of cases outside this range and whether this cut-off introduces selection bias. METHODS: Subjects were prospectively enrolled and normal and neoplastic tissues were snap-frozen. All subjects had grade 2 to 3 HGSC. Subjects that received neoadjuvant chemotherapy were excluded. PNN was determined by estimating the fraction of neoplastic nuclei relative to non-neoplastic nuclei on a representative hematoxylin and eosin stained frozen section from the primary neoplasm. Germline BRCA mutation status was determined with Sanger or BROCA sequencing. RESULTS: PNN was <70% in 101 (33%) of 306 cases. PNN was significantly higher among subjects without optimal cytoreduction (P=0.018). 55 subjects had germline BRCA1/BRCA2 mutations. HGSC associated with BRCA2 but not BRCA1 mutations had significantly lower PNN compared to HGSC in non-carriers (54% vs. 70%, P=0.018). Overall survival was not significantly different between subjects with <70% or ≥70% PNN (median survival 51.8 vs. 46.6months, P=0.858). CONCLUSIONS: One-third of HGSC has PNN <70%. Higher PNN is associated with suboptimal cytoreduction, while lower PNN is associated with inherited BRCA2 mutations. Our findings suggest a nonrandom distribution of PNN that may reflect cancer biology. Further studies exploring the stromal microenvironment are needed. Molecular analyses of HGSC selected for high PNN exclude a significant fraction of patients.

Characterization of precursor lesions in the endometrium and fallopian tube epithelium of early-stage uterine serous carcinoma.

To determine if selected cases of uterine serous carcinoma (USC) arise from tubal rather than endometrial epithelium. Bilateral fallopian tubes from 38 women with pure USC were entirely submitted for histopathologic examination using the protocol Sectioning and Extensively Examining the FIMbria (SEE-FIM). Non-neoplastic endometrium was extensively sampled. Immunohistochemistry for p53 was performed on all paraffin blocks of fallopian tube and non-neoplastic endometrium. Endometrial intraepithelial carcinoma (EIC) was present in 22 cases (58%). Endometrial p53 foci were identified in 3 patients. There were 11 cases (29%) with fallopian tube involvement; 9 of 11 had tubal wall invasion or lymphatic involvement without serous tubal intraepithelial carcinoma (STIC) and were, therefore, classified as metastatic from the endometrium. STIC was identified in 3 patients (8%). There were 3 cases with tubal p53 foci in non-neoplastic epithelium. EIC was present in 58% of patients, further supporting EIC as a precursor lesion to USC. STIC was present in 8%, suggesting that the fallopian tube may in fact represent the primary lesion in a minority of patients with USC. This finding may account for the early multifocal disease distribution observed in these patients.

Mutations in 12 genes for inherited ovarian, fallopian tube, and peritoneal carcinoma identified by massively parallel sequencing.

Inherited loss-of-function mutations in BRCA1 and BRCA2 and other tumor suppressor genes predispose to ovarian carcinomas, but the overall burden of disease due to inherited mutations is not known. Using targeted capture and massively parallel genomic sequencing, we screened for germ-line mutations in 21 tumor suppressor genes in genomic DNA from women with primary ovarian, peritoneal, or fallopian tube carcinoma. Subjects were consecutively enrolled at diagnosis and not selected for age or family history. All classes of mutations, including point mutations and large genomic deletions and insertions, were detected. Of 360 subjects, 24% carried germ-line loss-of-function mutations: 18% in BRCA1 or BRCA2 and 6% in BARD1, BRIP1, CHEK2, MRE11A, MSH6, NBN, PALB2, RAD50, RAD51C, or TP53. Six of these genes were not previously implicated in inherited ovarian carcinoma. Primary carcinomas were generally characterized by genomic loss of normal alleles of the mutant genes. Of women with inherited mutations, >30% had no family history of breast or ovarian carcinoma, and >35% were 60 y or older at diagnosis. More patients with ovarian carcinoma carry cancer-predisposing mutations and in more genes than previously appreciated. Comprehensive genetic testing for inherited carcinoma is warranted for all women with ovarian, peritoneal, or fallopian tube carcinoma, regardless of age or family history. Clinical genetic testing is currently done gene by gene, with each test costing thousands of dollars. In contrast, massively parallel sequencing allows such testing for many genes simultaneously at low cost.

53BP1 expression in sporadic and inherited ovarian carcinoma: Relationship to genetic status and clinical outcomes.

