Homologous Recombination Deficiency (HRD) Scoring, by Means of Two Different Shallow Whole-Genome Sequencing Pipelines (sWGS), in Ovarian Cancer Patients: A Comparison with Myriad MyChoice Assay.

High-grade serous ovarian cancer (HGSOC) patients carrying the BRCA1/2 mutation or deficient in the homologous recombination repair system (HRD) generally benefit from treatment with PARP inhibitors. Some international recommendations suggest that BRCA1/2 genetic testing should be offered for all newly diagnosed epithelial ovarian cancer, along with HRD assessment. Academic tests (ATs) are continuously under development, in order to break down the barriers patients encounter in accessing HRD testing. Two different methods for shallow whole-genome sequencing (sWGS) were compared to the reference assay, Myriad. All these three assays were performed on 20 retrospective HGSOC samples. Moreover, HRD results were correlated with the progression-free survival rate (PFS). Both sWGS chemistries showed good correlation with each other and a complete agreement, even when compared to the Myriad score. Our academic HRD assay categorized patients as HRD-Deficient, HRM-Mild and HRN-Negative. These three groups were matched with PFS, providing interesting findings in terms of HRD scoring and months of survival. Both our sWGS assays and the Myriad test correlated with the patient's response to treatments. Finally, our AT confirms its capability of determining HRD status, with the advantage of being faster, cheaper, and easier to carry out. Our results showed a prognostic value for the HRD score.

Expression of miR-132 and miR-212 in prostate cancer and metastatic lymph node: Case report and revision of the literature.

MicroRNAs (miRNAs) are a class of small, non-coding RNAs that act as key regulators in various physiological and pathological processes as prostate cancer (PCa). In this study we describe molecular evaluation of 132 and 212 miRNAs expression, by Real-time reverse-transcription PCR (qRT-PCR), in a Caucasian man 64-year-old with locally advanced PCa (PSA 160 ng/ml, Gleason score 4+3/ISUP Grade Group 3, clinical stage T3NXM0) who underwent radical retropubic prostatectomy plus extended pelvic lymphadenectomy (LAD) as first step of a multimodal therapeutic treatment. A normal prostate of a 67-year-old man removed by post mortem autopsy was used as a control in the study. The mRNA for this study was conducted on paraffined prostatic sections of: a) index case of PCa; b) metastatic lymph node of index case; c) normal prostate. MiRNA-132, miRNA- 212 and Glyceraldehyde 3-phosphate dehydrogenase (as reference gene) assays were obtained. Definitive specimen showed a pT3bN1R1 stage: acinar cells adenocarcinoma with involvement of the seminal vesicles, multifocal positive surgical margins, Gleason score 8 (4+4/ISUP Grade Group 4), metastases in 5/25 iliac lymph nodes. An increased expression of miRNA-132 and miRNA-212 in index case of prostatic adenocarcinoma compared to normal prostate tissue was found; moreover, a lower expression of miR-132 and miR-212 in metastatic lymph node compared to primitive PCa and normal prostate tissue was demonstrated. Although a greater number of patients should be evaluated, these data suggest that the biology of the primary PCa, in our clinical case, was different from metastatic lymph node.

Diagnostic test assessment. Validation study of an alternative system to detect microsatellite instability in colorectal carcinoma.

The American Society for Clinical Pathology (ASCP), College of American Pathologists (CAP), Association for Molecular Pathology (AMP), and the American Society of Clinical Oncology (ASCO) have been recently strongly recommended the evaluation of mismatch repair status (MMS) as molecular biomarkers in colorectal cancer for a better prognostic stratification of patients. This recommendation is emphasized by the recent evidence of Microsatellite Instability (MSI) as a predictive marker for chemotherapy and immunotherapy.In this scenario, the validation of molecular biomarker testing methods seems to be essential to design the most appropriate tailored therapy and the most suitable care strategy, respectively.In this study, we validated an alternative method based on capillary electrophoresis system label-free PCR (Qiaxcel system) to evaluate the MSI Bethesda Panel. We also parallel the results with a standard approach.Our data showed total concordance with the standard approach, with a highly time-efficient and easy procedure combined with high sensitivity for MSI detection.Alternative capillary electrophoresis based on label-free PCR such as the Qiaxel system is a very sensitive and specific method to detect MSI for the management of patients with colorectal cancer. This procedure is adequate and suitable in diagnostic routine for the evaluation of microsatellite repeats compared to standard procedures.

Cerebellar Metastasis From Ovarian Carcinoma Harboring PIK3CA-Activating Mutation: A "Clear" Explanation for Unexpected "Vertigo".

