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Centrosome anomalies contribute to tumorigenesis, but it remains unclear how they are generated in lethal cancer phenotypes. Here, it is demonstrated that human microsatellite instable (MSI) and BRAFV600E-mutant colorectal cancers with a lethal rhabdoid phenotype are characterized by inactivation of centrosomal functions. A splice site mutation that causes an unbalanced dosage of rootletin (CROCC), a centrosome linker component required for centrosome cohesion and separation at the chromosome 1p36.13 locus, resulted in abnormally shaped centrosomes in rhabdoid cells from human colon tissues. Notably, deleterious deletions at 1p36.13 were recurrent in a subgroup of BRAFV600E-mutant and microsatellite stable (MSS) rhabdoid colorectal cancers, but not in classical colorectal cancer or pediatric rhabdoid tumors. Interfering with CROCC expression in near-diploid BRAFV600E-mutant/MSI colon cancer cells disrupts bipolar mitotic spindle architecture, promotes tetraploid segregation errors, resulting in a highly aggressive rhabdoid-like phenotype in vitro. Restoring near-wild-type levels of CROCC in a metastatic model harboring 1p36.13 deletion results in correction of centrosome segregation errors and cell death, revealing a mechanism of tolerance to mitotic errors and tetraploidization promoted by deleterious 1p36.13 loss. Accordingly, cancer cells lacking 1p36.13 display far greater sensitivity to centrosome spindle pole stabilizing agents in vitro. These data shed light on a previously unknown link between centrosome cohesion defects and lethal cancer phenotypes providing new insight into pathways underlying genome instability.Implications: Mis-segregation of chromosomes is a prominent feature of chromosome instability and intratumoral heterogeneity recurrent in metastatic tumors for which the molecular basis is unknown. This study provides insight into the mechanism by which defects in rootletin, a centrosome linker component causes tetraploid segregation errors and phenotypic transition to a clinically devastating form of malignant rhabdoid tumor. Mol Cancer Res; 16(9); 1385–95. ©2018 AACR.

Supplementary Table 1. Clinicopathological, immunohistochemical and molecular features of the 12 rhabdoid colorectal cancers7 Supplementary Table 2. Somatic mutations of candidate genes (15 out of 20 are reported) shared by the two rhabdoid cancers at exome sequencing Supplementary Table 3. Rhabdoid colorectal cancers (RC): molecular alterations, centrosome aberrations and ploidy Supplementary Table 4. Rhabdoid tumours of infants (RI): molecular alterations, centrosome aberrations, and ploidy Supplementary Table 5: Clinicopathologic and molecular features of classical colorectal cancers analyzed Supplementary Table 6. Sequencing and Reverse transcription PCR primers Supplementary Table 6B. SMARCB1 regions covered by the custom panel used for next-generation target sequencing Supplementary Table 7. CROCC regions covered by the custom panel used for next-generation targeted sequencing Supplementary Table 8. Antibodies employed and working conditions for immunohistochemistry, immunofluorescence or western blot analysis of tissues and cell lines Supplementary Table 9. Rhabdoid colorectal cancer reported in literature between 1993-2015

The file contains supplementary Materials and Methods

S1. Whole exome sequencing analysis of two rhabdoid colorectal cancers (RC). S2. Non-silent somatic mutations in rhabdoid tumours are enriched in centrosome-microtubule components. S3. Effect of CROCC mutations on centrosome phenotype. S4. CROCC expression profiling in classical colorectal cancers (CRCs). S5. Allelic loss at the 1p36.13 locus correlates with chromosomal instability in colorectal cancer cells. S6. Centrosome and chromosome instability in colorectal cancer cells. S7. Mitotic errors and impaired cell cycle caused by stable CROCC depletion in RKO cells. S8. Invasive behavior of colorectal cancer cells triggered by CROCC depletion. S9. Mitotic aberrations suppressed by CROCC or microtubule stabilizing agents in metastatic T84 and HT29 colorectal cancer cells.

Epilepsy is a heterogenous group of disorders defined by recurrent seizure activity due to abnormal synchronized activity of neurons. A growing number of epilepsy cases are believed to be caused by genetic factors and copy number variants (CNV) contribute to up to 5% of epilepsy cases. However, CNVs in epilepsy are usually large deletions or duplications involving multiple neurodevelopmental genes. In patients who underwent seizure focus resection for treatment-resistant epilepsy, whole genome DNA methylation profiling identified 3 main clusters of which one showed strong association with receptor tyrosine kinase (RTK) genes. We identified focal copy number gains involving epidermal growth factor receptor (EGFR) and PDGFRA loci. The dysplastic neurons of cases with amplifications showed marked overexpression of EGFR and PDGFRA, while glial and endothelial cells were negative. Targeted sequencing of regulatory regions and DNA methylation analysis revealed that only enhancer regions of EGFR and gene promoter of PDGFRA were amplified, while coding regions did not show copy number abnormalities or somatic mutations. Somatic focal copy number gains of noncoding regulatory represent a previously unrecognized genetic driver in epilepsy and a mechanism of abnormal activation of RTK genes. Upregulated RTKs provide a potential avenue for therapy in seizure disorders.

Tumors develop through an evolutionary process in which genomic lesions accumulate in different coexisting subclones leading to intra-tumor heterogeneity (ITH). ITH allows the tumor to adapt to microenvironments and to resist therapies and has been shown to be a prognostic marker. In this work we show the characterization of ITH in Multiple Myeloma (MM) at single cell resolution from a pool of Circulating Multiple Myeloma Cells (CMMCs) and we study the relationship between number of sampled cells and diversity detected. Peripheral blood was collected from 3 patients affected by MM. Using CellSearch® system, blood was enriched for target CMMCs, with immuno-magnetic selection of CD138+, and immunofluorescently stained cells, positive for CD38-PE, DAPI and negative for CD45/CD19-APC, were enumerated. The samples were then analyzed and CMMCs were isolated with DEPArray platform. The DNA of each single cell (n=261) was amplified with Ampli1™ WGA kit and the WGA output was used for genome-wide single cell copy number alteration analysis with Ampli1 LowPass kit. Copy number profiling revealed from 2 to 3 coexisting subclones per patient, with minor clones size going from 6.5% to 33%. ITH was assessed by determining the Shannon index, which showed a range of values from 0.44 (low diversity) to 1.01 (high diversity) depending on source sample. We evaluated the effect of number of cells/profiles on Shannon index by bootstrapping (200 times) a number of copy number profiles ranging from 2 to the total number of profiles available per patient. Results showed that the mean Shannon index almost reaches a plateau at 15-20 cells/profiles in the case of lower diversity samples and at about 30 cells/profiles for the highest diversity sample. Moreover, as few as 10 cells allowed to differentiate low vs. high diversity samples. Cell enrichment and sorting by CellSearch and DEPArray platforms allow to quantify ITH at diversity level with high precision even from a limited number of initial single-cells and provide a promising method to objectively measure heterogeneity, as required in order to study its clinical implications. Citation Format: Alberto Ferrarini, Claudio Forcato, Valentina del Monaco, Mario Terracciano, Marianna Garonzi, Massimiliano Pellicano, Genny Buson, Francesca Fontana, Paola Tononi, Nicolo Manaresi. Evaluation of intra-tumor heterogeneity by single-cell copy number profiling of circulating multiple myeloma cells using Shannon index [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6087.

