The Combined Immunohistochemical Expression of GLI1 and BCOR in Synovial Sarcomas for the Identification of Three Risk Groups and Their Prognostic Outcomes: A Study of 52 Patients.

Synovial sarcoma (SS) is a rare soft-tissue tumor characterized by a monomorphic blue spindle cell histology and variable epithelial differentiation. Morphologically, SSs may be confused with other sarcomas. Systemic treatment is more effective for patients with high-risk SSs, patients with advanced disease, and younger patients. However, further studies are required to find new prognostic biomarkers. Herein, we describe the morphological, molecular, and clinical findings, using a wide immunohistochemical panel, of a series of SS cases. We studied 52 cases confirmed as SSs by morphological diagnosis and/or molecular studies. Clinical data (gender, age, tumor size, tumor location, resection margins, adjuvant treatment, recurrences, metastasis, and survival) were also retrieved for each patient. All the available H&E slides were examined by four pathologists. Three tissue microarrays (TMAs) were constructed for each of the tumors, and a wide immunohistochemical panel was performed. For time-to-event variables, survival analysis was performed using Kaplan-Meier curves and log-rank testing, or Cox regression. Statistical significance was considered at p < 0.05. The mean age of our patients was 40.33, and the median was 40.5 years. We found a predominance of males versus females (1.7:1). The most frequent morphological subtype was monophasic. TRPS1, SS18-SSX, and SSX-C-terminus were positive in 96% of cases. GLI1 expression was strong in six and focal (cytoplasmic) in twenty patients. Moreover, BCOR was expressed in more than half of SSs. Positive expression of both proteins, BCOR and GLI1, was correlated with a worse prognosis. Multivariate analysis was also performed, but only BCOR expression appeared to be significant. The combination of GLI1 and BCOR antibodies can be used to group SSs into three risk groups (low, intermediate, and high risk). We hypothesize that these findings could identify which patients would benefit from receiving adjuvant treatment and which would not. Moreover, these markers could represent therapeutic targets in advanced stages. However, further, larger series of SSs and molecular studies are necessary to corroborate our present findings.

The hallmarks of ovarian cancer: proliferation and cell growth.

Epithelial ovarian cancer (EOC) is a heterogeneous group of diseases with distinct biological and clinical behaviour. Despite the differences between them, the capability of tumour cells to continuously proliferate and avoid death is maintained among histotypes. This ability is the result of alterations at different levels, causing the deregulation of cell cycle and proliferative-related pathways. Even if the leading role is played by RB and TP53, changes in other molecular pathways are involved in the development of EOC. This ability can be exploited to generate in vitro and in vivo models resembling the conditions of tumour development in a patient. In vivo models, such as patient-derived xenografts (PDX) or genetically engineered mouse models (GEMM), represent a fundamental tool in the study of the molecular mechanisms implicated in each EOC biotype for testing new therapeutic approaches. Herein we describe the major proliferation-related pathways and its disruption found in EOC and how these features can be used to establish in vivo models for translational research.

A shared origin from a common ancestor: A case report of two histologically different tumors.

The origin of tumors has been under discussion over the years. Different theories have been suggested to explain this phenomenon. Among them, the Cancer-Stem Cells model, is one of the most outstanding. In this study, we reported a case of a 72-year-old man who presented two histologically different tumors with a 7-years gap, a Penile Squamous Cell Carcinoma and a Pleomorphic Undifferentiated Sarcoma, that share some molecular features. Phonotypical differences were showed and confirmed at histological and IHC levels. Molecular analysis showed an HPV infection in the carcinoma. Additionally, sequencing results revealed common (CDKN2A and TERT) and exclusive (FBXW7 and TP53) genetic alterations in both tumors (Table 1). The possible germline origin of common mutations was discarded after negative germline testing. Here we describe, for the first time a clinical case of a possible origin of two histologically different tumors from a common ancestor based on molecular data. Even if different hypothesis appear as possible, the Cancer Stem Cell-based model appears as the most suitable.

Tumor Microenvironment and Its Clinicopathologic and Prognostic Association in Cutaneous and Noncutaneous Angiosarcomas.

