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Simple Summary Circulating tumor cells (CTCs) are rare cells found in the bloodstream of oncologic patients with a central role in the metastatic spread. In this study, we aim at exploring their heterogeneity levels in metastatic breast cancer patients focusing on single cell single nucleotide variant (SNV) and copy number aberration (CNA) analyses. Our results show high levels of heterogeneity, especially concerning SNVs. Further analysis revealed the presence of CNAs associated with breast tumorigenesis, while longitudinal CNA profiling was demonstrated to track clonal selection of CTCs during treatment. Despite this heterogeneity, we found a group of CTCs from different patients sharing common genomic aberrations, such as losses on 15q. Collectively, our findings demonstrate that single-cell molecular analyses could be exploited in future to better address therapeutic strategies. Further investigations to better characterize this mixed population are needed to understand its role in MBC. Circulating tumor cells' (CTCs) heterogeneity contributes to counteract their introduction in clinical practice. Through single-cell sequencing we aim at exploring CTC heterogeneity in metastatic breast cancer (MBC) patients. Single CTCs were isolated using DEPArray NxT. After whole genome amplification, libraries were prepared for copy number aberration (CNA) and single nucleotide variant (SNV) analysis and sequenced using Ion GeneStudio S5 and Illumina MiSeq, respectively. CTCs demonstrate distinctive mutational signatures but retain molecular traces of their common origin. CNA profiling identifies frequent aberrations involving critical genes in pathogenesis: gains of 1q (CCND1) and 11q (WNT3A), loss of 22q (CHEK2). The longitudinal single-CTC analysis allows tracking of clonal selection and the emergence of resistance-associated aberrations, such as gain of a region in 12q (CDK4). A group composed of CTCs from different patients sharing common traits emerges. Further analyses identify losses of 15q and enrichment of terms associated with pseudopodium formation as frequent and exclusive events. CTCs from MBC patients are heterogeneous, especially concerning their mutational status. The single-cell analysis allows the identification of aberrations associated with resistance, and is a candidate tool to better address treatment strategy. The translational significance of the group populated by similar CTCs should be elucidated.