Gatekeeper mutations are identified in only 50% of the cases at resistance to Anaplastic Lymphoma Kinase (ALK)-tyrosine kinase inhibitors (TKIs). Circulating tumor cells (CTCs) are relevant tools to identify additional resistance mechanisms and can be sequenced at the single-cell level. Here, we provide in-depth investigation of copy number alteration (CNA) heterogeneity in phenotypically characterized CTCs at resistance to ALK-TKIs in ALK-positive non-small cell lung cancer. Single CTC isolation and phenotyping were performed by DEPArray or fluorescence-activated cell sorting following enrichment and immunofluorescence staining (ALK/cytokeratins/CD45/Hoechst). CNA heterogeneity was evaluated in six ALK-rearranged patients harboring ≥ 10 CTCs/20 mL blood at resistance to 1st and 3rd ALK-TKIs and one presented gatekeeper mutations. Out of 82 CTCs isolated by FACS, 30 (37%) were ALK+/cytokeratins-, 46 (56%) ALK-/cytokeratins+ and 4 (5%) ALK+/cytokeratins+. Sequencing of 43 CTCs showed highly altered CNA profiles and high levels of chromosomal instability (CIN). Half of CTCs displayed a ploidy >2n and 32% experienced whole-genome doubling. Hierarchical clustering showed significant intra-patient and wide inter-patient CTC diversity. Classification of 121 oncogenic drivers revealed the predominant activation of cell cycle and DNA repair pathways and of RTK/RAS and PI3K to a lower frequency. CTCs showed wide CNA heterogeneity and elevated CIN at resistance to ALK-TKIs. The emergence of epithelial ALK-negative CTCs may drive resistance through activation of bypass signaling pathways, while ALK-rearranged CTCs showed epithelial-to-mesenchymal transition characteristics potentially contributing to ALK-TKI resistance. Comprehensive analysis of CTCs could be of great help to clinicians for precision medicine and resistance to ALK-targeted therapies.

Background: Combination therapy with dabrafenib + trametinib demonstrated robust activity in patients (pts) with BRAFV600E-mutant advanced non-small cell lung cancer (NSCLC), but its resistance mechanisms are poorly known. Liquid biopsy components such as circulating tumor cells (CTCs) and cell-free (cf) tumor DNA can provide a comprehensive genomic picture of tumor content. Molecular profiling of single CTCs from pts with BRAF-V600Emutant NSCLC was performed to carry out a pilot study to identify resistance mutations at failure to dabrafenib + trametinib and to compare the mutations detected on CTCs to the mutations found on cfDNA and tumor biopsies. Patients and Methods: Eight pts with advanced BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib were prospectively enrolled between Jul 2018 and Mar 2019 at Gustave Roussy (IDRCB2008-A00585-50). Bloods samples were collected. Matched tissue-cfDNA and CTCs were available in 3 pts and matched tissue-CTCs for 4 pts. Single CTC isolation strategy included RosetteSep enrichment, immunofluorescent staining (Hoechst/CD45/cytokeratins) and fluorescence activated cell-sorting. The process to identify CTC mutations included Ampli1 whole-genome amplification, quality controls, multiplex targeted PCR with the Ampli1 CHPCustomBeta cancer panel developed by (Menarini Silicon Biosystems) and next-generation sequencing (NGS). The cfDNA was analyzed using InVisionFirst-Lung. Tissue samples were analyzed using targeted NGS in the MATCH-R trial (Recondo G; NPJ Precis Oncol 2020). Results: Single CTCs were isolated from 7 pts. As baseline characteristics, the median age was 66 years, 5 (71%) were smoker; all the pts with adenocarcinoma histology. Most of the pts received dabrafenib + trametinib as 2nd line (86%). The median of CTCs isolated by patient was 20 (8-28). A wide spectrum of mutations in CTCs was observed at treatment failure that were involved in the main cancer pathways, including MAPK (n=1; NRAS), tyrosine kinase receptors (n=5; EGFR, ALK, FLT3, HER2,…), signal transduction (n=4; IDH1, EZH2,⋯), and DNA repair (n=2; AKT1, ATM,⋯). In the same CTC, several mutations were observed in 5/7 patients, commonly involving more than one cancer pathways. A higher degree of mutational diversity was observed in CTCs compared to tumor tissue biopsies and cfDNA. In the 3 patients with an available tumor/liquid biopsy, only 1 shared mutations between CTCs and matched tumor and cfDNA. Conclusion: Single CTC profiling reveals a wide spectrum of therapeutic resistance mutations not detected by other analyses in pts with BRAFV600E-mutant NSCLC at failure to dabrafenib + trametinib. Integration of single CTC sequencing to tumor and cfDNA analysis, provides important perspectives to assess heterogeneous resistance mechanisms and to guide precision medicine in BRAFV600E- NSCLC. Citation Format: Laura Mezquita, Marianne Oulhen, Agathe Aberlenc, Marc Deloger, Aurélie Honoré, Marianna Garonzi, Genny Buson, Claudio Forcato, Yann Lecluse, Mihaela Aldea, Maud NgoCamus, Claudio Nicotra, Karen Howarth, Ludovic Lacroix, Luc Friboulet, Benjamin Besse, Nicolò Manaresi, David Planchard, Françoise Farace. Resistance mechanisms to BRAF inhibition identified by single circulating tumor cell and cell-free tumor DNA molecular profiling in BRAF-mutant non-small cell lung cancer [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 598.

Background: Clonal evolution and heterogeneity are among the factors that make treatment of Multiple Myeloma (MM) challenging, and disease monitoring requires invasive bone marrow biopsies. Tumor heterogeneity has previously been demonstrated at copy-number level, showing patterns of recurring Copy-Number Alterations (CNA). Conversely, Loss-of-Heterozygosity (LoH) profiling has been investigated less extensively. Presence of genomic patterns of LoH has been associated with Homologous Recombination Deficiency (HRD) in MM, suggesting a role for therapy with PARP inhibitors. Here we report the genome-wide LoH profiling in single-CMMCs isolated from enriched peripheral blood of four different MM patients using a non-invasive approach that combines CellSearch® and DEPArray™ technology. Methods: CMMCs were obtained from peripheral blood of four MM patients using CellSearch for enrichment and DEPArray NxT for isolation. CMMCs enrichment was obtained using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. After single cell isolation, Ampli1™ Whole Genome Amplification (WGA) kit was used to amplify single-cell genomic DNA. Whole genome sequencing libraries were prepared from WGA products using Ampli1™ LowPass kit and low-pass sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBiosuite. Single-cell genome-wide LoH profiles were obtained by a custom algorithm based on the ratio between mono- and bi-allelic loci in non-overlapping segments of uniform copy-number. Results: Out of 315 cells analyzed from the four MM patients, 244 (77%) showed LoH events. Extensive LoH regions were detected in all patients, with 231 cells showing copy-neutral LoH events and 121 showing LoH events in copy-gain regions. Globally, 460 copy-neutral LoHs, and 292 LoHs in copy-gain regions were detected. Two highly conserved copy-neutral events (9p; 16q) were detected in one of the patients analyzed, suggesting they are early events. Interestingly, the first of the 2 events includes CDKN2A, a tumor suppressor which has been implicated in predisposition to MM. Notably, in a second patient, chromosome 11 is associated both to a copy-neutral LoH and to an LoH event in a copy-gain segment including the CCND1 gene, whose amplification in MM has been associated with multidrug resistance expression. Conclusions: Here we present a non-invasive workflow for the molecular characterization of single CMMCs isolated from peripheral blood. Specifically, we reported the presence of chromosomal or sub-chromosomal LoH events in all MM patients, suggesting a pervasive phenomenon in MM which deserves to be further explored as potential therapy target. Citation Format: Claudio Forcato, Alberto Ferrarini, Genny Buson, Paola Tononi, Marianna Garonzi, Valentina del Monaco, Andrea Raspadori, Steven Gross, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Analysis of low-pass sequencing data reveals extensive loss-of-heterozygosity in circulating multiple myeloma cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2702.

