Benchmarking and improving the performance of variant-calling pipelines with RecallME.

MOTIVATION: The steady increment of Whole Genome/Exome sequencing and the development of novel Next Generation Sequencing-based gene panels requires continuous testing and validation of variant calling (VC) pipelines and the detection of sequencing-related issues to be maintained up-to-date and feasible for the clinical settings. State of the art tools are reliable when used to compute standard performance metrics. However, the need for an automated software to discriminate between bioinformatic and sequencing issues and to optimize VC parameters remains unmet. RESULTS: The aim of the current work is to present RecallME, a bioinformatic suite that tracks down difficult-to-detect variants as insertions and deletions in highly repetitive regions, thus providing the maximum reachable recall for both single nucleotide variants and small insertion and deletions and to precisely guide the user in the pipeline optimization process. AVAILABILITY AND IMPLEMENTATION: Source code is freely available under MIT license at https://github.com/mazzalab-ieo/recallme. RecallME web application is available at https://translational-oncology-lab.shinyapps.io/recallme/. To use RecallME, users must obtain a license for ANNOVAR by themselves.

Circulating extracellular vesicles expressing PD1 and PD-L1 predict response and mediate resistance to checkpoint inhibitors immunotherapy in metastatic melanoma.

BACKGROUND: The immunotherapy with immune checkpoints inhibitors (ICI) has changed the life expectancy in metastatic melanoma (MM) patients. Nevertheless, several patients do not respond hence, the identification and validation of novel biomarkers of response to ICI is of crucial importance. Circulating extracellular vesicles (EVs) such as PD-L1+ EV mediate resistance to anti-PD1, instead the role of PD1+ EV is not fully understood. METHODS: We isolated the circulating EVs from the plasma of an observational cohort study of 71 metastatic melanoma patients and correlated the amount of PD-L1+ EVs and PD1+ EVs with the response to ICI. The analysis was performed according to the origin of EVs from the tumor and the immune cells. Subsequently, we analysed the data in a validation cohort of 22 MM patients to assess the reliability of identified EV-based biomarkers. Additionally we assessed the involvement of PD1+ EVs in the seizure of nivolumab and in the perturbation of immune cells-mediated killing of melanoma spheroids. RESULTS: The level of PD-L1+ EVs released from melanoma and CD8+ T cells and that of PD1+ EVs irrespective of the cellular origin were higher in non-responders. The Kaplan-Meier curves indicated that higher levels of PD1+ EVs were significantly correlated with poorer progression-free survival (PFS) and overall survival (OS). Significant correlations were found for PD-L1+ EVs only when released from melanoma and T cells. The multivariate analysis showed that high level of PD1+ EVs, from T cells and B cells, and high level of PD-L1+ EVs from melanoma cells, are independent biomarkers of response. The reliability of PD-L1+ EVs from melanoma and PD1+ EVs from T cells in predicting PFS was confirmed in the validation cohort through the univariate Cox-hazard regression analysis. Moreover we discovered that the circulating EVs captured nivolumab and reduced the T cells trafficking and tumor spheroids killing. CONCLUSION: Our study identified circulating PD1+ EVs as driver of resistance to anti-PD1, and highlighted that the analysis of single EV population by liquid biopsy is a promising tool to stratify MM patients for immunotherapy.

NR1H3 (LXRα) is associated with pro-inflammatory macrophages, predicts survival and suggests potential therapeutic rationales in diffuse large b-cell lymphoma.

The role of macrophages (Mo) and their prognostic impact in diffuse large B-cell lymphomas (DLBCL) remain controversial. By regulating the lipid metabolism, Liver-X-Receptors (LXRs) control Mo polarization/inflammatory response, and their pharmacological modulation is under clinical investigation to treat human cancers, including lymphomas. Herein, we surveyed the role of LXRs in DLBCL for prognostic purposes. Comparing bulk tumors with purified malignant and normal B-cells, we found an intriguing association of NR1H3, encoding for the LXR-α isoform, with the tumor microenvironment (TME). CIBERSORTx-based purification on large DLBCL datasets revealed a high expression of the receptor transcript in M1-like pro-inflammatory Mo. By determining an expression cut-off of NR1H3, we used digital measurement to validate its prognostic capacity on two large independent on-trial and real-world cohorts. Independently of classical prognosticators, NR1H3high patients displayed longer survival compared with NR1H3low cases and a high-resolution Mo GEP dissection suggested a remarkable transcriptional divergence between subgroups. Overall, our findings indicate NR1H3 as a Mo-related biomarker identifying patients at higher risk and prompt future preclinical studies investigating its mouldability for therapeutic purposes.

