Overview of Tumor Heterogeneity in High-Grade Serous Ovarian Cancers.

Ovarian cancers encompass a group of neoplasms originating from germinal tissues and exhibiting distinct clinical, pathological, and molecular features. Among these, epithelial ovarian cancers (EOCs) are the most prevalent, comprising five distinct tumor histotypes. Notably, high-grade serous ovarian cancers (HGSOCs) represent the majority, accounting for over 70% of EOC cases. Due to their silent and asymptomatic behavior, HGSOCs are generally diagnosed in advanced stages with an evolved and complex genomic state, characterized by high intratumor heterogeneity (ITH) due to chromosomal instability that distinguishes HGSOCs. Histologically, these cancers exhibit significant morphological diversity both within and between tumors. The histologic patterns associated with solid, endometrioid, and transitional (SET) and classic subtypes of HGSOCs offer prognostic insights and may indicate specific molecular profiles. The evolution of HGSOC from primary to metastasis is typically characterized by clonal ITH, involving shared or divergent mutations in neoplastic sub-clones within primary and metastatic sites. Disease progression and therapy resistance are also influenced by non-clonal ITH, related to interactions with the tumor microenvironment and further genomic changes. Notably, significant alterations occur in nonmalignant cells, including cancer-associated fibroblast and immune cells, during tumor progression. This review provides an overview of the complex nature of HGSOC, encompassing its various aspects of intratumor heterogeneity, histological patterns, and its dynamic evolution during progression and therapy resistance.

Histological patterns and intra-tumor heterogeneity as prognostication tools in high grade serous ovarian cancers.

OBJECTIVE: High grade serous ovarian carcinoma (HGSOC) is the most common type of malignant ovarian neoplasm and the main cause of ovarian cancer related deaths worldwide. Although novel biomarkers such as homologous recombination deficiency testing have been implemented into the clinical decision-making algorithm since diagnosis, morphological classification and immunohistochemistry analysis are essential for diagnostic purpose. This study aims at identifying histologic and clinical features that can be predictive of patients' prognosis. METHODS: Morphological and architectural characterization including SET (Solid-Endometroid-Transitional)/Classic features was carried out in a cohort of 234 patients analyzing 695 slides. From each slide tumor infiltrating lymphocyte (TILs), the presence of necrosis, the number of mitoses, the presence of psammoma bodies, giant cells and atypical mitoses were recorded. Morphological heterogeneity was quantified by the Shannon's diversity index (SDI) considering the percentage of each architectural pattern per patient's slide. RESULTS: The frequency of architectural patterns and morphological variables varied with respect of the surgical strategy (primary debulking surgery vs interval surgery after neoadjuvant chemotherapy). HGSOCs exhibiting SET features had a longer overall as well as progression free survival. Among SET features, pseudo-endometrioid and transitional like patterns had the best outcome, while it was heterogenous for solid pattern, that had better outcome for BRCA 1 negative and less heterogeneous tumors. In patients submitted to neoadjuvant chemotherapy a higher intratumor heterogeneity as defined by SDI was a negative independent prognostic factor. CONCLUSIONS: A comprehensive histological examination considering architectural patterns and their heterogeneity can help in prognostication of HGSOCs.

Cell-stiffness and morphological architectural patterns in clinical samples of high grade serous ovarian cancers.

High grade serous ovarian carcinoma (HGSOC) is recognized as the most frequent type of ovarian cancer and the main cause of ovarian cancer related deaths worldwide. Although homologous recombination deficiency testing has been adopted in the clinical workflow, morphological analysis remains the main diagnostic tool. In this study Atomic Force Microscopy (AFM) was tested in standard hematoxylin and eosin (H&E) stained sections to investigate the biomechanical properties of different architectural growing patterns of HGSOC. Our results showed that AFM was able to discriminate HGSOC morphological growing patterns as well as patients' stage. Micropapillary pattern, which has been associated to poor outcome, had lower Young's moduli. In addition stage IV HGSOC was significantly softer than stage III cancers. Based on our results, AFM analysis could represent an additional tool in HGSOC morphological diagnosis as the biomechanical proprieties of HGSOC were quantitatively associated to tumor staging and architectural pattern.