Background: Genome-wide analysis of Loss-of-heterozygosity (LoH) has been shown to be important in several contexts, including the assessment of BRCAness signature, a marker of homologous repair deficiency in several cancer types associated with efficacy of platinum therapy and PARP inhibitors. Methods to infer genome-wide LoH status require high-coverage whole-genome sequencing or low-pass sequencing of a large number of single cells. We present a method to infer genome-wide LoH status of as low as one single-cell from low-pass sequencing of libraries produced by Ampli1™ LowPass kit, at a fraction of the cost of current NGS-based methods. Methods: We used low-pass sequencing data generated from 294 single Circulating Tumor Cells (CTCs) from prostate, multiple myeloma and lung cancer patients, and from 24 single cells from 8 cell lines. DNA amplified with Ampli1™ WGA kit (based on ligation-mediated PCR) combined with Ampli1 LowPass kit for Illumina generates a reduced genome representation, whereby a significant fraction of fragments is expected to be covered by more than one read. We reasoned that, even at a mean coverage insufficient for variant calling (≤0.05x), a statistically significant decrease in loci showing 2 alleles should be expected in LoH regions. A pileup of mapped sequences at dbSNP loci was obtained (minor allele frequency ≥5%; read depth ≥2). Mono- and bi-allelic loci (pileup containing 1 or 2 different alleles, respectively), were counted in each uniform copy-number (CN) genome segment within a single chromosome arm. Regions with CN equal to cell ploidy were used as internal reference. A one-sided Fisher test on biallelic sites depletion (multiplicity corrected p-value threshold of 0.05) was used to call LoH on each region. Results: Compared with libraries obtained using random shearing, Ampli1 LowPass single-cell libraries showed an increase in mean coverage of covered genome from 1.1 to 1.9 starting from the same number of reads (1M) thus increasing the number of loci for LoH calling. Analysis of 10 patients' single cells with known LoH events, corresponding to regions at copy number 1, detected 1006 events out of 1142. Several (492) copy neutral LoH events or LoHs in copy gained regions were also detected. Representatives of such events were verified successfully by high pass sequencing of the same library. Performance was confirmed by analysis of 24 single cells from cell lines whose LoH status was inferred from CCLE database. Conclusions: This innovative method leverages reduced genome representation inherent in Ampli1 LowPass libraries, to compute genome-wide LoH along with copy number (CN) status, meeting the need for LoH/CN analysis at single-cell level on rare-cells such as CTCs. Citation Format: Alberto Ferrarini, Claudio Forcato, Marianna Garonzi, Genny Buson, Massimiliano Pellicano, Francesca Fontana, Nicolò Manaresi. Genome-wide loss-of-heterozygosity calling from low-pass sequencing of ligation-mediated whole genome amplified DNA in single tumor cells [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 1327.

Background Cancer of unknown primary (CUP) is a heterogeneous group of metastatic cancers whose primary site cannot be determined after standard clinical and pathological evaluation. CUP patients are generally treated with empirical chemotherapy and have poor prognosis. As reported in recent studies, CUPs present an average of 4 genetic alterations per tumor and these genetic alterations can be detected in circulating tumor DNA (Kato 2017). Thus, a potential improvement in CUP outcomes could derive from targeted therapies directed toward actionable mutations, and Circulating tumor cells (CTCs) can provide valuable information. Here, we describe a case of a 51-yr-old Caucasian female (Pt#71) with metastatic CUP. CTCs were detectable in her blood and analyzed for genetic alterations. Methods Using CellSearch and DEPArray NxT, n=3 CTCs and n=1 leukocyte as a control were isolated from patient's peripheral blood. Single-cell whole genome amplification was obtained with Ampli1 WGA Kit and libraries were generated with Ampli1 OncoSeek Panel and Ampli1 LowPass kits. In parallel, formalin-fixed paraffin embedded (FFPE) tissue derived from a lymph node biopsy was analyzed with two different methods. One curl was sent for comprehensive genomic profile to FoundationOne assay testing (Roche). From another curl, DNA was extracted and directly processed with DEPArray LibPrep and DEPArray OncoSeek Panel kits. After sequencing on Illumina MiSeq platform, raw data were analyzed on MSBiosuite platform (Menarini Silicon Biosystems). Results CTCs genome-wide characterization allowed the detection of sub-chromosomal losses, including a LOH region comprising the APC gene, and patterns of extensive gains. Moreover, amplification signals were detected in correspondence of FGFR2 and CCNE1 genes. Targeted sequencing on both CTCs and bulk tumor DNA confirmed the FGFR2 amplification in all the samples and detected a somatic variant in APC gene (APC:p.T1556Nfs*3). FoundationOne assay on FFPE biopsy reported the same amplification on FGFR2 and CCNE1 genes, along with a somatic variant in ARID1A gene (ARID1A:p.R1276*), a gene not present in the OncoSeek Panel. On the other hand, APC somatic variant was not identified by FoundationOne, probably due to contamination by normal cells and/or tumor heterogeneity. Conclusions The comprehensive genomic profile of tumor FFPE tissue and CTCs was highly overlapping and allowed the characterization of genetic alterations in this CUP case, revealing potentially actionable mutations and copy number alterations. The advantage of isolating and analyzing single CTCs is clearly shown by the non-invasive procedure combined with a precise detection of druggable alterations due to cell purity. Citation Format: Elisa Porcellini, Francesco Gelsomino, Noemi Laprovitera, Mattia Riefolo, Marianna Garonzi, Paola Tononi, Francesca Fontana, Antonia D'Errico, Maria Pantaleo, Nicolò Manaresi, Andrea Ardizzoni, Manuela Ferracin. Isolation and genetic characterization of circulating tumor cells from cancer of unknown primary [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 5361.

Background The study describes the Salmonella Rissen phage ϕ1 isolated from the ϕ1-sensitive Salmonella Rissen strain RW. The same phage was then used to select the resistant strain RRϕ1+, which can harbour or not ϕ1. Results Following this approach, we found that ϕ1, upon excision from RW cells with mitomycin, behaves as a temperate phage: lyses host cells and generates phage particles; instead, upon spontaneous excision from RRϕ1+ cells, it does not generate phage particles; causes loss of phage resistance; switches the O-antigen from the smooth to the rough phenotype, and favors the transition of Salmonella Rissen from the planktonic to the biofilm growth. The RW and RRϕ1+ strains differ by 10 genes; of these, only two (phosphomannomutase_1 and phosphomannomutase_2; both involved in the mannose synthesis pathway) display significant differences at the expression levels. This result suggests that phage resistance is associated with these two genes. Conclusions Phage ϕ1 displays the unusual property of behaving as template as well as lytic phage. This feature was used by the phage to modulate several phases of Salmonella Rissen lifestyle. Electronic supplementary material The online version of this article (10.1186/s12866-018-1360-z) contains supplementary material, which is available to authorized users.

Background Characterization of individual CTCs helps investigating intra-tumour heterogeneity, and provides longitudinal information about temporal evolution of genomic lesions following therapeutic evolutionary pressure, missed by one-time, bulk, single-biopsy analysis. Here we present the validation of a complete workflow to detect hotspot mutations and CNAs in single cells. It combines ligation-mediated PCR (LM-PCR) Whole Genome Amplification (Ampli1™ WGA kit), best-in-class in terms of low allelic drop-out (ADO) and reproducibility of amplification bias, with a tailored, WGA-aware, next generation sequencing (NGS) targeted cancer panel (Ampli1™ OncoSeek) and a fully-automated cloud-based platform for bioinformatic analysis (MSBiosuite). Methods Single-cells (n=24) of breast, prostate, lung and neuroblastoma cancer cell lines spiked in healthy-donor blood, alongside 15 single White Blood Cells (WBCs) from 5 healthy donors and CTCs from prostate, lung and breast patients were enriched with CellSearch® System, sorted with DEPArray™ NxT technology and WGA’ed with Ampli1™ WGA kit (Menarini Silicon Biosystems). NGS reference materials (Seraseq™ Breast CNV and Seraseq™ Lung & Brain CNV) with known CNAs were also WGA’ed. WGA products were used as input for the Ampli1™ OncoSeek Panel assay (Menarini Silicon Biosystems), a single-tube, Illumina®-compatible kit that covers 60 clinically relevant genes including more than 1500 mutation hotspots and CNAs for a subset of 19 genes. WGA-tailored primer pairs design and their concentrations were optimized so that targeted amplicons for sequence variants are sufficiently represented. Redundant amplicons were included for robustness of focal CNA detection. Bioinformatic analysis was performed with an assay-specific, cloud-based pipeline (MSBiosuite, Menarini Silicon Biosystems). Results Results on Seraseq NGS reference materials with known copy number gains (3, 6, 12) on 6 genes showed accurate detection of expected CNAs and high linearity of response (R2 = 0.97 ± 0.04). We observed low ADO rate (12.7% ± 4.2%). The Ampli1™ OncoSeek Panel assay detected known mutations and CNAs from cell lines at high sensitivity and the analysis of polymorphic variants in WBCs showed high agreement between biological replicates (overall agreement = 0.94 ± 0.06). Sequencing of CTCs from patients is ongoing and will be presented at the conference. Conclusions Here we presented a complete solution to detect hotspot mutations and focal CNAs that meets the need for accurate tumour characterization at single-cell level, suitable for individual CTC analysis. Citation Format: Paola Tononi, Valentina del Monaco, Alberto Ferrarini, Genny Buson, Marianna Garonzi, Claudio Forcato, Andrea Raspadori, Nicolò Manaresi. Validation of a targeted sequencing workflow for sequence variants and focal copy number alterations (CNAs) in single circulating tumor cells (CTCs) [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 2517.