Background: Fusions (Fs) are major molecular biological abnormalities in acute leukemias (ALs), and all well-known Fs in leukemias are founder variations are routinely used as molecular markers for the diagnosis, classification, risk stratification, and targeted therapy but there is a considerable part of ALs that are not screened for other known/unknown transcripts. For example in B-Other B Acute Lymphoblastic Leukemia (B-ALL) [Ph-/-/-; negative for t(9;22); t(1;19); t(4;11); 61% of adult B-ALL], many chimeric genes have been identified leading to a refined classification of B-ALL and to, in some cases, tailored therapies. At this point, a RNA-seq approach is needed but challenging for many aspects, among them a not standardized and very heterogeneous F data analysis. Aims: We developed and validated our integrated pipeline in order to assess targetable biomarkers and to better classify patients (pts). Methods: we performed 1385 gene RNAseq (Illumina) of 224 adult AL samples (112 Ph-/-/-, 41 Ph+, 6 t(1;19), 16 T-LBL/T-ALL and 49 AML). Starting from Ph-/-/-, we developed a combined 4 tool analysis that is further implemented with a filtering strategy with a specific ALs fusion literature filter (Patent PCTEP2021-065692 and 749 ALL Fs database copyright) (Fig.1A). We validate our strategy using: RT-PCR, FISH SNP Arrays; MLPA and total RNA-seq. Results: From 3022 candidate Fs, we retained 160 of them (5.3%; excluding WHO canonical Fs) not otherwise detected, in 120 pts with a high Fs rate in T-LBL/T-ALL, Ph-/-/- and Ph+ (62.5%, 62.2% and 61% respectively) denoting that ALs are not deeply characterized (Fig 1B). The lower rate was found in AML (26.5%), but we were able to identify new ETV6 rearrangement (r) and a FISH cryptic F in a 46, XY pt (TBL1XR1-MECOM). We validated 98 Fs that have been already reported in the literature and 43 novel F transcripts. We obtained a validation rate of 81%. The majority of fused samples (65.2%) had only one detectable F while a smaller group was characterized by multiple Fs. Many of these Fs were previously described in B/T-ALL and AML (e.g. KMT2A-MLLT1, ABL1/2-RCSD1, IGH-MYC, NUP214-SET, ETV6-MECOM). In our bigger sub-cohort (Ph-/-/-), 44 Fs out of 109 (40.3%) were never been reported in Ph- ALL cases. In 15 Ph-like pts, we identified and validate 11 new transcripts. Ph-/-/- F detection help to sub-classify our fused pts in Ph-/-/- subgroups (ZNF384r-11.8%; Ph-like-19.1%; DUX4r- 4.4%; HLFr, MLLr and BCL2/MYC in 2.9%; MEF2Dr-1.5%). Conclusions: we identified pivotal transcripts in all ALs and an unexpected high rate of secondary Fs in adult ALs subgroups (52.4%) that are not characterized with conventional diagnostic methods. The use of an NGS approach and a powerful pipeline permit us to detect Fs useful for a better classification, prognostic identification (e.g. TBL1XR1-MECOM) and in some cases to find targetable Fs (e.g. ABL1-2/RCSD1, NUMA1-CSF1R, ZMYM2-FLT3). Supported by: L3P2505. Citation Format: Anna Ferrari, Silvia Vitali, Eugenio Fonzi, Andrea Ghelli Luserna Di Rora, Chiara Domizio, Cristina Papayannidis, Maria Teresa Bochicchio, Giovanni Marconi, Daniele Dall'Olio, Michela Tebaldi, Michela Rondoni, Barbara Giannini, Fabio Giglio, Crescenza Pasciolla, Monica Fumagalli, Sara Galimberti, Gastone Castellani, Daniel Remondini, Giorgia Simonetti, Giovanni Martinelli. Fusion landscape in acute leukemias: A submerged world of not routinely characterized transcripts [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 6163.

Background: Neuroendocrine neoplasms (NENs) are a subgroup of poorly characterized rare tumors with challenging management. Identification of clinically useful biomarkers for patient stratification and treatment tailoring is urgently needed. This study proposes the use of near-patient models based on autologous extracellular matrix (aECM)-scaffolds and zebrafish (ZF) xenografts integrated with next generation multi-omics analysis for the phenotype and genotype mapping of NEN cells. The aim is to uncover molecular signatures associated with disease aggressiveness and to detect oncogenic drivers for targeted treatment. Methods: Primary tumor cells isolated from surgical specimens of NEN patients were cultured in aECM scaffolds or injected into ZF embryos. Autologous ECM-scaffolds were synthesized starting from the patients’ matrices and recellularized with primary cells derived from matched patients. In aECM scaffolds we evaluated tumor cell proliferation and neuroendocrine differentiation. In ZF we evaluated cell angiogenic ability and distant invasiveness. For each primary culture, we performed exome and transcriptome analysis of the original tumor tissue compared to the matched healthy counterpart. Results: We demonstrated that ZF xenografting faithfully recreates the behavior that tumor cells exhibit in patients. Specifically, we derived a primary culture from an ileal grade 1 neuroendocrine tumor from both the primary and the metastatic lesions. When injected into ZF embryos, cells derived from the primary tumor displayed low invasive ability with a metastatic rate of only 23%. Conversely, cells derived from the metastatic lesion were able to invade the ZF circulation and form distant metastases in 79% of embryos. Consecutively, we derived three primary cultures of neuroendocrine Merkel cell carcinoma (MCC). The 3 MCC cultures into ZF embryos showed high aggressiveness with metastatic rates of 81%, 50% and 64%. For one culture, angiogenic ability was also observed. RNA-sequencing of these aggressive MCC specimens highlighted the involvement of the following pathways: ECM remodeling, TNF-ⲁ and PI3K-Akt signaling, epithelial-mesenchymal transition and regulation of apoptotic processes. Next, we demonstrated that aECM-scaffolds morphologically resemble a native tumor tissue, and tumor cells, once seeded on them, preserved the expression of neuroendocrine markers. Conclusions: Here we developed innovative neuroendocrine tumor models that are expected to generate new knowledge on the disease molecular determinants. All identified genomic and genetic alterations will be correlated with the clinical outcomes of enrolled patients, to validate their clinical significance and to ultimately introduce a biomarker-based approach for NEN patients. Citation Format: Chiara Calabrese, Giacomo Miserocchi, Silvia Vanni, Alessandro De Vita, Alberto Bongiovanni, Chiara Spadazzi, Claudia Cocchi, Nicoletta Ranallo, Federica Pieri, Michela Tebaldi, Flavia Foca, Giorgio Ercolani, Davide Cavaliere, Carlo Alberto Pacilio, Giovanni Martinelli, Laura Mercatali, Chiara Liverani. Autologous ECM-composed scaffolds and zebrafish PDXs for individualized theranostics in rare neuroendocrine neoplasms [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3076.