Halting the vicious cycle within the multiple myeloma ecosystem: blocking JAM-A on bone marrow endothelial cells restores angiogenic homeostasis and suppresses tumor progression.

Interactions of malignant multiple myeloma (MM) plasma cells (MM-cells) with the microenvironment control MM-cell growth, survival, drug-resistance and dissemination. As in MM microvascular density increases in the bone marrow (BM), we investigated whether BM MM endothelial cells (MMECs) control disease progression via the junctional adhesion molecule A (JAM-A). Membrane and cytoplasmic JAM-A levels were upregulated in MMECs in 111 newly diagnosed (NDMM) and 201 relapsed-refractory (RRMM) patients compared to monoclonal gammopathy of undetermined significance (MGUS) and healthy controls. Elevated membrane expression of JAM-A on MMECs predicted poor clinical outcome. Mechanistically, addition of recombinant JAM-A to MMECs increased angiogenesis whereas its inhibition impaired angiogenesis and MM growth in 2D and 3D in vitro cell culture and chorioallantoic membrane-assays. To corroborate these findings, we treated MM bearing mice with JAM-A blocking mAb and demonstrated impaired MM progression corresponding to decreased MM-related vascularity. These findings support JAM-A as an important mediator of MM progression through facilitating MM-associated angiogenesis. Collectively, elevated JAM-A expression on bone marrow endothelial cells is an independent prognostic factor for patient survival in both NDMM and RRMM. Blocking JAM-A restricts angiogenesis in vitro, in embrio and in vivo and represents a suitable druggable molecule to halt neoangiogenesis and MM progression.

Long Non-Coding RNA Landscape in Prostate Cancer Molecular Subtypes: A Feature Selection Approach.

Prostate cancer is one of the most common malignancies in men. It is characterized by a high molecular genomic heterogeneity and, thus, molecular subtypes, that, to date, have not been used in clinical practice. In the present paper, we aimed to better stratify prostate cancer patients through the selection of robust long non-coding RNAs. To fulfill the purpose of the study, a bioinformatic approach focused on feature selection applied to a TCGA dataset was used. In such a way, LINC00668 and long non-coding(lnc)-SAYSD1-1, able to discriminate ERG/not-ERG subtypes, were demonstrated to be positive prognostic biomarkers in ERG-positive patients. Furthermore, we performed a comparison between mutated prostate cancer, identified as "classified", and a group of patients with no peculiar genomic alteration, named "not-classified". Moreover, LINC00920 lncRNA overexpression has been linked to a better outcome of the hormone regimen. Through the feature selection approach, it was found that the overexpression of lnc-ZMAT3-3 is related to low-grade patients, and three lncRNAs: lnc-SNX10-87, lnc-AP1S2-2, and ADPGK-AS1 showed, through a co-expression analysis, significant correlation values with potentially druggable pathways. In conclusion, the data mining of publicly available data and robust bioinformatic analyses are able to explore the unknown biology of malignancies.

A Promising Role of TGF-ß Pathway in Response to Regorafenib in Metastatic Colorectal Cancer: A Case Report.

Colorectal cancer (CRC) is one of the most common cancer types around the world. The prognosis of patients with advanced diseases is still poor in spite of currently available therapeutic options. Regorafenib is an oral tyrosine kinase inhibitor (TKI) approved to treat refractory metastatic colorectal cancer (mCRC). We investigated Somatic mutations in several genes involved in immunological response and cancer progression in both long/short responder mCRC patients who underwent third-line therapy with regorafenib to identify predictive biomarkers of response using Ion Torrent PGM sequencing and bioinformatic tools. We found Somatic mutations in TGFBR1, TGFBR2, and TGFBR3 genes in primary tumor and metastases samples of long-responder patients. Furthermore, our bioinformatic results show that they were mainly enriched in immune response, cell junction, and cell adhesion in long responder patients, particularly in primary tumor and metastatic sites. These data suggest that the TGF-b pattern could be the leading actor of a prolonged response to this drug.