OBJECTIVES: 53BP1, a critical mediator of the DNA damage response, functions by regulating the balance between homologous recombination (HR) and the more error-prone non-homologous endjoining (NHEJ). Deletion of 53BP1 in brca1 (but not brca2) null cells partially restores HR and reverses sensitivity to poly-ADP-ribose polymerase inhibitors (PARPi). We characterized 53BP1 and BRCA1 expression and their association with clinical outcomes in sporadic and inherited ovarian carcinomas. METHODS: We evaluated 53BP1 and BRCA1 protein expression using immunohistochemistry in 248 ovarian carcinomas and mRNA expression in 89 cases with quantitative reverse transcriptase PCR. All subjects were comprehensively characterized for germline mutations in BRCA1 and BRCA2. RESULTS: BRCA1-mutated (but not BRCA2-mutated) ovarian carcinomas had significantly higher 53BP1 protein expression than wildtype carcinomas. 53BP1 message levels were significantly associated with BRCA1 message levels in wildtype and BRCA1-mutated but not BRCA2-mutated carcinomas. In wildtype carcinomas, lower 53BP1 message predicted improved survival (p=0.02, median survival 74 vs. 41months, HR 0.49, 95% CI 0.27-0.88). Survival was not impacted by BRCA1 message level. 53BP1 expression was not associated with primary platinum resistance. In 54 paired primary and recurrent cases, 53BP1 protein expression was equally likely to decrease or increase, and there was no association between decreased 53BP1 at recurrence and the development of platinum resistance. CONCLUSIONS: BRCA1-mutated ovarian carcinomas have higher 53BP1 protein expression than wildtype or BRCA2-mutated carcinomas, in opposition to previous findings in breast carcinomas. Higher 53BP1 protein, which promotes NHEJ, could explain the frequent chromosomal aberrations that are characteristic of BRCA1-mutated ovarian carcinomas. In wildtype ovarian carcinomas, decreased 53BP1 message predicts improved survival, but message and protein expression were not associated.

Characteristics of women with ovarian carcinoma who have BRCA1 and BRCA2 mutations not identified by clinical testing.

OBJECTIVES: Few studies have comprehensively tested all ovarian cancer patients for BRCA1 and BRCA2 (BRCA1/2) mutations. We sought to determine if clinically identified mutation carriers differed in clinical characteristics and outcomes from mutation carriers not identified during routine clinical care. METHODS: We included women with ovarian, tubal or peritoneal carcinoma. BROCA, an assay using targeted capture and massively parallel sequencing was used to identify mutations in BRCA1/2 and 19 other tumor suppressor genes. We identified subjects with BRCA1/2 mutations using BROCA that had not previously received standard genetic testing (BROCA, n=37) and compared them to subjects with BRCA1/2 mutations identified during routine clinical care (known, n=70), and to those wildtype for 21 genes using BROCA (wildtype, n=291). RESULTS: BROCA mutation carriers were older than known carriers, median age of 58 (range 41-77), vs. 51 (range 33-76, p=0.003, Mann-Whitney). 58/70 (82.9%) of known carriers had a strong family history, compared with 15/37 (40.5%) of BROCA carriers, p<0.0001, (Fisher's Exact). Median overall survival was significantly worse for BROCA mutation carriers compared to known mutation carriers, (45 vs. 93months, p<0.0001, HR 3.47 (1.79-6.72), Log-rank test). The improved survival for BRCA1/2 mutation carriers (known and BROCA) compared with wildtype cases (69 vs. 44months, p=0.0001, HR 0.58 (0.43-0.77), Log-rank test) was driven by known mutation carriers. CONCLUSIONS: Older age, absence of a strong family history, and poor survival are all associated with decreased clinical identification of inherited BRCA1/2 mutations in women with ovarian cancer. Using age and family history to direct genetic testing will miss a significant percentage of mutation carriers. Testing should be initiated at the time of diagnosis to maximize identification of mutations and minimize survival bias.

Loss of function germline mutations in RAD51D in women with ovarian carcinoma.

OBJECTIVE: RAD51D, a gene in the Fanconi Anemia-BRCA homologous recombination pathway, has recently been shown to harbor germline mutations responsible for ovarian carcinoma in multiply affected families. We aimed to extend these results to ovarian carcinoma in the general population. METHODS: We sequenced RAD51D in germline DNA from 360 individuals with primary ovarian, peritoneal or fallopian tube carcinoma who were not selected for age of cancer onset or family history. We also sequenced RAD51D in 459 probands from 226 high risk breast cancer families who were wild type for 21 breast and ovarian cancer genes. RESULTS: Of 360 cases, three (0.8%) carried loss-of-function mutations in RAD51D. All three subjects had ovarian carcinoma; one was also diagnosed with a synchronous endometrial carcinoma. Only one of the three subjects had a family history of breast or ovarian cancer. Combined with previous data for this series, 23.9% of women with unselected ovarian, fallopian tube, or peritoneal carcinoma carried a germline loss-of-function mutation in any of 13 tumor suppressor genes. Among the 449 women and 10 men with familial breast cancer, none carried a loss of function mutation in RAD51D. CONCLUSIONS: These data support the previous observation that loss-of-function mutations in RAD51D predispose to ovarian carcinoma but not to breast carcinoma. We conclude that inherited ovarian cancer is highly heterogeneous genetically, and that approximately one in four ovarian carcinoma patients carry a germline mutation in a known tumor suppressor gene that confers high risk.