PVs and LPVs in BRCA1/2 genes are correlated to a high risk of developing breast cancer and/or ovarian cancer (Hereditary Breast and Ovarian Cancer syndrome, HBOC); additionally, in recent years, an increasing number of BRCA 1/2 variants have been identified and associated with pancreatic cancer. Epidemiologic studies have highlighted that inherited factors are involved in 10% to 20% of PCs, mainly through deleterious variants of BRCA2. The frequency of BRCA1/2 germline alterations fluctuates quite a lot among different ethnic groups, and the estimated rate of PVs/LPVs variants in Italian HBOC families is not very accurate, according to different reports. The aim of our study is to describe the prevalence of a BRCA2 PV observed in a selected cohort of HBOC patients and their relatives, whose common origin is the eastern coast of Emilia Romagna, a region of Italy. This study provides insight into the frequency of the variant detected in this area and provides evidence of an increased risk of pancreatic and breast cancer, useful for genetic counseling and surveillance programs.

To improve the success rate of current preclinical drug trials, there is a growing need for more complex and relevant models that can help predict clinical resistance to anticancer agents. Here, we present a three‐dimensional (3D) technology, based on biomimetic collagen scaffolds, that enables the modeling of the tumor hypoxic state and the prediction of in vivo chemotherapy responses in terms of efficacy, molecular alterations, and emergence of resistance mechanisms. The human breast cancer cell lines MDA‐MB‐231 (triple negative) and MCF‐7 (luminal A) were treated with scaling doses of doxorubicin in monolayer cultures, 3D collagen scaffolds, or orthotopically transplanted murine models. Lineage‐specific resistance mechanisms were revealed by the 3D tumor model. Reduced drug uptake, increased drug efflux, and drug lysosomal confinement were observed in triple‐negative MDA‐MB‐231 cells. In luminal A MCF‐7 cells, the selection of a drug‐resistant subline from parental cells with deregulation of p53 pathways occurred. These cells were demonstrated to be insensitive to DNA damage. Transcriptome analysis was carried out to identify differentially expressed genes (DEGs) in treated cells. DEG evaluation in breast cancer patients demonstrated their potential role as predictive biomarkers. High expression of the transporter associated with antigen processing 1 (TAP1) and the tumor protein p53‐inducible protein 3 (TP53I3) was associated with shorter relapse in patients affected by ER+ breast tumor. Likewise, the same clinical outcome was associated with high expression of the lysosomal‐associated membrane protein 1 LAMP1 in triple‐negative breast cancer. Hypoxia inhibition by resveratrol treatment was found to partially re‐sensitize cells to doxorubicin treatment. Our model might improve preclinical in vitro analysis for the translation of anticancer compounds as it provides: (a) more accurate data on drug efficacy and (b) enhanced understanding of resistance mechanisms and molecular drivers.