OBJECTIVES: We explored features of the angiosarcoma (AS) tumor microenvironment to discover subtypes that may respond to immunotherapy. METHODS: Thirty-two ASs were included. Tumors were studied by histology, immunohistochemistry (IHC), and gene expression profile using the HTG EdgeSeq Precision Immuno-Oncology Assay. RESULTS: Comparing cutaneous and noncutaneous ASs, the second group showed 155 deregulated genes, and unsupervised hierarchical clustering (UHC) delineated two groups: the first mostly cutaneous AS and the second mainly noncutaneous AS. Cutaneous ASs showed a significantly higher proportion of T cells, natural killer cells, and naive B cells. ASs without MYC amplification revealed a higher immunoscore in comparison with ASs with MYC amplification. PD-L1 was significantly overexpressed in ASs without MYC amplification. UHC showed 135 deregulated genes differentially expressed when comparing ASs from the non-head and neck area with patients who had AS in the head and neck area. ASs from the head and neck area showed high immunoscore. PD1/PD-L1 content was significantly more highly expressed in ASs from the head and neck area. IHC and HTG gene expression profiling revealed a significant correlation between PD1, CD8, and CD20 protein expression but not PD-L1. CONCLUSIONS: Our HTG analyses confirmed a high degree of tumor and microenvironment heterogeneity. Cutaneous ASs, ASs without MYC amplification, and ASs located in the head and neck area seem to be the most immunogenic subtypes in our series.

The Scarface Score: Deciphering Response to DNA Damage Agents in High-Grade Serous Ovarian Cancer-A GEICO Study.

Genomic Instability (GI) is a transversal phenomenon shared by several tumor types that provide both prognostic and predictive information. In the context of high-grade serous ovarian cancer (HGSOC), response to DNA-damaging agents such as platinum-based and poly(ADP-ribose) polymerase inhibitors (PARPi) has been closely linked to deficiencies in the DNA repair machinery by homologous recombination repair (HRR) and GI. In this study, we have developed the Scarface score, an integrative algorithm based on genomic and transcriptomic data obtained from the NGS analysis of a prospective GEICO cohort of 190 formalin-fixed paraffin-embedded (FFPE) tumor samples from patients diagnosed with HGSOC with a median follow up of 31.03 months (5.87-159.27 months). In the first step, three single-source models, including the SNP-based model (accuracy = 0.8077), analyzing 8 SNPs distributed along the genome; the GI-based model (accuracy = 0.9038) interrogating 28 parameters of GI; and the HTG-based model (accuracy = 0.8077), evaluating the expression of 7 genes related with tumor biology; were proved to predict response. Then, an ensemble model called the Scarface score was found to predict response to DNA-damaging agents with an accuracy of 0.9615 and a kappa index of 0.9128 (p < 0.0001). The Scarface Score approaches the routine establishment of GI in the clinical setting, enabling its incorporation as a predictive and prognostic tool in the management of HGSOC.

Phase 2 Trial (POLA Study) of Lurbinectedin plus Olaparib in Patients with Advanced Solid Tumors: Results of Efficacy, Tolerability, and the Translational Study.

We hypothesized that the combination of olaparib and lurbinectedin maximizes DNA damage, thus increasing its efficacy. The POLA phase 1 trial established the recommended phase 2 dose of lurbinectedin as being 1.5 mg (day 1) and that of olaparib as being 250 mg/12 h (days 1-5) for a 21-day cycle. In phase 2, we explore the efficacy of the combination in terms of clinical response and its correlation with mutations in the HRR genes and the genomic instability (GI) parameters. Results: A total of 73 patients with high-grade ovarian (n = 46), endometrial (n = 26), and triple-negative breast cancer (n = 1) were treated with lurbinectedin and olaparib. Most patients (62%) received ≥3 lines of prior therapy. The overall response rate (ORR) and disease control rate (DCR) were 9.6% and 72.6%, respectively. The median progression-free survival (PFS) was 4.54 months (95% CI 3.0-5.2). Twelve (16.4%) patients were considered long-term responders (LTR), with a median PFS of 13.3 months. No clinical benefit was observed for cases with HRR gene mutation. In ovarian LTRs, although a direct association with GI and a total loss of heterozygosity (LOH) events was observed, the association did not reach statistical significance (p = 0.055). Globally, the total number of LOHs might be associated with the ORR (p =0.074). The most common grade 3-4 toxicities were anemia and thrombocytopenia, in 6 (8.2%) and 3 (4.1%) patients, respectively. Conclusion: The POLA study provides evidence that the administration of lurbinectedin and olaparib is feasible and tolerable, with a DCR of 72.6%. Different GI parameters showed associations with better responses.

A phase I dose-finding, pharmacokinetics and genotyping study of olaparib and lurbinectedin in patients with advanced solid tumors.