Biomarkers of progestin therapy resistance and endometrial hyperplasia progression.

OBJECTIVE: We sought to identify biomarkers associated with progestin therapy resistance and persistence/progression of endometrial hyperplasia. STUDY DESIGN: We performed a nested case-control study among women with complex (n = 73) and atypical (n = 41) hyperplasia treated with oral progestin, followed up 2-6 months for persistence/progression. We evaluated index endometrial protein expression for progesterone receptor isoform A, progesterone receptor isoform B (PRB), PTEN, Pax-2, and Bcl-2. Odds ratios and 95% confidence intervals (CIs) were estimated. RESULTS: Among women with atypical hyperplasia, high PRB expression was associated with 90% decreased risk of persistence/progression (95% CI, 0.01-0.8). High expression of progesterone receptor A and PRB suggested decreased risk of persistence/progression (odds ratio, 0.1; 95% CI, 0.02-1.0). These findings were not observed among women with complex hyperplasia. No associations were found with PTEN, Pax-2, and Bcl-2 protein expression. CONCLUSION: PRB expression shows promise as a biomarker of progestin response. Further research is warranted to understand how PRB expression may guide treatment decisions.

PAX2 loss by immunohistochemistry occurs early and often in endometrial hyperplasia.

Immunohistochemical markers to assist in the diagnosis and classification of hyperplastic endometrial epithelial proliferations would be of diagnostic use. To examine the possible use of PAX2 as a marker of hyperplastic endometrium, cases of normal endometrium, simple and complex hyperplasia without atypia, atypical hyperplasia, and International Federation of Gynecology and Obstetrics (FIGO) grade 1 endometrioid carcinomas were stained for PAX2. Two hundred and six endometrial samples were available for interpretation of PAX2 staining. The percentage of cases with complete PAX2 loss (0% of cells staining) increased with increasing severity of hyperplasia: 0% of normal proliferative and secretory endometrium (n=28), 17.4% of simple hyperplasia (n=23), 59.0% of complex hyperplasia (n=83), 74.1% of atypical hyperplasia (n=54), and 73.3% of FIGO grade 1 endometrioid cancers (n=15). Partial loss of PAX2 expression did occur in normal endometrium (17.9%) but in smaller proportions of tissue and was less frequent than in simple hyperplasia (47.8% with partial loss), complex hyperplasia (32.5%), atypical hyperplasia (22.2%), and FIGO grade 1 carcinomas (20.0%). Uniform PAX2 expression was rare in complex (8.4%) and atypical hyperplasia (3.7%) and carcinoma (6.7%). When evaluating loss of PAX2 in histologically normal endometrium adjacent to lesional endometrium in a given case, statistically significant differences in staining were observed for simple hyperplasia (P=0.011), complex hyperplasia (P<0.001), atypical hyperplasia (P<0.001), and FIGO grade 1 endometrioid cancer (P=0.003). In summary, PAX2 loss seems to occur early in the development of endometrial precancers and may prove useful in some settings as a diagnostic marker in determining normal endometrium from complex and atypical hyperplasia and low-grade carcinomas. However, it is not useful in distinguishing between these diagnostic categories.

MiR-221 and MiR-222 alterations in sporadic ovarian carcinoma: Relationship to CDKN1B, CDKNIC and overall survival.

MicroRNAs are often aberrantly expressed in human neoplasms and are postulated to play a role in neoplastic initiation and progression. miR-221 and miR-222 negatively regulate expression of CDKN1B (p27) and CDKN1C (p57), two cell cycle regulators expressed in ovarian surface epithelium and down-regulated in ovarian carcinomas. We characterized miR-221 and miR-222 expression in 49 sporadic high grade ovarian carcinomas and determined whether somatic mutation or epigenetic alterations explained the differences in expression of these miRNAs. We correlated these findings with protein expression of CDKN1B and CDKN1C as assessed by immunohistochemistry. Expression of miR-221 and miR-222 were closely correlated with each other (P = 0.0001). Interestingly, a lower ratio of miR-221 to miR-222 expression was significantly correlated with worse overall survival (P = 0.01) and remained a significant predictor of overall survival in multivariate analysis using the covariate adequacy of surgical cytoreduction (P = 0.03). Higher miR-222 and miR-221 expression were significantly associated with decreased CDKN1C expression (P = 0.009 and 0.01). In contrast, CDKN1B expression was not associated with miR-221 or miR-222 expression. Neither somatic mutations nor methylation of the studied region explained the alterations in miR-221 and miR-222 expression in most carcinomas.