Brain metastasis is a rare and generally late manifestation of an advanced-stage, high-grade serous ovarian carcinoma. Nowadays, the improved control of intra-abdominal disease by surgery and platinum-based chemotherapy results in a longer survival, allowing distant metastasis to implant and grow in the brain parenchyma. Herein, we describe a unique case of a cerebellar metastasis from clear cell ovarian carcinoma that initially presented as a FIGO Stage IC cancer. Surprisingly, 6 mo after surgery, the patient was in good condition with complete disappearance of symptoms and no evidence of recurrence. This relatively good biologic behavior may be explained by the presence of a PIK3CA-activating mutation in exon 9 which as previously reported in the literature, may be associated with better prognosis. To the best of our knowledge, this is the first reported case of cerebellar metastasis from ovarian clear cell carcinoma. In the presence of neurological symptoms, both clinicians and pathologists must be aware of this rare possibility, to assure correct patient management and effective therapeutic options. Generally, the prognosis of epithelial ovarian cancer patients with brain metastases is poor. PIK3CA mutations could be a good prognostic indicator in clear cell carcinomas.

miRNAs as Candidate Biomarker for the Accurate Detection of Atypical Endometrial Hyperplasia/Endometrial Intraepithelial Neoplasia.

Endometrial cancer is the most common gynecologic malignancy in developed countries. Estrogen-dependent tumors (type I, endometrioid) account for 80% of cases and non-estrogen-dependent (type II, non-endometrioid) account for the rest. Endometrial cancer type I is generally thought to develop via precursor lesions along with the increasing accumulation of molecular genetic alterations. Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia is the least common type of hyperplasia but it is the type most likely to progress to type I cancer, whereas endometrial hyperplasia without atypia rarely progresses to carcinoma. MicroRNAs are a class of small, non-coding, single-stranded RNAs that negatively regulate gene expression mainly binding to 3'-untranslated region of target mRNAs. In the current study, we identified a microRNAs signature (miR-205, miR-146a, miR-1260b) able to discriminate between atypical and typical endometrial hyperplasia in two independent cohorts of patients. The identification of molecular markers that can distinguish between these two distinct pathological conditions is considered to be highly useful for the clinical management of patients because hyperplasia with an atypical change is associated with a higher risk of developing cancer. We show that the combination of miR-205, -146a, and -1260b has the best predictive power in discriminating these two conditions (>90%). With the aim to find a biological role for these three microRNAs, we focused our attention on a common putative target involved in endometrial carcinogenesis: the oncosuppressor gene SMAD4. We showed that miRs-146a,-205, and-1260b directly target SMAD4 and their enforced expression induced proliferation and migration of Endometrioid Cancer derived cell lines, Hec1a cells. These data suggest that microRNAs-mediated impairment of the TGF-ß pathway, due to inhibition of its effector molecule SMAD4, is a relevant molecular alteration in endometrial carcinoma development. Our findings show a potential diagnostic role of this microRNAs signature for the accurate diagnosis of Endometrial hyperplasia with atypia/Endometrial Intraepithelial Neoplasia and improve the understanding of their pivotal role in SMAD4 regulation.

Clear cell carcinoma of the endometrium: an immunohistochemical and molecular analysis of 45 cases.

The aim of the present paper is to study a cohort of pure selected endometrial clear cell carcinomas (ECCCs) from an immunohistochemical and molecular perspective to provide new data about the molecular profile of this disease. In detail, 45 consecutive patients with a proven diagnosis of pure ECCC, according to World Health Organization criteria, were included into the study. We determined the incidence of KRAS, BRAF, and PIK3CA mutations as well as the immunohistochemical expression of mismatch repair (MMR) proteins (MLH1, MSH2, MSH6, PMS2), estrogen, progesterone receptors, p16, and p53. Immunohistochemical analyses for α-methylacyl-coenzyme-A racemase and Napsin A were performed to support the diagnosis of ECC. All cases were positive for at least 1 of the 2 markers. In detail, 34 of 45 (75.5%) cases were positive for α-methylacyl-coenzyme-A racemase, and 40 of 45 (88.8%) cases showed positive staining for Napsin A. All selected cases exhibited negative immunostain for estrogen receptor and progesterone receptor, a "patchy" immunostain for p16, and a "wild-type" staining pattern for p53. Fifteen patients (15/45; 33.3%) showed loss of 1 or more MMR proteins by immunohistochemistry. Seven patients showed dual loss of MSH2 and MSH6, 4 patients (8.8%) showed isolated loss of MSH6, and the remaining 4 patients showed isolated loss of PMS2, respectively. Pyrosequencing analysis revealed the presence of 5 of 45 mutations (11%) at codon 12 in exon 2 of KRAS (3/5 p.G12D, 60%; 2/5 p. G12V, 40%) and 5 of 45 (11%) mutations in PIK3CA gene, of which 3 of 5 (60%) were in exon 9 of PIK3CA (2 p.E542K and 1 p.Q546K) and 2 of 5 (40%) were in exon 20 (p.H1047R). Two synchronous mutations affecting exon 9 of PIK3CA (p.Q546K) and exon 2, codon 12 of KRAS (p.G12D) were found. No mutations were detected in the hot spot region of BRAF. In conclusion, we provided detailed immunohistochemical and molecular data in a series of ECC, demonstrating a high incidence (33%) of MMR deficiencies detected by immunohistochemistry as well as a synchronous mutation affecting PIK3CA and KRAS genes. A more extensive interrogation of the genomic features of a much larger series of clear cell carcinomas will be required to define the genomic landscape of this subtype and to determine whether there are molecular alterations that are unique to, or significantly enriched in, clear cell tumors compared to other subtypes.