Introduction: Multiple Myeloma (MM) evolution and heterogeneity is interesting for its potential translational relevance. Recent studies using bulk sequencing of Smoldering MM cells obtained from Bone Marrow (BM) report recurring CNA patterns. By analyzing single-CMMCs isolated from enriched peripheral blood, we show here, for the first time in MM, evidence of frequent convergent lesions, i.e. alterations developed independently across different evolutionary branches, including CNAs often found in MM and previously reported as common truncal alterations. Methods: Peripheral blood samples (4.0 ml) were obtained from n=3 patients with MM. CMMCs were enriched with CellSearch® AutoPrep® using a custom kit with anti-CD138 or anti-CD138/CD38 antibody-conjugated ferrofluids for positive enrichment and CD38-PE, CD19/CD45-APC immunofluorescent staining for detection. Cell enumeration was based on the co-localization of nuclear DAPI staining and CD38-PE on CellSearch CTAII®. Single CMMCs (CD38+/CD19- and CD45-/DAPI+) and White Blood Cells (WBCs: CD38-/CD19+ or CD45+/DAPI+) were then isolated with DEPArray NxT system. Single-cell genomic DNA was amplified using Ampli1™ Whole Genome Amplification (WGA) kit, Illumina®-compatible libraries were obtained using Ampli1™ LowPass kit and the process was automated on a Hamilton STARLet Liquid handler. Multiplexed, low-pass whole-genome sequencing was performed on HiSeq 2500 Illumina® platform. Genome-wide single-cell Low-Pass Copy Number Alteration (LPCNA) analysis was performed using the cloud-based bioinformatic suite MSBio Suite (Menarini Silicon Biosystems). Results: 186/215 (86%) single CMMC in total passed QC criteria and were included in the analysis. Single WBCs were also included as normal controls. Cumulatively, CNA profiles of single CMMCs showed patterns typical of MM, including 1q gain, 13 monosomy, sub-chromosomal gain or trisomy 3, 5, 7, 9, 11, 15, 21. Of these, intra-patient single-cell profiling surprisingly revealed -in all three patients- convergent lesions, i.e. alterations developed independently across different evolutionary branches, along with conserved (common truncal), and divergent alterations (found only in specific sub-clusters). In addition, we found evidence that 1q gain, 13q deletion and 6p gain were actually subclonal, in contrast with recent publications reporting them as truncal early-onset lesions. Conclusion: Single CMMCs CNA profiling reveals patterns of frequent convergent alterations developed independently through branched evolution, undetectable through bulk sequencing. Citation Format: Claudio Forcato, Andrea Raspadori, Alberto Ferrarini, Mario Terracciano, Valentina del Monaco, Marianna Garonzi, Carrie Morano, Steven Gross, Genny Buson, Chiara Bolognesi, Francesca Fontana, Gianni Medoro, Mark Connelly, Nicolò Manaresi. Genome-wide copy number profiling of single circulating multiple myeloma cells (CMMCs) reveals intra-patient convergent copy-number alterations (CNAs) [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 2911.

Background: Classical Hodgkin Lymphoma (cHL) is generally highly curable with standard frontline therapies, although about 20% of the patients relapse or become refractory after initial treatment. cHL hallmark is the presence of morphologically characteristic malignant Hodgkin and Reed-Sternberg (HRS) cells that represent only a small fraction (about 1%) of the surrounding non-malignant environment. Genetic alterations of HRS cells are potentially a precious source of information to develop new treatments or prognostic biomarkers. In this perspective, low tumor cellularity, worsened by DNA degradation of FFPE samples, poses technical challenges to unravel malignant cells genetic alterations. Hereby we present new insights on purified HRS single cells obtained through highly automated platforms, providing precise observation of tumor genetic alterations. Methods: FFPE tissue sections from 5 cHL patients were dissociated down to single cell suspensions. Cells were immunofluorescently labeled using anti-CD30-FITC and anti-PD-L1-PE antibodies. HRS cells, along with normal leukocytes, were selected on the basis of morphological and immunofluorescence criteria, and isolated using DEPArray™ NxT (Menarini Silicon Biosystems, MSB). Customized, high-throughput automated protocols were developed and implemented on STARLet liquid handler (Hamilton Life Sciences) to amplify isolated purified single cells genomic DNA and to generate genome-wide copy-number alterations (CNAs) profiles using Ampli1™ WGA and Ampli1™LowPass kits (MSB), respectively. Results: More than 150 HRS cells were isolated from the 5 patient samples, from which CNA profiles were obtained. HRS cells presented extensive gains and losses across the whole genome, while leukocytes displayed flat profiles as expected. HRS cells clustered coherently with patients, revealing a high degree of heterogeneity of CNA profiles among different patients. However some commonalities across the patients genomes were identified. In particular, gains and amplification were detected in PD-L1, PD-L2 and JAK2 region (9p24), as well as gains and losses in regions where REL and other genes involved in NF-kB pathway map. Conclusions: Leveraging on high throughput automated platforms and single cells isolation, the described method enabled cHL genome-wide genetic analysis at a single cell level, overcoming the intrinsic limitations of low-frequency of HRS and DNA degradation due to FFPE samples. Furthermore, unprecedented data on single HRS cells were described, opening up to a new approach to understand tumor diversity and to potentially develop personalized therapeutic strategies for cHL patients. Citation Format: Andrea Raspadori, Paola Tononi, Chiara Mangano, Marianna Garonzi, Claudio Forcato, Chiara Bolognesi, Genny Buson, Francesca Fontana, Gianni Medoro, Nicolò Manaresi. Hodgkin and Reed-Sternberg cells genome-wide copy number alteration analysis at single cell level by high-throughput automated platforms [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 3410.

DNA sequencing technologies are evolving at a prodigious rate. First-generation approaches have now been largely replaced by second-generation technologies (still known as “next generation sequencing” (NGS) even though they are now current and commonplace), and third-generation technologies (sometimes called “next-next generation sequencing”) are starting to arrive. This has led to global boom in whole genome or exome sequencing, boosting the discovery of sequence variants associated with disease that will eventually be translated into new diagnostic, prognostic, and therapeutic targets for individual patients in “precision medicine.” Acknowledgement of disease predisposition and specific therapeutic behavior for each individual addresses a more preventive approach. Adoption of such novel means represents an anticipation-relevant outcome as it can affect our healthcare on many different levels, ranging from a simple lifestyle adjustment to a well-defined clinical guideline. In this chapter we summarize current and emerging sequencing technologies for clinical applications, and some of the challenges that lie ahead.