Although targeting of cell metabolism is a promising therapeutic strategy in acute myeloid leukemia (AML), metabolic dependencies are largely unexplored. We aimed to classify AML patients based on their metabolic landscape and map connections between metabolic and genomic profiles. Combined serum and urine metabolomics improved AML characterization compared with individual biofluid analysis. At intracellular level, AML displayed dysregulated amino acid, nucleotide, lipid, and bioenergetic metabolism. The integration of intracellular and biofluid metabolomics provided a map of alterations in the metabolism of polyamine, purine, keton bodies and polyunsaturated fatty acids and tricarboxylic acid cycle. The intracellular metabolome distinguished three AML clusters, correlating with distinct genomic profiles: NPM1-mutated(mut), chromatin/spliceosome-mut and TP53-mut/aneuploid AML that were confirmed by biofluid analysis. Interestingly, integrated genomic-metabolic profiles defined two subgroups of NPM1-mut AML. One was enriched for mutations in cohesin/DNA damage-related genes (NPM1/cohesin-mut AML) and showed increased serum choline + trimethylamine-N-oxide and leucine, higher mutation load, transcriptomic signatures of reduced inflammatory status and better ex-vivo response to EGFR and MET inhibition. The transcriptional differences of enzyme-encoding genes between NPM1/cohesin-mut and NPM1-mut allowed in silico modeling of intracellular metabolic perturbations. This approach predicted alterations in NAD and purine metabolism in NPM1/cohesin-mut AML that suggest potential vulnerabilities, worthy of being therapeutically explored.

// Gianluca Tedaldi 1 , Michela Tebaldi 1 , Valentina Zampiga 1 , Rita Danesi 2 , Valentina Arcangeli 3 , Mila Ravegnani 2 , Ilaria Cangini 1 , Francesca Pirini 1 , Elisabetta Petracci 4 , Andrea Rocca 5 , Fabio Falcini 2 , Dino Amadori 5 and Daniele Calistri 1 1 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 2 Romagna Cancer Registry, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 3 Department of Medical Oncology, Ospedale Infermi, Rimini, Italy 4 Unit of Biostatistics and Clinical Trials, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy 5 Department of Medical Oncology, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS, Meldola, Italy Correspondence to: Daniele Calistri, email: daniele.calistri@irst.emr.it Keywords: hereditary breast and ovarian cancer, multiple-gene panel, next-generation sequencing, bilateral breast cancer, cancer predisposition Received: October 19, 2016 Accepted: March 14, 2017 Published: April 03, 2017 ABSTRACT As new genes predisposing to breast (BC) and ovarian cancer (OC) are constantly emerging, the use of panels of genes analyzed by Next-Generation Sequencing (NGS) is increasing in clinical diagnostics. The identification of a large number of new germline mutations allows for deeper knowledge of cancer predisposition, although raising many questions about patient management. BC and OC patients recruited by our counseling service between 2012-2015 were included in this study. DNA was extracted from peripheral blood and a panel of 94 genes involved in hereditary tumors was analyzed by NGS. Patient clinical features of BC and OC and cancer family history were collected and compared to the patient genetic profile. A total of 255 women were analyzed, 57 of whom had a pathogenic mutation in BRCA1/2 genes, and 17 carried pathogenic mutations in other genes, such as PALB2 , ATM , BRIP1 , RAD51D , MSH6 , PPM1D , RECQL4 , ERCC3 , TSC2 , SLX4 and other Fanconi anemia genes. Patients with a pathogenic mutation in genes other than BRCA1 and BRCA2 showed no significant difference from the BRCA1/2 -mutated carriers with respect to age at diagnosis and clinical features, suggesting that mutations in other genes could pose a high risk of cancer development. These patients had a much higher percentage of bilateral breast cancer (BBC) and a lower rate of OC than BRCA -mutated patients and patients with no pathogenic mutations: as a consequence, the surveillance protocol should be customized to the patient genetic characteristics.