Rapid Serological Assays and SARS-CoV-2 Real-Time Polymerase Chain Reaction Assays for the Detection of SARS-CoV-2: Comparative Study.

BACKGROUND: Real-time polymerase chain reaction (RT-PCR) testing for the identification of viral nucleic acid is the current standard for the diagnosis of SARS-CoV-2 infection, but technical issues limit its utilization for large-scale screening. Serological immunoglobulin M (IgM)/IgG testing has been proposed as a useful tool for detecting SARS-CoV-2 exposure. OBJECTIVE: The objective of our study was to compare the results of the rapid serological VivaDiag test for SARS-CoV-2-related IgM/IgG detection with those of the standard RT-PCR laboratory test for identifying SARS-CoV-2 nucleic acid. METHODS: We simultaneously performed both serological and molecular tests with a consecutive series of 191 symptomatic patients. The results provided by a new rapid serological colorimetric test for analyzing IgM/IgG expression were compared with those of RT-PCR testing for SARS-CoV-2 detection. RESULTS: Of the 191 subjects, 70 (36.6%) tested positive for SARS-CoV-2 based on RT-PCR results, while 34 (17.3%) tested positive based on serological IgM/IgG expression. Additionally, 13 (6.8%) subjects tested positive based on serological test results, but also tested negative based on RT-PCR results. The rapid serological test had a sensitivity of 30% and a specificity of 89% compared to the standard RT-PCR assay. Interestingly, the performance of both assays improved 8 days after symptom appearance. After 10 days had passed since symptom appearance, the predictive value of the rapid serological test was higher than that of the standard molecular assay (proportion of positive results: 40% vs 20%). Multivariate analysis showed that age >58 years (P<.01) and period of >15 days after symptom onset (P<.02) were significant and independent factors associated with serological test positivity. CONCLUSIONS: The rapid serological test analyzed in this study seems limited in terms of usefulness when diagnosing SARS-CoV-2 infection. However, it may be useful for providing relevant information on people's immunoreaction to COVID-19 exposure.

Standardization of CTC AR-V7 PCR assay and evaluation of its role in castration resistant prostate cancer progression.

BACKGROUND: Castration resistant prostate cancer (CRPC) represents the most aggressive status of this neoplastic disease, also characterized by the absence of biomarkers predictive of clinical outcome. New drugs as abiraterone or enzalutamide, affecting androgen receptor pathway at different levels, inhibit the proliferative advantage of prostate cancer cells with important long term benefits. Despite the advantages of this second-generation androgen deprivation therapy (ADT), resistance mechanisms, primitive or acquired, often develop. The existence of androgen receptor (AR) splice variants (AR-Vs), in particular AR-V7 expression detected in circulating tumor cells (CTCs), represents an example of acquired resistance, as evidenced in preclinical and clinical studies. Recent studies also have suggested the role of AR-V7 as a prognostic biomarker in mCRPC. In this field, hot topics are the methodology used to isolate CTC and the assay for AR-V7 measurement. Our study aims to develop a standardized operating procedure (SOP) to evaluate AR-V7 in CRPC. METHOD: The application of a realized cell based Reference Sample as Standardized Quality Control tool for CTC-AR-V7 assay has been shown. Then the development, the performance evaluation and contextualization in a clinical setting of this standardized operating procedure (SOP) have been reported to evaluate the prognostic biomarker AR-V7 in metastatic prostate cancer. RESULTS AND CONCLUSIONS: The standardized procedure has high sensitivity and specificity and enables the detection and quantification of the spliced variant with respect to the full length AR (AR-FL) mRNA in CTC DNA purified from the blood of patients with CRPC. This procedure has been further validated in a consecutive series of patients with mCRPC, confirming its role as prognostic biomarker.

A Comparative Assessment of Quality of Life in Patients with Multiple Myeloma Undergoing Autologous Stem Cell Transplantation Through an Outpatient and Inpatient Model.