The poly (ADP-Ribose) polymerase (PARP) inhibitor olaparib has shown antitumor activity in patients with ovarian or breast cancer with or without BRCA1/2 mutations. Lurbinectedin is an ecteinascidin that generates DNA double-strand breaks. We hypothesized that the combination of olaparib and lurbinectedin maximizes the DNA damage increasing the efficacy. A 3 + 3 dose-escalation study examined olaparib tablets with lurbinectedin every 21 days. The purpose of this phase I study is to determine the dose-limiting toxicities (DLTs) of the combination, to investigate the maximum tolerated dose (MTD), the recommended phase II dose (RP2D), efficacy, pharmacokinetics, in addition to genotyping and translational studies. In total, 20 patients with ovarian and endometrial cancers were included. The most common adverse events were asthenia, nausea, vomiting, constipation, abdominal pain, neutropenia, anemia. DLT grade 4 neutropenia was observed in two patients in dose level (DL) 5, DL4 was defined as the MTD, and the RP2D was lurbinectedin 1.5 mg/m2 + olaparib 250 mg twice a day (BID). Mutational analysis revealed a median of 2 mutations/case, 53% of patients with mutations in the homologous recombination (HR) pathway. None of the patients reached a complete or partial response; however, 60% of stable disease was achieved. In conclusion, olaparib in combination with lurbinectedin was well tolerated with a disease control rate of 60%. These results deserve further evaluation of the combination in a phase II trial.

The Frequency and Prognostic Significance of the Histologic Type in Early-stage Ovarian Carcinoma: A Reclassification Study by the Spanish Group for Ovarian Cancer Research (GEICO).

The frequency and prognostic significance of the histologic type in early-stage ovarian cancer (OC) is not as well established as in advanced stages. In addition, histologic typing based only on morphologic features may be difficult, especially in high-grade tumors. In this study, we have analyzed a prospective cohort of 502 early-stage OCs to investigate their frequency, immunohistochemical characteristics, and survival of the 5 main histologic types. Histotype was assigned according to not only the morphologic features but also according to the expression pattern of WT1, p53, Napsin A, and progesterone receptors. In addition, an extended panel including p16, ß-catenin, HER2, Arid1A, HINF1B, CK7, CDX2, and CK20 was used to refine the diagnosis in difficult cases. In this series, the frequency of the 5 major histologic types was as follows: endometrioid carcinoma, 32.7%; clear cell carcinoma, 25.1%; high-grade serous carcinoma (HGSC), 24.7%; mucinous carcinoma, 10.2%; low-grade serous carcinoma, 4.6%; and others, 2.8%. The combination of morphology and immunohistochemistry allowed the reclassification of 23% of OCs. The lowest concordance was found between samples initially diagnosed as endometrioid, but finally classified as high-grade serous tumors (22% error rate). Endometrioid carcinoma was the most favorable histologic type, whereas HGSC and low-grade serous carcinoma had the worst prognosis. Clear cell carcinoma with abnormal p53 immunostaining pattern also had poor prognosis. Although histologic grade was not a prognostic factor among early-stage endometrioid OCs, distinction between grade 3 endometrioid OC and HGSC is recommended, taking into account differences in prognosis and molecular alterations that can guide different treatments.

Prognostic Impact of let-7e MicroRNA and Its Target Genes in Localized High-Risk Intestinal GIST: A Spanish Group for Research on Sarcoma (GEIS) Study.

MicroRNAs (miRNAs) are small non-coding RNAs that negatively regulate gene expression at the post-transcriptional level, and they have been described as being associated with tumor prognosis. Here, miRNA profiling was planned to explore new molecular prognostic biomarkers in localized intestinal high-risk GIST. Paraffin tumor blocks of 14 and 86 patients were used in the discovery and expansion sets, respectively. GeneChip miRNA v3.0 was employed to identify the miRNAs differentially expressed between relapsed and non-relapsed patient samples, which were validated in the expansion set, by qRT-PCR. RT2 Profiler PCR Array was used for the screening of let-7e targets. Expression levels were correlated with relapse-free survival and overall survival. In the discovery set, 39 miRNAs were significantly deregulated, let-7e and miR-550 being the most underexpressed and overexpressed miRNAs in the relapsed group, respectively. In the expansion set, the underexpression of let-7e or the overexpression of 4 of its target genes (ACVR1B, CASP3, COL3A1, and COL5A2) were statistically associated with worse relapse-free survival. The expression of let-7e and 4 of its target genes are potential prognostic biomarkers in high-risk localized intestinal GIST. The expression of these genes is a potential molecular tool useful for a more accurate prognosis in this subset of GIST patients.

Molecular Heterogeneity of Endometrioid Ovarian Carcinoma: An Analysis of 166 Cases Using the Endometrial Cancer Subrogate Molecular Classification.