The main gene involved in gastric cancer (GC) predisposition is CDH1, the pathogenic variants of which are associated with diffuse-type gastric cancer (DGC) and lobular breast cancer (LBC). CDH1 only explains a fraction (10&ndash, 50%) of patients suspected of DGC/LBC genetic predisposition. To identify novel susceptibility genes, thus improving the management of families at risk, we performed a multigene panel testing on selected patients. We searched for germline pathogenic variants in 94 cancer-related genes in 96 GC or LBC Italian patients with early-onset and/or family history of GC. We found CDH1 pathogenic variants in 10.4% of patients. In 11.5% of cases, we identified loss-of-function variants in BRCA1, BRCA2, PALB2, and ATM breast/ovarian cancer susceptibility genes, as well as in MSH2, PMS2, BMPR1A, PRF1, and BLM genes. In 78.1% of patients, we did not find any variants with clear-cut clinical significance, however, 37.3% of these cases harbored rare missense variants predicted to be damaging by bioinformatics tools. Multigene panel testing decreased the number of patients that would have otherwise remained genetically unexplained. Besides CDH1, our results demonstrated that GC pathogenic variants are distributed across a number of susceptibility genes and reinforced the emerging link between gastric and breast cancer predisposition.

IntroductionGiven the COVID-19 coronavirus emergency, a special focus is needed on the impact of this rapidly spreading viral infection on cancer patients. No prophylactic treatments for COVID-19 have been clearly proven and found. In this pandemic context, cancer patients constitute a particularly fragile population that would benefit the best from such treatments, a present unmet need. TMPRSS2 is essential for COVID-19 replication cycle and it is under androgen control. Estrogen and androgen receptor dependent cues converge on TMPRSS2 regulation through different mechanisms of action that can be blocked by the use of hormonal therapies. Androgen receptor (AR) signaling in the TMPRSS2 regulation is emerging as an important determinant of SARS-CoV-2 susceptibility. Materials and MethodsIn our study, we analyzed AR and TMPRSS2 expression in 17352 normal and 9556 cancer tissues from public repositories and stratified data according to sex and age. Gene expression data were retrieved from TCGA and Genotype-Tissue Expression (GTEx) portal. Differences in gene expression were tested with Mann-Whitney U-test. ResultsThe emerging picture is that some patient groups may be particularly susceptible to SARS-CoV-2 infection and may benefit from anti-androgen or anti-estrogen based therapies. Indeed, while young individuals showed a coherent reduction of AR expression in tumors as compared to healthy tissues regardless of gender, elderly females showed increased AR levels in tumors compared to normal tissues. Coherently with AR overexpression, we also observed a significant increase in TMPRSS2 gene expression in tumors from elderly females with respect to age-matched normal tissues. These results highlight that cancers of elderly females have typically higher expression of AR and of TMPRSS2 identifying a population of cancer patients that may be particularly susceptible to COVID-19 infection. These findings are relevant to choose proper treatments in order to protect cancer patients from concomitant SARS-CoV-2 contagion and related symptoms and put forward the idea that hormonal therapies could be used as prophylactic agents against COVID-19. ConclusionsWe believe that there is enough body of evidence to foresee a prophylactic use of hormonal therapies against COVID-19 and this hypothesis can be easily tested on cohorts of breast and prostate cancer patients who follow those regimens. In case of pandemic, if the protective effect of hormonal therapies will be proven on cancer patients, the use of specific hormonal therapies could be extended to other oncological groups and to healthy individuals to decrease the overall risk of infection by SARS-CoV-2. Citation Format: Sara Bravaccini, Eugenio Fonzi, Michela Tebaldi, Davide Angeli, Giovanni Martinelli, Fabio Nicolini, Paola Parrella, Massimiliano Mazza. Estrogen and androgen receptor inhibitors: Unexpected allies in the fight against COVID-19 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 709.

