Search

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.

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.

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.