Imiquimod treatment of vulvar melanoma in situ invading the urethra.

The primary treatment of both in situ and invasive vulvar melanoma is wide local excision of the primary neoplasm. However, this can be a surgical challenge for size, multifocal presentation with proximity to urethra or anus and tendency for local recurrence. The data on adjuvant therapy for vulvar MIS is very limited. A 69-year-old patient with melanoma of the vulva underwent a simple vulvectomy with positive margins in peri-clitoral area, followed by modified radical vulvectomy and bilateral inguinofemoral sentinel lymph node dissection with negative margins. She was later diagnosed with MIS of the vulva on different locations and had multiple wide local excisions over several years. One lesion was close to the urethra and a complete excision was difficult. Topical imiquimod × 16 weeks (5% cream) was given. The regimen was augmented from 3 to 5 times weekly. Complete resolution was found at 16 weeks and patient was disease free for 4 years. Recently however, a vaginal melanoma was detected. Imiquimod appeared to be beneficial in the treatment of melanoma in situ of the vulva/ vagina when surgical options were not feasible producing local control of disease with the remaining risk for local and distant metastasis. Metastasis can appear years later, therefore long-term follow-up of patients treated with topical imiquimod is needed.

Methylation and protein expression of DNA repair genes: association with chemotherapy exposure and survival in sporadic ovarian and peritoneal carcinomas.

BACKGROUND: DNA repair genes critically regulate the cellular response to chemotherapy and epigenetic regulation of these genes may be influenced by chemotherapy exposure. Restoration of BRCA1 and BRCA2 mediates resistance to platinum chemotherapy in recurrent BRCA1 and BRCA2 mutated hereditary ovarian carcinomas. We evaluated BRCA1, BRCA2, and MLH1 protein expression in 115 sporadic primary ovarian carcinomas, of which 31 had paired recurrent neoplasms collected after chemotherapy. Additionally, we assessed whether promoter methylation of BRCA1, MLH1 or FANCF influenced response to chemotherapy or explained alterations in protein expression after chemotherapy exposure. RESULTS: Of 115 primary sporadic ovarian carcinomas, 39 (34%) had low BRCA1 protein and 49 (42%) had low BRCA2 expression. BRCA1 and BRCA2 protein expression were highly concordant (p < 0.0001). MLH1 protein loss occurred in 28/115 (24%) primary neoplasms. BRCA1 protein loss in primary neoplasms was associated with better survival (p = 0.02 Log Rank test) and remained significant after accounting for either stage or age in a multivariate model (p = 0.04, Cox proportional hazards). In paired specimens, BRCA1 protein expression increased in 13/21 (62%) and BRCA2 protein expression increased in 15/21 (71%) of recurrent carcinomas with low or intermediate protein in the paired primary. In contrast MLH1 expression was rarely decreased in recurrent carcinomas (1/33, 3%). Similar frequencies of MLH1, BRCA1, and FANCF promoter methylation occurred in primary carcinomas without previous chemotherapy, after neoadjuvant chemotherapy, or in recurrent neoplasms. CONCLUSION: Low BRCA1 expression in primary sporadic ovarian carcinoma is associated with prolonged survival. Recurrent ovarian carcinomas commonly have increased BRCA1 and/or BRCA2 protein expression post chemotherapy exposure which could mediate resistance to platinum based therapies. However, alterations in expression of these proteins after chemotherapy are not commonly mediated by promoter methylation, and other regulatory mechanisms are likely to contribute to these alterations.

Patterns and duration of primary and recurrent treatment in ovarian cancer patients with germline BRCA mutations.

The objectives of this study were to describe the patterns and duration of primary and recurrent treatment in patients with ovarian cancer (OC) harboring germline BRCA1 and BRCA2 (BRCA) mutations. A retrospective review of BRCA mutation carriers with advanced, high-grade OC diagnosed between 2004 and 2014 with at least 3 years of follow-up (or until death) was undertaken. Descriptive statistics were calculated and a Swimmer's Plot used to depict disease course. Forty BRCA mutation carriers (26 BRCA1, 14 BRCA2) were identified. Mean age was 54 (range 32-77). All had cytoreductive surgery and received platinum chemotherapy. Median platinum-free interval was 11.9 months (IQR 3.6-21.9). Among 28 patients who recurred, median number of treatment lines was 4 (IQR 3-6), with a median of 2 (IQR 2-3) platinum lines. On average, patients who recurred spent 32% (IQR 20-43%) of their time after diagnosis receiving cytotoxic chemotherapy and 54% (IQR 42-67%) of the time on some cancer-directed therapy, including maintenance. Median overall survival was 79.1 months from diagnosis and 25.4 months after first recurrence. In conclusion, beyond first-line therapy, there was treatment and outcome heterogeneity for BRCA-mutated OC. After OC diagnosis, patients spent close to half their life on treatment.