Discrimination of multifocal primary lung cancers from lung metastases is crucial to allow for an appropriate clinical management. We report here a case of multifocal lung adenocarcinomas with different morphological and molecular patterns. Radical surgery of one lung nodule was performed at the time of diagnosis, and subsequently on two other lung nodules. At the time of distant relapse, biopsy was repeated for molecular characterization. The patient was treated with EGFR tyrosine kinase inhibitor according to the detection of EGFR exon 21 mutation in metastatic sample and in one of the three lung tumors, characterized by lower mutated allele frequency. The progression free survival was three months according to radiological criteria and the treatment was provided for six months, until clinical progression. Following the assessment of EGFR mutations by pyrosequencing, tumor samples were analyzed by a 30-gene next generation sequencing (NGS) panel, allowing to study intra- and inter-tumor heterogeneity and to confirm the three lung tumors as independent. Different molecular profiles of synchronous tumors and identical EGFR, PIK3CA and TP53 mutations in one of three primary lung tumors and the metachronous metastasis were identified. In conclusion, morphological and molecular characterization of multiple lung nodules by NGS may help to define synchronous and metachronous adenocarcinomas, thus affecting surgical indication and systemic treatment. Intratumor heterogeneity may be associated with differential sensitivity to targeted treatment.

HER2-positive advanced gastric cancer patients frequently develop resistance to trastuzumab through mechanisms still poorly understood. In breast cancer, other members of the HER-family are known to be involved in trastuzumab-resistance, as is overexpression of the scaffold protein IQGAP1. In the present work, we investigated acquired resistance to trastuzumab in gastric cancer experimental models. Trastuzumab-resistant (HR) subclones derived from 3 HER2-overexpressing gastric cancer cells were generated and characterized for alterations in HER2-signaling mechanisms by next-generation sequencing, immunohistochemical, western blot and qRT-PCR techniques, and molecular modeling analysis. All subclones showed a reduced growth rate with respect to parental cell lines but each had a different resistance mechanism. In NCI N87 HR cells, characterized by a marked increase in HER2-signaling pathways with respect to the parental cell line, trastuzumab sensitivity was restored when IQGAP1 expression was silenced. AKG HR subclone showed higher HER3 protein expression than the parental line. High nuclear HER4 levels were observed in KKP HR cells. In conclusion, our study revealed that high IQGAP1 expression leads to resistance to trastuzumab in gastric cancer. Furthermore, 2 new mutations of the HER2 gene that may be involved in acquired resistance were identified in AKG HR and KKP HR subclones.