Chromosome number alterations, aneuploidy, is a hallmark of cancer. It occurs in about 15% of acute myeloid leukemia (AML) cases, is generally preserved throughout disease progression (Bochtler et al. Leukemia 2015) and correlates with adverse prognosis (Breems et al. JCO 2008, Papaemmanuil et al. NEJM 2016). This evidence highlights the need of understanding the molecular mechanisms that promote and sustain aneuploidy in AML, in order to define novel potential therapeutic targets. In the NGS-PTL project we profiled the genomic landscape of 536 hematological samples by whole exome sequencing (WES, Illumina). Among them, we analyzed 88 and 68 samples from aneuploid (A-) FLT3-wildtype AML (isolated trisomy and monosomy, complex and monosomal karyotype) and euploid (E-) AML (normal and complex karyotype, A-AML showed an increased genomic instability, as confirmed by a higher mutation load compared with E-AML (median number of variants: 22 (range: 2-95) and 11 (range: 3-45), respectively, pA substitutions, compared with the C>T transition-related signature, which is prevalent in AML. A-AML was associated with mutations and/or heterozygous deletion of TP53 (p We show here for the first time the molecular mechanisms promoting and maintaining aneuploidy in AML. Our results indicate that p53 deficiency, either caused by somatic mutations, copy number loss, impaired DNA damage response and enhanced PLK1 signaling synergize with APCgain, RAD50 structural or functional loss and forced progression through mitosis, to override cell cycle and mitotic checkpoints and allow the formation of daughter cells with an aberrant chromosome number. These mechanisms cooperate with recurrent mutations of genes involved in protein ubiquitination and proteasome-mediated protein catabolic process in A-AML, indicative of the attempt of aneuploid cells to override the proteotoxic stress due to the unbalanced protein load generated by the aneuploid condition. This evidence provides the rationale for exploiting proteasome inhibition (Velcade), p53 reactivation (MDM2/4 inhibitor) and targeting of the cell cycle (CHK1/2 inhibitor) downstream to p53 (WEE1 inhibitor) as strategies for novel combination therapies against aggressive aneuploid AML, which are under clinical investigation in our Institution and may serve as a model for aneuploid tumors. GS and AP: equal contribution. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:Ariad: Consultancy, Speakers Bureau; MSD: Consultancy; Celgene: Consultancy, Speakers Bureau; Roche: Consultancy, Speakers Bureau; Genentech: Consultancy; Novartis: Speakers Bureau; BMS: Speakers Bureau; Pfizer: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau.

BRCA1 is one of the most important gene associated with familial breast and ovarian cancer susceptibility and is involved in the DNA damage repair and cell cycle arrest. Alterations in BRCA1 and their consequences are well characterized in breast and ovarian tumors, while little is known about its role in Acute Myeloid Leukemia (AML). We aimed to investigate the frequency and the interplay of BRCA1 alterations and patterns of somatic mutations (SNVs) and copy number variations (CNVs) of in AML patients. We genotyped 118 AML samples at either diagnosis or relapse by Single Nucleotide Polymorphism (SNP) array (SNP6.0 and Cytoscan HD, Affymetrix) to detect CNVs. In addition, we performed Whole-Exome Sequencing (WES, 100 bp paired-end, Illumina) of 56 genotyped AML patients to detect SNVs and small indels (MuTect and Varscan 2.0). Differences in survival were assessed using Kaplan-Meier survival analysis and Long-Rank test, or Breslow when indicated. We detected BRCA1 loss in 14 out of 118 patients (12%), ranging from 1,6 Kb to the loss of the entire chromosome 17, with none of the BRCA1 loss patients having a normal karyotype. Moreover, BRCA1 losses significantly co-occurred with PALB2 and RAD50 losses. Notably, the PALB2 loss (chromosome 16) mostly involved exon 12 and was detected also in patients without the BRCA1 loss, with a frequency of 11 out of 118 cases (9%), suggesting a pivotal and previously undescribed role in AML pathogenesis. BRCA1 loss patients were enriched for copy number alterations in genes involved in the regulation of mitotic recombination, DNA repair and positive regulation of kinase activity compared with BRCA1 wild-type cases (p Finally, BRCA1 and PALB2 alterations defined group of patients with poor overall survival (OS). In particular, patients with alterations of either BRCA1 or PALB2 had a worst prognosis compared to patients without alterations in the two genes (p=0.009 and p=0.001, respectively). Patients harboring PALB2 loss had a poorer prognosis compared with patients with complex karyotype (p=0.021, Breslow test). In addition, double BRCA1 and PALB2 loss, single BRAC1 or PALB2 hits and lack of alterations in BRCA1 and PALB2 defined three different classes of risks in our AML cohort (p In summary, we define for the first time a novel subgroup of AML patients characterized by alterations targeting the DDR pathway, and in particular the BRCA1 and PALB2 genes. Our data suggest that a signature of genes involved in the DDR and cell cycle regulation synergize and led to the uncontrolled proliferation, independently of DNA damage accumulation. Our results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Acknowledgments: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), Fondazione del Monte di Bologna e Ravenna, FP7 NGS-PTL project. Disclosures Guadagnuolo: CellPly S.r.l.: Employment. Martinelli:Novartis: Speakers Bureau; Genentech: Consultancy; Roche: Consultancy, Speakers Bureau; BMS: Speakers Bureau; Celgene: Consultancy, Speakers Bureau; Amgen: Consultancy, Speakers Bureau; MSD: Consultancy; Pfizer: Consultancy, Speakers Bureau; Ariad: Consultancy, Speakers Bureau.

Next Generation Sequencing (NGS) studies identified 9 functional categories of mutations in acute myeloid leukemia (AML), with >99% of cases having at least one of those mutations (Ley et al. NEJM 2013). However, multiple genetic hits participate to AML pathogenesis, and metabolic dysregulations, as the one induced by IDH1/2 mutations, play oncogenic functions (Ward et al. Cancer Cell 2010). Aim of the study was to define novel functional categories of AML mutations affecting relevant and druggable biological processes, with focus on genetic determinants of metabolic plasticity. Out of 455 whole exome sequencing (WES) cases from onco-hematological patients collected in the NGS-PTL project, we analyzed 37 AML cases, belonging to our cohort of 239 FLT3-WT samples (886 AML total). We performed 100 bp paired-end WES (HiSeq2000, Illumina) and mapped the sequenced reads with Burrows-Wheeler Aligner. Variants where called with MuTect or GATK for single nucleotide variant (SNV) and indels detection, respectively (>90% confidence). Gene expression profiling was performed using HTA2.0 microarray (Affymetrix) on 56 bone marrow samples, including AML (≥80% blasts) and healthy controls. By WES analysis, we detected an average of 26 somatic variants per patient (range, 7 to 65). Gene ontology annotation identified 8 novel relevant functional categories of mutated genes: transcription, translation and post-translational modifications, protein degradation, cytoskeleton, cell cycle, DNA damage, cell survival and metabolism. Since metabolic pathways are promising targets for tailored therapies (e.g. IDH1/2 and glutaminase inhibitors), we focused our analysis on them. We identified 82 variants (74 SNVs, 2 frameshift and 4 nonframeshift deletions, 2 stopgains) targeting 70 genes involved in metabolism, with 78% of patients carrying at least one mutation in a metabolic gene and 35 variants rated as damaging by CONDEL algorithm. Among mutations in metabolic genes, the most represented pathways according to Recon X database were amino acids, lipids, CoA and nucleotides metabolism, transport and bioenergetics pathways. Notably, IMPDH2, a mediator of MYC-induced proliferation involved in nucleotide interconversion, was mutated and overexpressed in our AML cohort (p=0.01), suggesting a potential oncogenic function. Moreover, ALDH2, a regulator of hematopoietic stem cell functions which is involved in multiple metabolic pathways and associates with metabolic remodeling, was mutated and 2-fold downregulated in AML blasts. Seven genes were mutated in 5-8% of samples: RETSAT, HSPG2, CHPF, ABCA2, ND1, APOBR, NAAA. Among them, RETSAT, HSPG2, CHPF mutations were also predicted as “drivers” by DOTS-Finder tool. Bioenergetics pathways were affected by mutations in glycolysis and gluconeogenesis (GPI, ITPA), oxidative phosphorylation (ND1, ND4, ND5, CYTB), pentose phosphate pathway (H6PD, PGLS). Patients carrying mutations in the bioenergetics pathway showed a strong trend towards reduced overall survival, which did not associate with unfavorable molecular mutations. In conclusion, metabolism is the most represented class of mutated genes (8.6% of variants) in our FLT3-WT AML cohort after signaling, leading us to propose a novel functional category. Our data suggest that, along with mutations in established oncogenes and tumor suppressors involved in metabolic control (KRAS, TP53, MYC pathway), a number of genetic determinants participate to leukemia metabolic plasticity and oncogenic mutations of metabolic enzymes may drive leukemogenesis, impact on patient's survival and become novel targets for personalized therapies. Acknowledgements: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. GS and AP equally contributed to this work. Citation Format: Giorgia Simonetti, Antonella Padella, Ilaria Iacobucci, Italo Do Valle, Gabriele Fontanarosa, Elisa Zago, Francesca Griggio, Marianna Garonzi, Simona Bernardi, Cristina Papayannidis, Maria Chiara Abbenante, Giovanni Marconi, Giorgio Melloni, Laura Riva, Viviana Guadagnuolo, Mariachiara Fontana, Samantha Bruno, Elisa Zuffa, Eugenia Franchini, Annalisa Astolfi, Carmen Baldazzi, Elisa Dan, Barbara Sinigaglia, Michele Cavo, Nicoletta Testoni, Emanuela Ottaviani, Pier Giuseppe Pelicci, Marco Sazzini, Alberto Ferrarini, Massimo Delledonne, Daniel Remondini, Giovanni Martinelli. European Network NGS-PTL preliminary data: Whole exome sequencing identifies mutations of ALDH2, RETSAT, HSPG2, CHPF and other metabolic genes as a novel functional category in acute myeloid leukemia. [abstract]. In: Proceedings of the AACR Special Conference: Metabolism and Cancer; Jun 7-10, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(1_Suppl):Abstract nr A27.