Real-World Data on NGS Diagnostics: a survey from the Italian Society of Pathology (SIAPeC) NGS Network.

Next Generation Sequencing (NGS) is increasingly used in diagnostic centers for the assessment of genomic alterations to select patients for precision oncology. The Italian Society of Anatomic Pathology and Diagnostic Cytopathology (SIAPEC) through the Molecular Pathology and Predictive Medicine Study Group (PMMP) has been following the progressive development of centers that have adopted NGS technology in diagnostics over time. In July 2017, a study network on massive parallel sequencing was activated in Italy and recognized as the NGS SIAPeC National Network by the SIAPeC Scientific Society Board. Since then, activities have been implemented within the network that provide for alignment of laboratories through diagnostic concordance analysis and monitoring of centers adhering to the Network. Recently, considering the growing need for extended genomic analyses, the PMMP distributed a national survey to assess activities related to the use of genomic diagnostics in oncology within the NGS SIAPEC National Network.Thirty centers participated in the survey. Eighty percent of the centers are laboratories within Pathology Departments. The distribution of laboratories in the country, the diagnostic laboratory/population ratio, the staff dedicated, the type and number of sequencing and mechatronics platforms available, the genomic panels utilized, and the type and number of diagnostic tests carried out in the last year in each center, are reported.The centers were also asked whether they participated in a multidisciplinary Molecular Tumor Board (MTB) for management of patients. Thirty percent of the centers had a MTB that was ratified by regional decree. The professionals most frequently involved in the core team of the MTB are the pathologist, oncologist, molecular biologist, geneticist, pharmacologist, and bioinformatician.The data from this survey indicate that NGS diagnostics in Italy is still heterogeneous in terms of geographical distribution and the characteristics of laboratories and diagnostic test performed. The implementation of activities that favors harmonization, the logistics and the convergence of biological material in reference centers for molecular analyses is a priority for the development of a functional laboratory network.

Recent advancements in the study of breast cancer exosomes as mediators of intratumoral communication.

Breast cancer is a heterogeneous disease, with a morbidity rate of 27.8% and a mortality rate of 15% among women population worldwide. Understanding how this cancer develops and the mechanisms behind tumor progression and chemoresistance is of utmost importance. Exosomes mediate communication in a population of heterogeneous tumoral cells. They have a cargo composed of oncogenes and oncomiRs which change the transcriptomic scenario of their targeted cells and activate numerous tumor-promoting signaling pathways. Exosomes secreted by breast cancer cells lead to enhanced cell proliferation, replicative immortality, angiogenesis, invasion, migration, and chemoresistance. Studying exosomes from this perspective offers more in depth understanding of breast malignancy and may aid in the future development of early diagnostic, prognostic and therapeutic options. We present the latest findings in this area and offer practical solutions which may further stimulate the much-needed research of exosome in breast cancer.

A Novel HPLC-Based Method to Investigate on RNA after Fixation.

RNA isolated from fixed and paraffin-embedded tissues is widely used in biomedical research and molecular pathology for diagnosis. In the present study, we have set-up a method based on high performance liquid chromatography (HPLC) to investigate the effects of different fixatives on RNA. By the application of the presented method, which is based on the Nuclease S1 enzymatic digestion of RNA extracts followed by a HPLC analysis, it is possible to quantify the unmodified nucleotide monophosphates (NMPs) in the mixture and recognize their hydroxymethyl derivatives as well as other un-canonical RNA moieties. The results obtained from a set of mouse livers fixed/embedded with different protocols as well from a set of clinical samples aged 0 to 30 years-old show that alcohol-based fixatives do not induce chemical modification of the nucleic acid under ISO standard recommendations and confirm that pre-analytical conditions play a major role in RNA preservation.

Reliability of miRNA Analysis from Fixed and Paraffin-Embedded Tissues.