Background: The genetically heterogeneous and poor survival group of Philadelphia negative (Ph-) B-ALL group that doesn’t have the most recurrent adult rearrangements (t(9;22); t(1;19); t(4;11)) is collectively referred to as “triple negative” (Ph-/-/-). CRLF2 is frequently altered in adult B-ALL, especially in Ph-like pts (50-75% of cases). Alterations that lead, in the majority of cases, to a CRLF2 overexpression. Adult pts with CRLF2 upregulated have poor outcome and novel strategies are needed to improve it. Aims: Understand the genomic background of all Ph-/-/- ALL and subsequently clustering Ph-/-/- considering CRLF2 overexpression event, in order to define new biomarkers in these subgroups. Pts and Methods: Ph-/-/- pts were sequenced by WES (44pts/93 samples) and 92 B-Other pts were analyzed with NGS target seq (NTS) to validate the 8 most mutated genes (Fig1A). GEP were performed on 55 Ph-/-/-, 29 B-ALL Ph+ and on 7 donors. We cluster triple negative GEP data with our validated pipeline, based on CRLF2 upregulation and in a top ten-gene list. Ph-/-/- ALL samples were then characterized for the presence of gene fusions, Copy Number Alterations (CNAs) and mutations using different approaches (Pancancer and/or RNASeq; dMLPA-MRC-Holland; SNP Array; 454 Junior and PCR). Results: WES analysis identified some recurrent mutated genes (NRAS, PAX5, KRAS, PTPN11, EP400, JAK2, TP53, CREBBP) previously reported to be involved in B-ALL, confirming the pivotal roles of these gene in ALL. For the first time we described a little known gene PKHD1L1 as highly mutated (7.2%). TP53 was the most mutated gene (Fig1A) and that between these gene is the only one associated to a worst OS (p=0.004). Combining our new Ph-/-/- GEP clustering, WES, NTS, Fusion and CNA results we identify a defined 2-clusters-subdivision (Gr1 and Gr2). The Gr2 (14.1% of all B-ALL) is characterized by CTGF, CRLF2 and CD200 (Gr2=3C-up; Fig 1B) co-overexpression. The Gr2 GEP is similar to Ph+ one (Fig1C). Gr1 represents 46.9% of all B-ALL. Fusion, CNA and mutational screening done, detected that 3C-Up group has a higher frequency of Ph-like associated lesions (primarily CRLF2, JAK2, IL7R mut or del), that mainly affect JAK-STAT pathway. Also IKZF1 and EBF1 deletions are significantly associated to Gr2 (p=0.003; p=0.016). RAS pathway genes are highly affected in Gr1. We also validated not previously described fusions. Notably p53 pathway is enriched in both groups but with different deregulated genes: CHEK2 is upreg in the group1 and CDK6 in the Gr2. Preliminary data seems to confirm an higher effect on cell viability of a TKI on Gr2 primary cell pt (vs Gr1 pt). Conclusions: we identified a new signature, related to CRLF2 high expression, to classify Ph-/-/- ALL B. This new subclassification identifies new potential therapeutic targets with available drugs (α-CTGF, α-CD200, CDK2, CHK2 and CDK6 inhibitors; TKIs already effective on Ph+ and Ph-like) to test. Citation Format: Anna Ferrari, Silvia Vitali, Valentina Robustelli, Andrea Ghelli Luserna di Rorà, Eugenio Fonzi, Simona Righi, Carmen Baldazzi, Michela Tebaldi, Samanta Salvi, Cristina Papayannidis, Giovanni Marconi, Mariachiara Fontana, Enrica Imbrogno, Antonella Padella, Giorgia Simonetti, Alessandra Santoro, Jesus María Hernández-Rivas, Maria Teresa Bochicchio, Fabiana Mammoli, Benedetta Giannini, Nicoletta Testoni, Daniele Calistri, Massimiliano Bonafè, Gastone Castellani, Elena Sabattini, Daniel Remondini, Giovanni Martinelli. “3c-up” a new adult Philadelphia negative acute lymphoblastic leukemia subgroup: Novel molecular markers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 2140.

Li-Fraumeni syndrome (LFS) is an autosomal dominant disorder occurring at a young age that predisposes individuals to multiple forms of cancer and to a heterogeneous spectrum of malignancies. We describe the clinical history of a patient who had 5 primary malignant cancers and a familiar history consistent with LFS. We analyzed the genomic DNA of the proband and her relatives by next-generation sequencing (NGS) technology using an enrichment protocol for the simultaneous sequencing of 94 genes involved in hereditary cancers. Genetic analysis of the proband revealed a TP53 germline mutation in exon 5 determining a nucleotide alteration at codon 175 (R175H), a hot spot mutation site related to LFS and a reported pathogenic mutation. The proband daughter's and brother's DNA did not carry the TP53 mutation but they had some rare variants in common with the proband, in addition to other variants with a still unclear role. In conclusion, we identified a TP53 mutation in a patient with multiple primary tumors and a family history characterized by a severe susceptibility to cancer. The genetic analysis by targeted NGS led to the identification of the genetic background and to the exclusion of a cancer risk for the family members. Targeted NGS represents an efficient approach for the identification of mutations in families with a heterogeneous phenotype.