Outpatient autologous stem cell transplantation (ASCT) has proven to be feasible in terms of physical morbidity and mortality outcomes, but little data exist on the impact of this procedure on quality of life (QoL). The purpose of this prospective, observational, longitudinal cohort study was to compare the effects of inpatient (n = 76) and outpatient (n = 64) modes of care on QoL in patients with multiple myeloma who underwent ASCT. Patients were treated according to their preference for the inpatient or outpatient model. QoL was assessed using the Functional Assessment of Cancer Therapy-Bone Marrow Transplantation (FACT-BMT) at baseline (7 days before ASCT; T1) and at days +7 (T2) and +30 (T3) after ASCT. Overall, inpatients achieved higher mean values at each time point (86.05 ± 15.54 at T1, 89.23 ± 19.19 at T2, and 87.96 ± 13.6 at T3) compared with outpatients (85.62 ± 14.51 at T1, 87.42 ± 23.41 at T2, and 83.98 ± 20.2 at T3), although the differences did not reach statistical significance. Inpatients showed higher mean scores than outpatients in physical well-being (7.67 ± 5.7, 15.44 ± 6.34, and 12.96 ± 6.03, respectively, versus 5.89 ± 4.33, 13.92 ± 7.05, and 8.84 ± 6.33, respectively; P < .05). Mean scores on social/family well-being were significantly higher in the outpatient group compared with the inpatient group (22.93 ± 13.29, 21.14 ± 5.31, and 21.64 ± 4.58, respectively, versus 20.59 ± 3.79, 19.52 ± 5.12, and 20.01 ± 3.97, respectively; P = .003). There were no significant between-group differences with respect to functional well-being and emotional status. Among adults at a single institution undergoing ASCT for MM, the use of outpatient care compared with standard transplantation care did not result in improved QoL during transplantation. Further research is needed for replication and to assess longer-term outcomes and implications.

The search for a melanoma-tailored chemotherapy in the new era of personalized therapy: a phase II study of chemo-modulating temozolomide followed by fotemustine and a cooperative study of GOIM (Gruppo Oncologico Italia Meridionale).

BACKGROUND: It is frequently asked whether chemotherapy can still play a role in metastatic melanoma considering the effectiveness of the available drugs today, including antiCTLA4/antiPD1 immunotherapy and antiBRAF/antiMEK inhibitors. However, only approximately half of patients respond to these drugs, and the majority progress after 6-11 months. Therefore, a need for other therapeutic options is still very much apparent. We report the first large trial of a sequential full dose of fotemustine (FM) preceded by a low dose of temozolomide (TMZ) as a chemo-modulator in order to inactivate the DNA repair action of O(6)-methylguanine DNA-methyltransferase (MGMT). Primary endpoints were overall response and safety. We also evaluated specific biological parameters aiming to tailor these chemotherapies to selected patients. METHODS: A total of 69 consecutive patients were enrolled. The main features included a median age of 60 years (21-81) and M1c stage, observed in 74% of the patients, with brain metastases in 15% and high LDH levels in 42% of the patients. The following schedule was used: oral TMZ 100 mg/m2 on days 1 and 2 and FM iv 100 mg/m2 on day 2, 4 h after TMZ; A translational study aiming to analyse MGMT methylation status and base-excision repair (BER) gene expression was performed in a subset of 14 patients. RESULTS: We reported an overall response rate of 30.3% with 3 complete responses and a disease control rate of 50.5%. The related toxicity rate was low and mainly of haematological types. Although our population had a very poor prognosis, we observed a PFS of 6 months and an OS of 10 months. A non-significant correlation with response was found with the mean expression level of the three genes involved in the BER pathway (APE1, XRCC1 and PARP1), whereas no association was found with MGMT methylation status. CONCLUSION: This schedule could represent a good alternative for patients who are not eligible for immune or targeted therapy or whose previous therapies have failed. TRIAL REGISTRATION: EUDRACT 2009-016487-36l ; date of registration 23 June 2010.

GATK hard filtering: tunable parameters to improve variant calling for next generation sequencing targeted gene panel data.