Endometrioid ovarian carcinoma (EOC) has clinical and biological differences compared with other histologic types of ovarian carcinomas, but it shares morphologic and molecular features with endometrioid endometrial carcinoma. To analyze the molecular heterogeneity of EOC according to the new molecular classification of endometrial cancer and to evaluate the prognostic significance of this molecular classification, we have analyzed 166 early-stage EOC by immunohistochemistry for mismatch repair proteins and p53 expression, and by Sanger sequencing for the exonuclease domain of polymerase epsilon (POLE EDM). In addition, we have carried out next-generation sequencing analysis of tumors with POLE EDM mutations to confirm the ultramutated profile. Eight tumors carried POLE EDM mutations and were classified as ultramutated (5%), 29 showed mismatch repair deficiency and were classified as hypermutated (18%), 16 tumors had a mutated pattern of p53 expression and were classified as p53 abnormal (11%), and 114 tumors did not have any of the previous alterations and were classified as no specific type (66%). Five tumors showed >1 classification criteria. The frequencies of ultramutated and hypermutated tumors were lower in EOC compared with the frequency reported in endometrial cancer. Subrogate molecular groups differed in both morphologic features (histologic grade, squamous and morular metaplasia, and necrosis) and immunohistochemical expression of several biomarkers (ARID1A, nuclear ß-catenin, estrogen receptors, Napsin A, and HINF1B). In addition, the number of CD8 tumor-infiltrating lymphocytes was higher in ultramutated and hypermutated tumors. The most commonly mutated genes in the ultramutated group were ARID1A (100%), PIK3R1, PTEN, BCOR, and TP53 (67% each), whereas no mutations were detected in KRAS. Although the prognosis did not differ among subgroups in the multivariate analysis, a trend toward a better prognosis in POLE-mutated and a worse prognosis in p53 abnormal tumors was observed. In addition, this classification could have important therapeutic implications for the use of immunotherapy in tumors classified as ultramutated and hypermutated.

Mismatch Repair Deficiency in Ovarian Carcinoma: Frequency, Causes, and Consequences.

Mismatch repair deficiency (MMRD) is involved in the initiation of both hereditary and sporadic tumors. MMRD has been extensively studied in colorectal cancer and endometrial cancer, but not so in other tumors, such as ovarian carcinoma. We have determined the expression of mismatch repair proteins in a large cohort of 502 early-stage epithelial ovarian carcinoma entailing all the 5 main subtypes: high-grade serous carcinoma, endometrioid ovarian carcinoma (EOC), clear cell carcinoma (CCC), mucinous carcinoma, and low-grade serous carcinoma. We studied the association of MMRD with clinicopathologic and immunohistochemical features, including tumor-infiltrating lymphocytes in EOC, the histologic type in which MMRD is most frequent. In addition, MLH1 promoter methylation status and massive parallel sequencing were used to evaluate the proportion of sporadic and Lynch syndrome-associated tumors, and the most frequently mutated genes in MMRD EOCs. MMRD occurred only in endometriosis-associated histologic types, and it was much more frequent in EOC (18%) than in CCC (2%). The most frequent immunohistochemical pattern was loss of MLH1/PMS2, and in this group, 80% of the cases were sporadic and secondary to MLH1 promoter hypermethylation. The presence of somatic mutations in mismatch repair genes was the other mechanism of MMRD in sporadic tumors. In this series, the minimum estimated frequency of Lynch syndrome was 35% and it was due to germline mutations in MLH1, MSH2, and MSH6. ARID1A, PTEN, KTM2B, and PIK3CA were the most common mutated genes in this series. Interestingly, possible actionable mutations in ERRB2 were found in 5 tumors, but no TP53 mutations were detected. MMRD was associated with younger age and increased tumor-infiltrating lymphocytes. Universal screening in EOC and mixed EOC/CCC is recommended for the high frequency of MMRD detected; however, for CCC, additional clinical and pathologic criteria should be evaluated to help select cases for analysis.

Prognostic classification of endometrial cancer using a molecular approach based on a twelve-gene NGS panel.

Endometrial Cancer (EC) is one of the most common malignancies in women in developed countries. Molecular characterization of different biotypes may improve clinical management of EC. The Cancer Genome Atlas (TCGA) project has revealed four prognostic EC subgroups: POLE, MSI; Copy Number Low (CNL) and Copy Number High (CNH). The goal of this study was to develop a method to classify tumors in any of the four EC prognostic groups using affordable molecular techniques. Ninety-six Formalin-Fixed Paraffin-embedded (FFPE) samples were sequenced following a NGS TruSeq Custom Amplicon low input (Illumina) protocol interrogating a multi-gene panel. MSI analysis was performed by fragment analysis using eight specific microsatellite markers. A Random Forest classification algorithm (RFA), considering NGS results, was developed to stratify EC patients into different prognostic groups. Our approach correctly classifies the EC patients into the four TCGA prognostic biotypes. The RFA assigned the samples to the CNH and CNL groups with an accuracy of 0.9753 (p < 0.001). The prognostic value of these groups was prospectively reproduced on our series both for Disease-Free Survival (p = 0.004) and Overall Survival (p = 0.030).Hence, with the molecular approach herein described, a precise and suitable tool that mimics the prognostic EC subtypes has been solved and validated. Procedure that might be introduced into routine diagnostic practices.