Ovarian Clear Cell Carcinoma: From Morphology to Molecular Biology.

Ovarian clear cell carcinoma (oCCC) is a distinctive subtype of ovarian carcinoma, with peculiar genetic and environmental risk factors, precursor lesions, molecular events during oncogenesis, patterns of spread, and response to treatment. Because of low response to chemotherapy and poor prognosis in advanced stages, there is growing interest in investigating the molecular pathways involved in oCCC development, in order to individualize novel/molecular targeted therapies. Until now, the main molecular genetic changes associated with oCCC remain to be identified, and, although several molecular changes have been reported in clear cell tumors, most studies have analyzed a limited number of cases; therefore, the true prevalence of those changes is not known. The present review will present the clinicopathologic features of oCCC, from morphology to molecular biology, discussing the diagnostic and treatment challenges of this intriguing ovarian carcinoma.

MGMT promoter methylation status in Merkel cell carcinoma: in vitro versus invivo.

PURPOSE: Expression of O6-methylguanine-DNA methyltransferase (MGMT) in Merkel cell carcinoma (MCC) is very variable; thus, we tested whether this may be due to differential methylation of the MGMT gene promoter. METHODS: Quantitative analysis of MGMT mRNA and protein expression, as well as MGMT promoter methylation status, was performed in a series of tissue samples of MCC tumors, representing both primary and metastatic lesions, as well as in six MCC cell lines. RESULTS: These analyses revealed a very heterogeneous MGMT mRNA and protein expression in MCC both in vivo and in vitro. However, neither the MGMT mRNA nor protein expression correlated with the sensitivity of MCC cell lines toward the alkylating agent dacarbazine in vitro. Notably, increased methylation at the promoter of the MGMT gene was observed in 2/6 (33%) of the MCC cell lines; however, MGMT promoter methylation was absent in all MCC tissue samples. According to our results, albeit aberrant methylation of MGMT gene promoter can be observed in in vitro propagated MCC cell lines, it seems to be absent or very rare in MCC lesions in situ. CONCLUSION: Thus, the evaluation of this marker has no or only little significance for predicting response to therapy or for improving efficacy of demethylating agents in the treatment of MCC. Microenvironmental factors may play a role in explaining the different results between MCC cell lines and MCC samples.

FOXL2 molecular status in adult granulosa cell tumors of the ovary: A study of primary and metastatic cases.

Granulosa cell tumors (GCTs) of the ovary are uncommon neoplasms, accounting for ~5% of all malignant ovarian tumors. GCTs are a relatively homogeneous group of tumors, categorized into two distinct subtypes, juvenile GCT and adult GCT (AGCT), likely arising from a limited set of molecular events usually involving the disruption of pathways that regulate granulosa cell proliferation. In the present study, the presence of forkheadbox L2 (FOXL2) c.402C>G mutation was investigated in a series of 42 samples of primary and metastatic AGCT of the ovary. The samples consisted of 37 primary and 5 metastatic ovarian AGCTs from 37 patients. FOXL2 mutational status was evaluated using a pyrosequencing approach on 2.5-µm sections of formalin-fixed paraffin-embedded tissue. FOXL2 c.402C>G mutation was found in 33/37 (89.2%) primary AGCTs and in 4/5 (80.0%) metastases, with the molecular status of the metastases recapitulating that of the primary tumors (4 mutated cases and 1 wild-type case). Overall, FOXL2 mutation is present in the majority of primary and metastatic AGCTs, and could be used as a valid tool in the diagnosis of the disease and in cases of metastatic lesions from an unknown primary origin. Moreover the concordance of FOXL2 molecular status in primary and associated metastases suggests its early appearance and genomic stability in AGCT tumorigenesis.

Molecular status of PI3KCA, KRAS and BRAF in ovarian clear cell carcinoma: an analysis of 63 patients.