BACKGROUND: NGS technology represents a powerful alternative to the standard Sanger sequencing in the context of clinical setting. The proprietary software that are generally used for variant calling often depend on preset parameters that may not fit in a satisfactory manner for different genes. GATK, which is widely used in the academic world, is rich in parameters for variant calling. However the self-adjusting parameter calibration of GATK requires data from a large number of exomes. When these are not available, which is the standard condition of a diagnostic laboratory, the parameters must be set by the operator (hard filtering). The aim of the present paper was to set up a procedure to assess the best parameters to be used in the hard filtering of GATK. This was pursued by using classification trees on true and false variants from simulated sequences of a real dataset data. RESULTS: We simulated two datasets, with different coverages, including all the sequence alterations identified in a real dataset according to their observed frequencies. Simulated sequences were aligned with standard protocols and then regression trees were built up to identify the most reliable parameters and cutoff values to discriminate true and false variant calls. Moreover, we analyzed flanking sequences of region presenting a high rate of false positive calls observing that such sequences present a low complexity make up. CONCLUSIONS: Our results showed that GATK hard filtering parameter values can be tailored through a simulation study based-on the DNA region of interest to ameliorate the accuracy of the variant calling.

VEGF and TWIST1 in a 16-biomarker immunoprofile useful for prognosis of breast cancer patients.

This study concerns the expression of biomarkers involved in diverse pathways, such as progression, DNA repair mechanisms and angiogenesis to establish an immunoprofile capable of characterizing sporadic versus familial breast cancers (BCs). The aim was to identify a patient subgroup with a different clinical outcome, which could then be directed towards new targeted therapies. Hierarchical cluster analysis (HCA) was carried out using the immunohistochemical score from tissue microarray sections of an initial cohort of 183 (88 sporadic and 95 familial) patients with invasive BC. For the survival analysis, only those patients with complete follow-up were considered. The HCA revealed a 16-protein immunoprofile, nine of which represent the core, as was also found when familial and sporadic BCs were analysed individually. The 16-biomarker immunoprofile was able to identify a group of patients (Group 1) with a more aggressive tumour phenotype. Survival analyses showed that VEGF+ /TWIST1- patients with familial BC of Group 1 tended to demonstrate a lower DFS than the VEGF- /TWIST1+ sporadic BC patients of Group 2 (p = 0.052). Moreover, the entire cohort of VEGF+ /TWIST1- patients showed a statistically worse DFS than the patients with VEGF- /TWIST1+ expression (p = 0.034). In conclusion, we found that tumour stratification based on an immunoprofile is useful to predict the patient clinical behaviour. In particular, our study indicates that the clustering of tumors on the basis of this immunoprofile suggests the possibility to differentiate familial from sporadic BCs and to clinically select those patients who are more likely to benefit from inhibition of the VEGF pathway.

BRCA1-2 diagnostic workflow from next-generation sequencing technologies to variant identification and final report.

The BRCA1-BRCA2 genes predispose to hereditary breast and ovarian cancer, and the germline and mutational status of these genes defines a target population that can benefit from PARP inhibitor treatments. To respond to the increasing number of BRCA1-BRCA2 tests, it is necessary to shift to high-throughput technologies that are reliable and less time consuming. Different methodological platforms are dedicated to this purpose with different approaches and algorithms for analysis. Our aim was to set up a cost-effective and low time-consuming BRCA1-BRCA2 mutation detection workflow using the Ion Torrent PGM technology. A retrospective cohort of 40 patients with familial breast/ovarian cancer previously tested by Sanger sequencing and a prospective cohort of 72 patients (validation set) were analyzed. The validation set included 64 patients affected by familial breast/ovarian cancer and eight sporadic ovarian cancer cases, who are potential candidates for PARPi treatments. A complete and standardized workflow easily usable and suitable in a certified laboratory has been proved and validated. This includes all steps from library preparation to the final report. The use of next-generation sequencing will be of benefit for patients enrolled in the genetic counseling process and, moreover, will enhance the process of selecting patients eligible for personalized treatments. © 2016 Wiley Periodicals, Inc.

Nuclear PARP1 expression and its prognostic significance in breast cancer patients.