Background Although non muscle invasive bladder cancer (NMIBC) generally has a good long-term prognosis, up to 80% of patients will nevertheless experience local recurrence after the primary tumor resection. The search for markers capable of accurately identifying patients at high risk of recurrence is ongoing. We retrospectively evaluated the methylation status of a panel of 24 tumor suppressor genes (TIMP3, APC, CDKN2A, MLH1, ATM, RARB, CDKN2B, HIC1, CHFR, BRCA1, CASP8, CDKN1B, PTEN, BRCA2, CD44, RASSF1, DAPK1, FHIT, VHL, ESR1, TP73, IGSF4, GSTP1 and CDH13) in primary lesions to obtain information about their role in predicting local recurrence in NMIBC. Methods Formaldehyde-fixed paraffin-embedded (FFPE) samples from 74 patients operated on for bladder cancer were analyzed by methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA): 36 patients had relapsed and 38 were disease-free at the 5-year follow up. Methylation status was considered as a dichotomous variable and genes showing methylation ≥20% were defined as “positive”. Results Methylation frequencies were higher in non recurring than recurring tumors. A statistically significant difference was observed for HIC1 (P = 0.03), GSTP1 (P = 0.02) and RASSF1 (P = 0.03). The combination of the three genes showed 78% sensitivity and 66% specificity in identifying recurrent patients, with an overall accuracy of 72%. Conclusions Our preliminary data suggest a potential role of HIC1, GSTP1 and RASSF1 in predicting local recurrence in NMIBC. Such information could help clinicians to identify patients at high risk of recurrence who require close monitoring during follow up.

Objectives Urine cell-free (UCF) DNA has recently been proposed as a potential marker for early bladder cancer diagnosis. It is known that normal apoptotic cells produce highly fragmented DNA while cancer cells release longer DNA. Therefore, we verified the potential role of UCF DNA integrity in early bladder cancer diagnosis. Materials and methods UCF DNA was isolated from 51 bladder cancer patients, 46 symptomatic patients, and 32 healthy volunteers. To verify UCF DNA integrity, sequences longer than 250 bp, c-Myc , BCAS1 , and HER2 , were quantified by real time PCR. Results At the best cutoff value of 0.1 ng/μl, UCF DNA integrity analysis showed a sensitivity of 0.73 (95% CI 0.61–0.85), and a specificity of 0.84 (95% CI 0.71–0.97) in healthy individuals and 0.83 (95% CI 0.72–0.94) in symptomatic patients. Receiver operating characteristic (ROC) curve analysis revealed an area under the curve of 0.834 (95% CI 0.739–0.930) for healthy individuals and 0.796 (95% CI 0.707–0.885) for symptomatic patients. Conclusions These preliminary data suggest that UCF DNA integrity is a potentially good marker for early noninvasive diagnosis of bladder cancer. Its diagnostic performance does not seem to vary significantly, even in an “at risk” population of symptomatic individuals.

Background CHEK2 is a multi-cancer susceptibility gene whose common germline mutations are known to contribute to the risk of developing breast and prostate cancer. Case presentation Here, we describe an Italian family with a high number of cases of breast cancer and other types of tumour subjected to the MLPA test to verify the presence of BRCA1, BRCA2 and CHEK2 deletions and duplications. We identified a new 23-kb duplication in the CHEK2 gene extending from intron 5 to 13 that was associated with breast cancer in the family. The presence and localisation of the alteration was confirmed by a second analysis by Next-Generation Sequencing. Conclusions This finding suggests that CHEK2 mutations are heterogeneous and that techniques other than sequencing, such as MLPA, are needed to identify CHEK2 mutations. It also indicates that CHEK2 rare variants, such as duplications, can confer a high susceptibility to cancer development and should thus be studied in depth as most of our knowledge of CHEK2 concerns common mutations.