In clinical practice, patients' tissues are fixed and paraffin-embedded in order to enable histological diagnosis. Nowadays, those tissues are also used for molecular characterization. Formalin is the most used fixative worldwide, and Bouin's solution in some worldwide institutions. Among molecular targets, micro RNAs (miRNAs), the single-stranded non-coding RNAs comprised of 18 to 24 nucleotides, have been demonstrated to be resistant to fixation and paraffin-embedding processes, with consequent possible application in clinical practice. In the present study, let-7e-5p, miR-423-3p, miR-92a-1-5p, miR-30d-5p, miR-155-5p, miR-200a-3p, and miR-429 were investigated in formalin and matched Bouin's solution-fixed tissues of high grade serous ovarian cancers by means of real-time and droplet digital PCR (ddPCR). Micro RNAs were detectable and analyzable in both formalin- and Bouin's-fixed specimens, but on average, higher Ct values and lower copies/µL were found in Bouin's-fixed samples. Data from formalin-fixed samples correlated significantly for most targets with Bouin's ones, except for let-7e-5p and miR-155-5p. This study shows that miRNAs are analyzable in both formalin- and Bouin's-fixed specimens, with the possibility, after proper data normalization, to compare miRNA-based data from formalin-fixed samples to those of Bouin's-fixed ones.

A Practical Approach to Tumor Heterogeneity in Clinical Research and Diagnostics.

This Pathobiology issue tries to better define the complex phenomenon of intratumor heterogeneity (ITH), mostly from a practical point of view. This topic has been chosen because ITH is a central issue in tumor development and has to be investigated directly in patient tissue and immediately applied in the treatment of the presenting patient. Different types of ITH should be considered: clonal genetic and epigenetic evolution, morphological heterogeneity, and tumor sampling, heterogeneity resulting from microenvironmental autocrine and paracrine interaction, and stochastic plasticity related to different functional cell efficiencies. For a higher level of reproducibility in clinical research and diagnostics, it is necessary to establish standardized analytical methods, including microdissection. In situ techniques can be pivotal to explore tumor microenvironment and can be improved with associated digital analysis. Liquid biopsies for plasma DNA analysis are at present the best method to study recurrent tumors with treatment adaptation, and widespread clinical use could be beneficial. The different types of tumor genomic instabilities could have pragmatic applications to rank ITH for clinical applications: treatment approaches differ in patients with a high nucleotide mutation rate and patients with high copy number alterations.

The "good-cop bad-cop" TGF-beta role in breast cancer modulated by non-coding RNAs.

BACKGROUND: Lack of early diagnosis methods and the development of drug resistance are among the main reasons for increased mortality rates within breast cancer patients. These two aspects are governed by specific pro-carcinogenic modifications, where TGBß-induced EMT is one of the leading actors. Endowment of the epithelial cells with mesenchymal characteristics allows them to migrate and invade secondary tissues in order to form malignant sites and also confers chemoresistance. TGFß which role switches from the tumor suppressor cytokine to the oncogenic one favoring the tumor microenvironment regulates this process. SCOPE OF REVIEW: This review aims to comprehensively present the updated TGFß-induced EMT in breast cancer, including the regulatory role of the non-coding RNAs with focus on the miR-200 family and newly discovered lncRNAs such as HOTAIRM1. Additionally, a new phenotype, P-EMT, also modulated by miR-200 and miR-34 families that form complex feedback loops with TGFß, SNAI1 and ZEB1/2 is presented under an updated form. MAJOR CONCLUSIONS: The hallmarks of EMT are becoming increasingly associated with aggressive forms of breast cancer and low survival rates among patients. Considering that this phenotypical switch can trigger drug resistance, invasion and metastasis, inhibition of EMT could represent an important milestone in mammary cancer treatment. GENERAL SIGNIFICANCE: The present review assembles the most recent data regarding TGFß induced EMT, including the input of non-coding RNAs, contributing to the possible development of new targeted treatment strategies for cancer patients.

A synonymous EGFR polymorphism predicting responsiveness to anti-EGFR therapy in metastatic colorectal cancer patients.