Introduction Immune-checkpoint blockade has emerged as an effective therapeutic strategy in solid tumor and in hematologic malignancies, including classical Hodgkin Lymphoma (cHL). cHL represents about 11% of all malignant lymphoma and it is generally highly curable with standard frontline therapies, although about 20% of the patients will relapse or become refractory after initial treatment. The hallmark of cHL is the presence of malignant Hodgkin and Reed-Sternberg Cells (HRS) that represent only a small fraction (about 1%) of the surrounding heterogeneous immune infiltrate. Despite this extensive inflammatory microenvironment, HRS are able to escape immune surveillance using several mechanisms, including the overexpression of PD-1 ligands (PD-Ls) that bind PD-1 on reactive T-cells, inhibiting their activity and proliferation and causing ultimately T-cell exhaustion. The PD-Ls expression is upregulated in a dose-dependent manner by copy number alterations of chromosome 9p24.1, a locus encoding for PD-L1/PD-L2 as well as JAK2, which further enhances PD-Ls expression through JAK2/STAT pathway. Here we present a method for the isolation and the genetic characterization of single purified HRS, which overcomes the limitations posed by the low tumor cellularity of cHL biopsies and gives an estimation of inter-tumor and intra-tumor heterogeneity which may be useful to guide immune treatment selection. Methods FFPE tissue sections from 4 cHL patients were dissociated down to single-cell suspension and stained using anti-CD30 and anti-PD-L1 antibodies. Since CD30 is not expressed exclusively by malignant cells, beyond the positivity to CD30 and PD-L1 HRS were selected according to morphological criteria, such as cell size and the presence of nuclei with ploidy higher than the surrounding lymphocytes. DEPArray™ NxT system (Menarini Silicon Biosystems) was used to isolate single target cells. After recovery, single cells were whole genome amplified (Ampli1™ WGA, Menarini Silicon Biosystems), and genome-wide copy-number alterations (CNAs) profiles were obtained using Ampli1™ LowPass kits (Menarini Silicon Biosystems) on Illumina® and Ion Torrent™ platforms. Results For each patient, at least 8 HRS cells and infiltrating lymphocytes were identified and isolated from lymphoid tissue using DEPArray™ NxT system. Copy-number analyses of recovered cells allowed us to precisely discriminate HRS, characterized by extensive gains and losses, from non-tumor cells, showing flat profiles as expected (Fig.1). Ploidy of HRS was automatically determined, based on best-fitting of profiles with underlying copy number levels. Hierarchical clustering showed that some alterations are highly conserved among patients, e.g. the region containing PD-L1/PD-L2/JAK2 has several copy gains in the majority of malignant cells. Interestingly, these alterations show high variable copy-number levels between different HRS even in the same patient, ranging from few copy-gains to amplifications, suggesting some level of heterogeneity. Different CNAs are also detected in regions containing genes belonging to pathways already known to be altered in cHL, like REL/NFKB and JAK/STAT pathways, which may be involved in the constitutive activation of proliferative and antiapoptotic phenotype of HRS. Conclusion Single HRS sorting combined with low-pass whole genome sequencing offer a valuable tool to uncover genetic alterations hidden by the massive cHL immune infiltrate and to estimate inter-tumor and intra-tumor heterogeneity in cHL patients. Considering that PD-Ls locus amplifications are associated with advanced stages of the disease and with a shorter progression free survival, the analysis of purified HRS could be helpful for patient stratification for the adoption of immune therapy. Disclosures Mangano: Menarini Silicon Biosystems: Employment. Edoardo:Menarini Silicon Biosystems: Employment. Garonzi:Menarini Silicon Biosystems: Employment. Lanzellotto:Menarini Silicon Biosystems: Employment. Papadopulos:Menarini Silicon Biosystems: Employment. Bolognesi:Menarini Silicon Biosystems: Employment. Buson:Menarini Silicon Biosystems: Employment. Ferrarini:Menarini Silicon Biosystems: Employment. Forcato:Menarini Silicon Biosystems: Employment. Fontana:Menarini Silicon Biosystems: Employment. Ceccolini:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fabbri:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Fici:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Gallerani:Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori (IRST) IRCCS: Employment. Simonelli:Menarini Silicon Biosystems: Employment. Medoro:Menarini Silicon Biosystems: Employment. Manaresi:Menarini Silicon Biosystems: Employment.

Genetic testing, which is now a routine part of clinical practice and disease management protocols, is often based on the assessment of small panels of variants or genes. On the other hand, continuous improvements in the speed and per-base costs of sequencing have now made whole exome sequencing (WES) and whole genome sequencing (WGS) viable strategies for targeted or complete genetic analysis, respectively. Standard WGS/WES data analytical workflows generally rely on calling of sequence variants respect to the reference genome sequence. However, the reference genome sequence contains a large number of sites represented by rare alleles, by known pathogenic alleles and by alleles strongly associated to disease by GWAS. It’s thus critical, for clinical applications of WGS and WES, to interpret whether non-variant sites are homozygous for the reference allele or if the corresponding genotype cannot be reliably called. Here we show that an alternative analytical approach based on the analysis of both variant and non-variant sites from WGS data allows to genotype more than 92% of sites corresponding to known SNPs compared to 6% genotyped by standard variant analysis. These include homozygous reference sites of clinical interest, thus leading to a broad and comprehensive characterization of variation necessary to an accurate evaluation of disease risk. Altogether, our findings indicate that characterization of both variant and non-variant clinically informative sites in the genome is necessary to allow an accurate clinical assessment of a personal genome. Finally, we propose a highly efficient extended VCF (eVCF) file format which allows to store genotype calls for sites of clinical interest while remaining compatible with current variant interpretation software.