Poly(adenosine diphosphate [ADP]-ribose) polymerase 1 (PARP1) plays important roles in DNA damage response pathways and is often overexpressed in various human tumors. Currently, the use of PARP inhibitors for breast cancer (BC) therapy is the subject of debate, and there is an urgent need to understand much the expression and prognostic role of the PARP1 protein. The aim was to investigate the clinicopathological and prognostic significance of PARP1 in BC patients. The PARP1 and breast cancer susceptibility gene 1 (BRCA1) protein expressions were evaluated in 114 BCs by immunohistochemistry. Disease-free survival (DFS) and overall survival (OS) were determined based on the Kaplan-Meier method. Our results showed that nuclear PARP1 expression was significantly associated with peritumoral vascular invasion (P = 0.046), chemotherapeutic treatment (P = 0.026), oestrogen receptor (ER; P = 0.013), human epidermal growth factor receptor 2 (HER2; P = 0.003) and BRCA1 (P < 0.001) expression. Survival analyses showed a significant association with clinical outcome in the subgroup of ER-negative patients (P = 0.017 for DFS and P = 0.048 for OS) and in the subgroup of patients treated with chemotherapeutic agents (P = 0.042 for DFS and P = 0.046 for OS). A significant correlation was also found for DFS in patients characterized by tumors without peritumoral vascular invasion (P = 0.022). More importantly, multivariate analyses revealed that high nuclear PARP1 expression was associated with decreased DFS (P = 0.012) and OS (P = 0.026). In conclusion, PARP1 expression may be used as an independent prognostic factor in BC patients. In addition, this study demonstrated that high PARP1 expression may represent a marker of poorer prognosis both for patients with worse clinical outcome and in less aggressive clinical conditions.

Combined microRNA and ER expression: a new classifier for familial and sporadic breast cancer patients.

BACKGROUND: The role of miRNAs in familial breast cancer (fBC) is poorly investigated as also in the BRCA-like tumors. To identify a specific miRNA expression pattern which could allow a better fBC classification not only based on clinico-pathological and immunophenotypical parameters we analyzed miRNA profile in familial and sporadic samples. Moreover since BRCA1 tumors and sporadic triple negative (TN) breast tumors share similarities regarding clinical outcomes and some histological characteristics, we focused on TN and not TN cases. METHODS: The sample set included fresh frozen tissue samples, including 39 female fBCs (19 BRCA-related and 20 BRCAX) and 12 male fBC (BRCAX). Moreover, we considered TN and non TN (NTN), 21 BRCA-related and 27 sporadic BCs. MiRNA profiling was performed through GeneChip miRNA v.1.0 Array (Affymetrix). ANOVA, hierarchical and consensus clustering analyses allowed identification of pattern of expression of miRNAs and pathway enrichment analysis, considering validated target genes, was carried out to achieve a deeper biological understanding. RESULTS: ANOVA test led to the identification of 53 deregulated miRNAs; hierarchical and consensus clustering of female fBCs (fFBCs) and male fBCs (fMBCs) highlighted the presence of 3 sample clusters named FBC1, FBC2 and FBC3. We found a correlation between ER-status and the three sample clusters. The three clusters are distinct by a different expression of two clusters of miRNAs (CLU1 and CLU2), which resulted to be different in targeted pathways. In particular, CLU1 targets cellular pathways and CLU2 is involved in epigenetic activities. Considering TN and NTN BRCA-related and sporadic tumors, a hierarchical clustering identified two clusters of miRNAs, which were not so different from CLU1 and CLU2, both in miRNA content and targeted pathways. CONCLUSIONS: Our results highlighted the importance of miRNA regulation to better clarify similarities and differences between familial and sporadic BC groups.

Genetic risk transmission in a family affected by familial breast cancer.

Breast Cancer is the most common malignancy among women. Family history is the strongest single predictor of breast cancer risk, and thus great attention has been focused on BRCA1 and BRCA2 genes whose mutations lead to a high risk of developing this disease. Today, only 25% of high- and moderate-risk genes are known, suggesting the importance of the discovery of new risk modifiers. Therefore, the investigation of new polygenic alterations is of great importance, especially if considered high- and moderate-risk variants. In this study, the transmission of BRCA1-2 polymorphisms in association with the transmission of polymorphisms in the genes NUMA1, CCND1, COX11, FGFR2, TNRC9 and SLC4A7 were examined in all members of a family with the BRCA2 c.6447_6448dup mutation. This is the first study about the transmission of high-risk polygenic variants in all members of a family with a strong history of breast cancer. The results about the possible polygenic variant associations that could increase and modify the risk suggested the importance to search new variants to better manage patients and their family members.

HOX gene methylation status analysis in patients with hereditary breast cancer.