Genetic factors are known to affect the efficiency of therapy with monoclonal antibodies (mAbs) targeting the epidermal growth factor receptor (EGFR) in patients with metastatic colorectal cancer (mCRC). At present, the only accepted molecular marker predictive of the response to anti-EGFR mAbs is the somatic mutation of KRAS and NRAS as a marker of resistance to anti-EGFR. However, only a fraction of KRAS wild-type patients benefit from that treatment. In this study, we show that the EGFR gene polymorphism rs1050171 defines, independently of RAS mutational status, a sub-population of 11 % of patients with a better clinical outcome after anti-EGFR treatment. Median PFS for patients with the GG genotype was 10.17 months compared to 5.37 of those with AG + AA genotypes. Taken together, our findings could be used to better define CRC populations responding to anti-EGFR therapy. Further studies in larger independent cohorts are necessary to validate the present observation that a synonymous polymorphism in EGFR gene impacts on clinical responsiveness.

Expression of the Endocannabinoid Receptor 1 in Human Stroke: An Autoptic Study.

OBJECTIVE: Stroke is one of the leading causes of disability and death in the world. The endocannabinoid (eCB) system is upregulated in several neurological diseases including stroke. A previous animal study demonstrated an increased expression of the endocannabinoid receptor 1 (CB1R) in the penumbra area surrounding the ischemic core, suggesting a crucial role in inflammation/reperfusion after stroke. Regarding the localization of CB1/CB2 receptors, animal studies showed that cortical neurons, activated microglia, and astroglia are involved. Our aim was to evaluate the cerebral expression of CB1R in the ischemic brain areas of 9 patients who died due to acute cerebral infarction in the middle cerebral artery territory. METHODS: The cerebral autoptic tissue was collected within 48 hours since death. Ischemic and contralateral normal-appearing areas were identified. After tissue preprocessing, 4-µm-thick cerebral sections were incubated with the primary CB1R antibodies (Cayman Chemical Company, Ann Arbor, MI). Thereafter, all cerebral sections were hematoxylin treated. In each section, the total cell number and CB1R-positive cells were counted and the CB1R-positive cell count ratio was calculated. For statistical analysis, Student's t-test was used. RESULTS: In normal tissue, CB1R-positive neurons were the majority; a few non-neuronal cells expressed CB1R. In the ischemic areas, a few neurons were detectable. A significant increase in total CB1R staining was found in the ischemic regions compared to contralateral areas. CONCLUSIONS: We found an increase in CB1R expression in the ischemic region (neuronal and non-neuronal cell staining), suggesting the inflammatory reaction to the ischemic insult. Whether such response might mediate neuroprotective actions or excitotoxicity-related detrimental effects is still unclear.

The proliferation marker Ki67, but not neuroendocrine expression, is an independent factor in the prediction of prognosis of primary prostate cancer patients.

BACKGROUND: Neuroendocrine markers, which could indicate for aggressive variants of prostate cancer and Ki67 (a well-known marker in oncology for defining tumor proliferation), have already been associated with clinical outcome in prostate cancer. The aim of this study was to investigate the prognostic value of those markers in primary prostate cancer patients. PATIENTS AND METHODS: NSE (neuron specific enolase), ChrA (chromogranin A), Syp (Synaptophysin) and Ki67 staining were performed by immunohistochemistry. Then, the prognostic impact of their expression on overall survival was investigated in 166 primary prostate cancer patients by univariate and multivariate analyses. RESULTS: NSE, ChrA, Syp and Ki67 were positive in 50, 45, 54 and 146 out of 166 patients, respectively. In Kaplan-Meier analysis only diffuse NSE staining (negative vs diffuse, p = 0.004) and Ki67 (≤ 10% vs > 10%, p < 0.0001) were significantly associated with overall survival. Ki67 expression, but not NSE, resulted as an independent prognostic factor for overall survival in multivariate analysis. CONCLUSIONS: A prognostic model incorporating Ki67 expression with clinical-pathological covariates could provide additional prognostic information. Ki67 may thus improve prediction of prostate cancer outcome based on standard clinical-pathological parameters improving prognosis and management of prostate cancer patients.

Management of stage II colon cancer - the use of molecular biomarkers for adjuvant therapy decision.