The endopeptidase Separase, encoded by the ESPL1 gene, plays a key role in faithful segregation of sister chromatids by cleaving the cohesin complex at the metaphase to anaphase transition. Its overexpression associates with aneuploidy and bad prognosis in solid tumors. Little is known in Acute Myeloid Leukemia (AML). We profiled the genomic landscape of 405 and 78 AML cases by SNP array (SNP 6.0 and Cytoscan HD, Affymetrix) and whole exome sequencing (100 bp, paired-end, Illumina), respectively. Bone marrow blasts from 61 patients were analyzed by gene expression profiling (HTA 2.0, Affymetrix). Separase expression was determined by Immunohistochemistry (1:600 antibody dilution Abnova, clone 6H6) in 44 AML and 4 control bone marrow specimens. One patient exhibited a nonsynonimous mutation in ESPL1 (1.3%), which was predicted to alter the protein function. Moreover, ESPL1 copy number gain was observed in 5/405 cases (1.2%): 2 hyperdiploid AML, one trisomy 12 and 2 cases with a short gain at 12q. Notably, protein level detection in one of the 12q-gain cases confirmed Separase overexpression. To determine the incidence of Separase overexpression, we performed Immunohistochemistry on additional 43 AML. Separase was overexpressed in 29/44 AML (66%, Separase-high), being comparable to control marrow biopsies in the remaining 15 samples (Separase-low). Sixty-two percent of Separase-high AML were aneuploid. However, no significative association was observed, as previously reported for mutations in the cohesin genes in AML. Separase overexpression correlated with increased patients’ age (median age 64 vs. 57 years, p=.01), 17-fold upregulation of CD34 (p=.004) and a trend towards reduced overall survival (6-years follow-up). Separase overexpression was not mutually esclusive with cohesin gene mutations, it co-occurred with NPM1 and FLT3 lesions and frequent mutations in genes involved in protein post-translational modification and ubiquitination (p=.04). Separase-low cases were enriched for mutations in RAS signaling pathway (NRAS, KRAS, NF1, RIT1, GRAP2, RALGDS; p=4.5x10-5) and in cell migration-related genes (LIMS2, S1PR1, PPIA, PLXNB1, FAT1). Separase-high cases also showed a defined transcriptomic profile, characterized by reduced expression of HOXA/B family genes, the DNA damage repair gene ATM, the p53 regulator MDM2 and forced expression of the cell cycle markers CDC20, AURKB, NUSAP1 and of MYC, independently of chromosome 8 gain. Taken together, our data suggest that genomic lesions targeting ESPL1 are a rare event in AML. However, Separase overexpression is a common feature and defines a new subset of AML cases with a distinct gene expression profile, which may benefit of innovative targeted therapies including CDC20 and bromodomain inhibitors. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Giorgia Simonetti, Antonella Padella, Simona Righi, Maria Chiara Fontana, Marco Manfrini, Cristina Papayannidis, Giovanni Marconi, Carmen Baldazzi, Marianna Garonzi, Alberto Ferrarini, Massimo Delledonne, Nicoletta Testoni, Elena Sabattini, Giovanni Martinelli. Separase overexpression defines a new subset of acute myeloma leukemia patients characterized by high CD34 and MYC levels [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3472. doi:10.1158/1538-7445.AM2017-3472

Partner and localizer of BRCA2 (PALB2) plays a key role in the DNA damage repair (DDR) by recruiting BRCA1, BRCA2 and RAD51. Alterations in PALB2 were described in hereditary breast cancer and Fanconi Anemia (FA). Little is known in Acute Myeloid Leukemia (AML). Aim of the study is to define the frequency and interplay of PALB2 alterations with patterns of somatic mutations and copy number variants (CNVs) in AML. We genotyped 233 AML samples by Single Nucleotide Polymorphism array (SNP 6.0 and Cytoscan HD, Affymetrix) to detect CNVs. We also performed Whole Exome Sequencing (WES, Illumina) of 56 cases to detect single nucleotide variants and small indels (MuTect and Varscan 2.0). Differences in survival were assessed by Kaplan-Meier survival analysis and Breslow test. We detected PALB2 loss in 12/233 patients (5%), with a minimal common region of deletion of 6.6 Kb including exon 12, which encodes for the domain of interaction with RAD51, BRCA2 and POLH. PALB2 deletion were correlated with CN loss of MYH11, CREBBP, PLK1 and FANCA on chromosome (chr) 16 (p≤.005), deletions of TP53, NF1 and BRCA1 on chr 17 (p≤.004), 5q deletions (p≤.001) and loss of XPO1 on chr 2 (p≤.006). PALB2-loss patients were enriched for alterations in genes involved in the protein kinase pathway (p=5×10-4), JUN kinase activity (p=.0006) and, notably, in genes belonging to the chromosome breakage pathway (p=.001; TP53, BRCA1 and BRCA2). To identify mutations that co-operates with CN alterations, we integrated WES data of 56 AML patients. No mutations in PALB2 were detected. However, we identified mutations in other DDR genes, including FANCE, BRCA2, TP53 and BRCA1. Of note, alterations in the DDR genes frequently co-occurred with both mutations and CN loss of TP53, leading to homozygous loss of function of TP53. In addition, KRAS, IDH1, TET2 and BCOR mutations were mutually exclusive with PALB2 loss. Preliminary gene expression profiling data revealed a 2-fold upregulation of FANCA in samples with DDR genes alterations compared with patients with no alterations (p=.04), while the other genes of the pathway were not affected. Finally, patients harboring PALB2 loss had a poorer prognosis compared complex karyotype cases (p=.021). We here define a new subset of AML patients, characterized by the synergistic loss of key DDR genes, which may in turn result in FANCA over-expression as a mechanism to overcome the DDR deficit. Our data suggest that the leukemogenic process associated PALB2 loss integrates defects in the DDR together with enforced proliferative stimuli leading to uncontrolled proliferation of cells, which potentially accumulate genetic lesions. These results may help improve patient stratification and define ad hoc therapeutic strategies for this aggressive leukemia type. Supported by: ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Antonella Padella, Giorgia Simonetti, Maria Chiara Fontana, Marco Manfrini, Giovanni Marconi, Anna Ferrari, Italo Faria do Valle, Marianna Garonzi, Cristina Papayannidis, Eugenia Franchini, Elisa Zuffa, Viviana Guadagnuolo, Samantha Bruno, Andrea Ghelli Luserna di Rorà, Emanuela Ottaviani, Daniel Remondini, Massimo Delledonne, Giovanni Martinelli. Co-occurrence of alterations in the DNA damage repair genes synergize with uncontrolled proliferation and associate with very-poor prognosis in acute myeloid leukemia patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4671. doi:10.1158/1538-7445.AM2017-4671