AIMS: To evaluate the incidence of PI3KCA, KRAS and BRAF mutations in primary ovarian clear cell carcinoma (OCCC). METHODS: 63 consecutive patients, with a proven diagnosis of OCCC, according to WHO criteria, were included into the study. Pyrosequencing analysis of all three genes hotspot regions were performed on 2.5 µm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. RESULTS: PI3KCA mutations were found in 20/63 (32%) cases; KRAS mutations were found in 8/63 (13%); no BRAF V600 mutations were found. In particular, 12/20 mutations (60%) of PI3KCA were found in the exon 20, whereas the remaining eight cases presented mutations in exon 9 (8/20; 40%). KRAS pyrosequencing analysis revealed higher incidence of codon 12 mutations (7/8; 90%) than codon 13 mutations (1/8; 10%). In five cases (5/66; 8%), synchronous mutations, affecting PI3KCA and KRAS genes, were found. No differences were found in the distribution of hotspot mutations, according to the stage. CONCLUSIONS: The high frequency of PI3KCA mutations, the low rate of mutations in KRAS and the absence of mutations in BRAF, indicate a molecular signature of OCCCs different from other ovarian carcinomas. Detection of driver mutations, such as PI3KCA and KRAS, may be the basis for a targeted therapy, although the clinical and therapeutic implications of these findings have to be supported by further studies.

Epidermal growth factor receptor exon 20 p.S768I mutation in non-small cell lung carcinoma: A case report combined with a review of the literature and investigation of clinical significance.

Epidermal growth factor receptor (EGFR) plays a significant role in non-small cell lung cancer (NSCLC), the most prevalent form of lung cancer worldwide. Therefore, EGFR may be a useful molecular target for personalized therapy utilizing tyrosine kinase inhibitors (TKIs). Somatic activating EGFR mutations may be used to identify tumors sensitive to the effects of small-molecule EGFR-TKIs (gefitinib and erlotinib), and alternative, less frequently observed mutations, including the majority of mutations identified within exon 20, may be associated with a lack of response to TKIs. However, due to the comparative rarity of EGFR exon 20 mutations, clinical information concerning the association between EGFR exon 20 mutations and responsiveness to TKIs has been limited within the relevant literature, particularly for certain rare mutations, including p.S768I. The current study reports the case of a patient with NSCLC harboring a p.S768I mutation in the EGFR gene [a substitution at codon 768 of exon 20 (c.2303G>T, p.S768I)], as well as a mutation at codon 719, exon 18 (p.G719A). The relevant literature concerning this rare EGFR somatic mutation is also reviewed.

Oral malignant melanoma: A report of two cases with BRAF molecular analysis.

Primary oral malignant melanoma is a rare condition, accounting for 1.3-1.4% of all melanomas, usually presenting with an aggressive clinical behavior. The present study reports the clinicopathological findings of two cases of oral malignant melanoma and discusses the epidemiology, diagnosis and current therapeutic approaches for this uncommon condition. In the first case the patient presented with a pigmented lesion located on the lower mucosal lip. The patient showed no nodal metastases and therefore, underwent a wedge resection. After seven months, the patient presented with neck lymph nodes and multiple visceral metastases. Molecular analysis of BRAF, using a pyrosequencing approach, revealed the presence of BRAF V600E mutation. The patient developed multiple visceral metastases, but refused treatment and was lost to follow-up. In the second case, no BRAF V600E mutation was found, but the patient exhibited a pigmented patch in the lower gingival mucosa, which was excised by surgical treatment. The patient was followed up by an oncologist, but did not undergo an additional therapy and is currently alive with no evidence of visceral metastases at one year following the diagnosis.

Mutational status of KRAS, NRAS, and BRAF in primary clear cell ovarian carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer with characteristic biological features and aggressive clinical behavior. OCCCs show a pattern of gene mutations different from other type I ovarian malignancies, notably a higher frequency of PIK3CA mutations. In low grade serous ovarian cancer, KRAS and BRAF mutations are frequent, but little data are available on the mutational status of these genes in OCCCs. To clarify this issue, we designed a clinicopathological study with the aim to establish the incidence of KRAS, NRAS, and BRAF hot spot mutations in OCCC. Between December 2006 and June 2012, 22 patients with a proven diagnosis of OCCC were admitted to our Institutions. In all cases, final diagnosis was established according to FIGO and WHO criteria. All women received complete surgical staging. The PyroMark Q24 system (Qiagen GmbH, Hilden, Germany) was used for pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions on 2.5-µm sections of formalin-fixed paraffin-embedded tissue from primary OCCC. Pyrosequencing analysis of KRAS, NRAS, and BRAF hot spot regions revealed the presence of mutations only at codon 12 in exon 2 of KRAS in 3 of 22 (14 %) cases. We found no mutations in the hot spot regions of NRAF (exons 2, 3, 4) or BRAF (exon 15). The median age of women with a KRAS mutated OCCC was 74 years. These OCCC were unilateral FIGO stage IA lesions in two cases associated with foci of endometriosis. We conclude that in 14 % of OCCCs, a KRAS mutation occurs in codon 2 exon 2. NRAS and BRAF mutations were not found.