BACKGROUND: There is uncertainty on the benefit of adjuvant chemotherapy in patients with stage II colorectal cancers. The aim of this study is to investigate the combined role of clinical, pathological and molecular parameters to identify those stage II patients who better benefit from adjuvant therapy. METHODS: We examined 120 stage II colon cancer patients. Of these, 60 patients received adjuvant 5-FU chemotherapy after surgery and the other 60 did not receive therapy. Immunohistochemical (IHC) analyses were performed to evaluate the expressions of Thymidylate synthetase (TYMS), TP53 (p53), ß-catenin (CTNNB1) and CD8. For TYMS, its mRNA expression levels were also investigated by real time qRT-PCR. The entire case study was characterized by the presence of a defect in the MMR (mismatch repair) system, the presence of the CpG island methylator phenotype (CIMP or CIMP-High) and for the V600E mutation in the BRAF gene. At the histo-pathological level, the depth of tumour invasion, lymphovascular invasion, invasion of large veins, host lymphocytic response and tumour border configuration were recorded. RESULTS: The presence of the V600E mutation in the BRAF gene was a poor prognostic factor for disease free and overall survival (DFS; hazard ratio [HR], 2.57; 95% CI: 1.03 -6.37; p = 0.04 and OS; HR, 3.68; 95% CI: 1.43-9.47; p < 0.01 respectively), independently of 5-FU treatment. Adjuvant therapy significantly improved survival in patients with high TYMS levels (p = 0.04), while patients with low TYMS had a better outcome if treated by surgery alone (DFS; HR, 6.07; 95% CI, 0.82 to 44.89; p = 0.04). In patients with a defect in the MMR system (dMMR), 5-FU therapy was associated to reduced survival (DFS; HR, 37.98; 95% CI, 1.04 to 1381.31; p = 0.04), while it was beneficial for CIMP-High associated tumours (DFS; HR, 0.17; 95% CI, 0.02 to 1.13; p = 0.05). CONCLUSIONS: Patients' characterization according to MMR status, CIMP phenotype and TYMS mRNA expression may provide a more tailored approach for adjuvant therapy in stage II colon cancer.

Impact of standardization in tissue processing: the performance of different fixatives.

Most tissues in clinical practice are formalin-fixed and paraffin-embedded for histological as well as molecular analyses. The reproducibility and uniformity of molecular analyses is strictly dependent on the quality of the biomolecules, which is highly influenced by pre-analytical processes. In this study, the effect of different fixatives was compared, including formalin, Bouin's solution, RCL2® and TAG-1™ fixatives, by stringent application of ISO standards in mouse liver tissue processing, including formalin-free transport of tissues and tissue grossing in a refrigerated environment. The effect of fixatives was studied in terms of nucleic acid quality at the time of tissue processing and after one year of tissue storage at room temperature in the dark. Furthermore, a microcomputed tomography (CT) scan analysis was applied to investigate the paraffin embedding. The results show that the application of ISO standards in tissue processing allows analysis of 400 bases amplicons from RNA and 1000 bases from DNA, even in extracts from formalin-fixed and paraffin-embedded tissues. However, after one year storage at room temperature in the dark, a degradation of the nucleic acids was observed. Nevertheless, extracts can still be analyzed, but for metachronous tests it is highly recommended to repeat the quantitation of housekeeping genes in order to standardize the extent of nucleic acid degradation.

AKT Isoforms Interplay in High-Grade Serous Ovarian Cancer Prognosis and Characterization.

High-grade serous ovarian cancer (HGSOC) is among the deadliest gynecological malignancies. The acquired resistance to platinum-based therapies and the intrinsic heterogeneity of the disease contribute to the low survival rate. To improve patients' outcomes, new combinatorial approaches able to target different tumor vulnerabilities and enhance the efficacy of the current therapies are required. AKT inhibitors are promising antineoplastic agents able to act in synergy with PARP inhibitors, but the spectrum of patients who can benefit from this combination is unclear, since the role of the three different isoforms of AKT is still unknown. Here, we study the expression of AKT isoforms on a retrospective cohort of archive tissue by RT-droplet digital PCR (ddPCR) analyzing their association with the clinicopathological features of patients. Based on AKT1/AKT2 and AKT1/AKT3 ratios, we define four AKT classes which were related to patients' survival, tumor morphology and BRCA1 expression. Moreover, our results show that high AKT3 expression levels were frequently associated with tumors having classic features, a low number of mitoses and the presence of psammoma bodies. Overall, our study obtains new insights on AKT isoforms and their associations with the clinicopathological features of HGSOC patients. These evidences could help to better define the subsets of patients who can benefit from AKT and PARP inhibitors therapy in future clinical trials.