Cancer development is related not only to genetic alterations but also to aberrant epigenetic changes that could lead to heritable gene patterns critical for neoplastic initiation and progression. Knowledge of epigenetic regulation in cancer cells is useful for both the understanding of carcinogenesis and for the possibility of using epigenetic drugs. HOX genes deregulation have a crucial role in oncogenesis process and tumor suppression. In this report, the methylation of HOXA1, HOXA9, HOXA10, HOXB13, HNF1B, OTX1, TLX1 genes have been analyzed in patients with hereditary breast cancer. This is the first study analyzing BRCA mutational status of patients with respect to methylation of HOX genes. HOXA10 has been found to be methylated in all patients analyzed but never in healthy subjects. With respect to clinical pathological information, hypermethylation of all studied genes, with the exception of OTX1, was significantly associated with absence of HER2 neu expression (P<0.05). Moreover, hypermethylation of HOXB13, HOXA10 and HOXA1 was associated with a high proliferation index (Mib1≥10%, P<0.05) and hypermethylation of HOXB13 and HOXA10 also with high expression of estrogen and progesterone receptors. These preliminary data suggest a possible involvement of HOX genes in familial breast cancer as marker helpful to identify high-risk patients.

Remote vs in-person BRCA1/2 non-carriers test disclosure: patients' choice during Covid-19 pandemic restriction.

During Covid-19 pandemic most hospitals have restricted in-person delivery of non-essential healthcare services, including genetic testing delivery, to slow the spread of the virus. Our Onco-Genetic Service also faced this challenging period and had to re-organize its clinical practice with the use of tele-health. Aim of the present paper is to understand whether and how Covid-19-related changes in medical practice influenced patients' satisfaction about the health service provided. 125 BRCA1/2 non carriers (109/125, 87.2% female and 16/125, 12.8% male) in Istituto Tumori "Giovanni Paolo II" of Bari were enrolled. All participants were asked to choose whether they prefer in-person or remote post-test counselling session. Basing on patients' choice, two groups of subjects were composed. One week after the post-test counselling session, participants were phone called and asked to complete: a socio-demographic form, a brief structured interview about their Covid-19 related worries and their satisfaction with the health service provided, Hospital Anxiety and Depression Scale and Fear of Covid-19 scale. Qualitative information about patients' choice were also collected. No significant difference about patients' satisfaction with the health service provided emerged between groups. Patients who preferred remote post-test counselling had higher anxiety, worries and fear-of Covid-19 than the others. All remote-counselling subjects preferred tele-genetics because of Covid-19 security, would choose it again and would recommend it to others. Cancer tele-genetics offers good guarantees of comfort and efficacy, but patients' choices are related to personal and psychological variables. The use of tele-genetics has to be a patient's choice.

Tumor Cellularity Assessment of Breast Histopathological Slides via Instance Segmentation and Pathomic Features Explainability.

The segmentation and classification of cell nuclei are pivotal steps in the pipelines for the analysis of bioimages. Deep learning (DL) approaches are leading the digital pathology field in the context of nuclei detection and classification. Nevertheless, the features that are exploited by DL models to make their predictions are difficult to interpret, hindering the deployment of such methods in clinical practice. On the other hand, pathomic features can be linked to an easier description of the characteristics exploited by the classifiers for making the final predictions. Thus, in this work, we developed an explainable computer-aided diagnosis (CAD) system that can be used to support pathologists in the evaluation of tumor cellularity in breast histopathological slides. In particular, we compared an end-to-end DL approach that exploits the Mask R-CNN instance segmentation architecture with a two steps pipeline, where the features are extracted while considering the morphological and textural characteristics of the cell nuclei. Classifiers that are based on support vector machines and artificial neural networks are trained on top of these features in order to discriminate between tumor and non-tumor nuclei. Afterwards, the SHAP (Shapley additive explanations) explainable artificial intelligence technique was employed to perform a feature importance analysis, which led to an understanding of the features processed by the machine learning models for making their decisions. An expert pathologist validated the employed feature set, corroborating the clinical usability of the model. Even though the models resulting from the two-stage pipeline are slightly less accurate than those of the end-to-end approach, the interpretability of their features is clearer and may help build trust for pathologists to adopt artificial intelligence-based CAD systems in their clinical workflow. To further show the validity of the proposed approach, it has been tested on an external validation dataset, which was collected from IRCCS Istituto Tumori "Giovanni Paolo II" and made publicly available to ease research concerning the quantification of tumor cellularity.