The human RECQ1 helicase is highly expressed in glioblastoma and plays an important role in tumor cell proliferation.

BACKGROUND: RecQ helicases play an essential role in the maintenance of genome stability. In humans, loss of RecQ helicase function is linked with predisposition to cancer and/or premature ageing. Current data show that the specific depletion of the human RECQ1 helicase leads to mitotic catastrophe in cancer cells and inhibition of tumor growth in mice. RESULTS: Here, we show that RECQ1 is highly expressed in various types of solid tumors. However, only in the case of brain gliomas, the high expression of RECQ1 in glioblastoma tissues is paralleled by a lower expression in the control samples due to the poor expression of RECQ1 in non-dividing tissues. This conclusion is validated by immunohistochemical analysis of a tissue microarray containing 63 primary glioblastomas and 19 perilesional tissue samples, as control. We also show that acute depletion of RECQ1 by RNAi results in a significant reduction of cellular proliferation, perturbation of S-phase progression, and spontaneous γ-H2AX foci formation in T98G and U-87 glioblastoma cells. Moreover, RECQ1 depleted T98G and U-87 cells are hypersensitive to HU or temozolomide treatment. CONCLUSIONS: Collectively, these results indicate that RECQ1 has a unique and important role in the maintenance of genome integrity. Our results also suggest that RECQ1 might represent a new suitable target for anti cancer therapies aimed to arrest cell proliferation in brain gliomas.

Gene expression changes in peripheral blood mononuclear cells in occupational exposure to nickel.

Allergic contact dermatitis is preceded by a clinically silent phase of sensitisation. In this study, we investigated whether the expression levels of six genes were related to nickel exposure and/or nickel sensitisation, and whether they could predict allergic manifestations to nickel. The mRNA expression level of six genes involved in cell growth (PIM1 and ETS2), metabolism/synthesis (HSD11B1 and PRDX4), apoptosis (CASP8) and signal transduction (CISH) was investigated by means of quantitative real-time RT-PCR in a cohort of 110 subjects, including healthy controls (n=51), nickel-exposed workers (n=23) and patients allergic to nickel (n=36). Our findings show that the expression levels of the analysed genes did not differ between allergic patients and healthy controls, while higher expression levels of ETS2 and CASP8 were detected in the nickel-exposed workers. Changes in ETS2 and CASP8 expression are likely to be related to nickel exposure rather than to allergy.

Evidence of multiple infectious agents in mycosis fungoides lesions.

The etiology of mycosis fungoides (MF) remains to be determined. Several studies have proposed a viral etiology with controversial results. In this case-control study we investigated the presence of Epstein-Barr virus (EBV) and the debated presence of Human T-cell lymphotrophic virus I (HTLV-I) sequences, by polymerase chain reaction on nucleic acid extracts from formalin-fixed paraffin-embedded skin biopsies. Moreover, by a multivariate approach we analyzed in the same case-control study also the contribution of two previously examined pathogens: Hepatitis C virus (HCV) and Borrelia burgdorferi (Bb). Significant differences in the frequency of infectious agents in cases and controls were detected for Bb, HTLV-I and EBV. In MF patients we found the concurrent presence of two or three of these pathogen sequences in 21 out of 83 cases, but only in 1 out of 83 healthy controls. Our results suggest that the persistence of multiple infectious agents may cause a long-term antigenic stimulation contributing to the malignant transformation of T lymphocytes, especially when associated with HTLV-I like sequences. However, these infectious agents do not seem to have effects on disease progression.