Whole exome and transcriptome sequencing (WES and RNAseq) technologies are able to provide a comprehensive analysis of the genomic aberrations acquired by malignant cells, of their synergistic effects and functional consequences. In particular, RNAseq enables the detection of gene fusions originating from rare chromosomal translocations that have been involved in the pathogenesis of Acute Myeloid Leukemia (AML). We performed WES and RNAseq of AML patients to identify novel genetic abnormalities playing a causative role in leukemia development. We collected bone marrow or peripheral blood samples of 31 patients. Sequencing was performed using the Illumina Hiseq2000 platform. WES raw data were analysed with Whole-Exome sequencing Pipeline web tool for variants detection (WEP). The presence of gene fusions was assessed in RNAseq data with deFuse and Chimerascan. Selected genes fusions and variants were validated by Sanger sequencing. By RNAseq we identified a rare gene fusion transcript involving the BCL11B gene, which been previously suggested to play an oncogenic role in AML. The gene encodes for a zinc-finger protein participating to chromatin remodelling and regulating the differentiation and apoptosis of hematopoietic cells. The fusion was identified in a patient with poorly differentiated leukemia phenotype and unfavourable karyotypic abnormalities: 46,XX, t(2;14)(q21;q32), t(11;12)(p15;q22), who received standard chemotherapy, underwent allogeneic bone marrow transplantation and is currently in complete remission. Differently from previous data, the BCL11B translocation was associated neither with FLT3-ITD nor DNMT3A mutations. WES analysis revealed mutations in the TET2 and WTAP genes, which are known to act as co-players in the leukemic transformation. The exome data of our AML cohort identified neither INDELs nor nonsynonymous mutations in the BCL11Bgene, suggesting that the oncogenic function of BCL11B is activated by chromosomal translocations. Gene expression profiling showed a 4-fold upregulation of BCL11B transcript in the patient’s blasts, compared to 53 AML samples with no chromosomal aberrations in the 14q32 region, according to cytogenetic analysis. The increased expression of BCL11B was associated with an upregulation of potential targets including the antiapoptotic protein SPP1. Our data suggest that chromosomal translocations involving the BCL11B gene are rare events in AML and associate with somatic mutations in the malignant transformation of myeloid lineage cells, potentially by altering the differentiation and apoptotic processes. Future studies will investigate putative fusion partners of BCL11Band elucidate the biological consequences of its upregulation in AML pathogenesis. The results highlight the molecular heterogeneity of AML and the need for high-resolution sequencing analysis of leukemic samples at diagnosis in order to tailor personalized therapies. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Disclosures Martinelli: Novartis: Consultancy, Speakers Bureau; BMS: Consultancy, Speakers Bureau; Pfizer: Consultancy; ARIAD: Consultancy.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to mast cell leukemia (MCL). Somatic mutations in the Kit receptor tyrosine kinase (most frequently, D816V) can be detected in >90% of patients with SM and are thought to play a key pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL patient who came to our attention in 2012 – a 63 year-old woman diagnosed with MCL, aleukemic variant (50-60% atypical mast-cells in the bone marrow [BM] smear; CD117+/CD2+/CD13+-/CD33+/CD59+ immunophenotype; serum tryptase, 2500 µg/L; no C-findings). The patient had received imatinib for 6 months, with no clinical benefit. The disease, however, had had an overall chronic clinical course for 6 more months until severe anemia occurred. The patient rapidly progressed and died after 21 months from diagnosis. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified MCs isolated from BM at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA sequencing on an HiSeq1000 (Illumina, San Diego CA). Cytoscan HD arrays (Affymetrix, Santa Clara CA) were also used to scan for chromosomal gains and losses as well as for loss of heterozigosity (LOH). Among the mutated genes detected by WES, SETD2 stood out among others because two distinct putatively inactivating heterozygous mutations were identified, a frameshift insertion of a C in exon 20 (NM_014159:c.7595_7596insC: p.Gly2515ArgfsTer5) and a nonsense mutation in exon 15 (NM_014159:c.G6753T:p.Glu2234Ter). The two mutations were found to hit distinct alleles, pointing to a loss-of-function event. Western Blotting (WB), however, showed that only the 2234 a.a. Setd2 truncated isoform resulting from the nonsense mutation, losing the highly conserved WW and SRI functional domains, was detectable in the sample. The SETD2 gene encodes a histone methyltransferase nonredundantly responsible for trimethylation of lysine 36 of histone H3, a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. SETD2 gene mutations have been described in a variety of cancers and, more recently, have been found to be cooperating events in acute leukemia initiation and progression. In yeast, deletion of the SRI domain abolishes Set2-RNA polymerase II (PolII) interaction causing transcription elongation defects and abolishes K36 methylation. The truncated SETD2 isoform was actually found to lose the ability to bind RNAPolII, as shown by co-immunoprecipitation. Accordingly, RNA-sequencing showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes rather than from canonical promoters. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the recently reported role of SETD2-dependent H3K36Me3 in homologous recombination (HR) repair and genome stability, Cytoscan HD arrays showed that LOH and several gains and losses at many chromosomal loci, undetectable at diagnosis, had been acquired at the time of progression. Haploinsufficiency of PSIP1 (recruiting HR machinery at double strand breaks) at 9p24.3 might have represented a cooperating event. Downmodulation of the Setd2 protein (in the presence of LOH but in the apparent absence of sequence variations other than polymorphisms) and reduced H3K36Me3 levels were detected in two more MCL cases, in which putative cooperative lesions were also identified. Results of WES and high resolution karyotyping of additional SM cases will be presented. Our findings point to epigenetic regulation and/or DNA repair as two candidate pathways deserving further investigation in an attempt to identify novel actors or mechanisms contributing to the pathogenesis and progression of SM, or novel modulators of disease phenotype. They also extend the recent observation that the molecular landscape of SM is much more complex than the initial finding of KIT mutations allowed to imagine. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12 (L. Bolondi) Disclosures Soverini: Novartis: Consultancy, Honoraria; Bristol-Meyers Squibb: Consultancy, Honoraria; Ariad: Consultancy, Speakers Bureau.

Transmembrane proteins allow cells to extensively communicate with the external world in a very accurate and specific way. They form principal nodes in several signaling pathways and attract large interest in therapeutic intervention, as the majority pharmaceutical compounds target membrane proteins. Thus, according to the current genome annotation methods, a detailed structural/functional characterization at the protein level of each of the elements codified in the genome is also required. The extreme difficulty in obtaining high-resolution three-dimensional structures, calls for computational approaches. Here we review to which extent the efforts made in the last few years, combining the structural characterization of membrane proteins with protein bioinformatics techniques, could help describing membrane proteins at a genome-wide scale. In particular we analyze the use of comparative modeling techniques as a way of overcoming the lack of high-resolution three-dimensional structures in the human membrane proteome.

Chromosome gain or loss, which is the hallmark of aneuploidy, occurs in about 10% of adult Acute Myeloid Leukemia (AML) cases (Farag et al. IJO 2002, Breems et al. JCO 2008), despite inducing a dramatic reduction of cellular fitness in non-malignant cells (Torres et al. Science 2007). The study aimed to identify AML-specific molecular mechanisms having a causative and/or tolerogenic role towards aneuploidy. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 38 aneuploid (A) and 34 euploid (E) AML cases, identified according to cytogenetic analysis and SNP array (CytoScan HD, Affymetrix). Variants were called with GATK, MuTect and VarScan. We also compared the transcriptomic profile of leukemic bone marrow cells from 21 A-AML and 28 E-AML cases (HTA 2.0, Affymetrix). A-AML showed a significantly higher mutation load compared with E-AML (median number of variants: 25 and 15, respectively, p Our data show a link between aneuploidy and genomic instability in AML and highlight novel molecular mechanisms for the acquisition and/or maintenance of the aneuploid phenotype. Deregulation of the cell cycle machinery and DNA damage/repair checkpoints, either through mutations, copy number and transcriptomic alterations, cooperate with leukemogenic pathways, as KRAS signaling, to develop A-AML and overcome the unfitness barrier. This evidence suggests that a number of A-AML patients may benefit from pharmacological reactivation of TP53 and inhibition of KRAS pathway. Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi). Citation Format: Giorgia Simonetti, Antonella Padella, Marco Manfrini, ĺtalo Faria do Valle, Cristina Papayannidis, Carmen Baldazzi, Maria Chiara Fontana, Viviana Guadagnuolo, Anna Ferrari, Elisa Zago, Marianna Garonzi, Simona Bernardi, Annalisa Astolfi, Maria Chiara Abbenante, Giovanni Marconi, Elisa Zuffa, Eugenia Franchini, Ilaria Iacobucci, Michele Cavo, Emanuela Ottaviani, Nicoletta Testoni, Alberto Ferrarini, Massimo Delledonne, Torsten Haferlach, Daniel Remondini, Giovanni Martinelli. A cell cycle-related genomic and transcriptomic signature distinguish aneuploid and euploid acute myeloid leukemia. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 90.

Introduction: Adult ALL represents a biologically and clinically heterogeneous group. Incidence and cure rates differ among children and adults. In adults, ALL is less common and generally carries a worse prognosis with shorter long-term survival probability. Although the remarkable progress made in the treatment of ALL in children and, with less efficacy, in adults, several ALL subtypes continue to have a poor prognosis. Aims: focus our attention on adult Ph-negative ALL pts using whole exome experiments to discover novel insights into the mechanisms involved in leukemogenesis and to develop genetic models that accurately define novel ALL subtypes based on the genomic profile of individual patients (pts). Patients and Methods: we performed the WES analysis of 72 samples of B-cell precursor ALL acute lymphoblastic leukemia (B-ALL) cases using the Illumina Hiseq2000 platform. All were adult patients (18-79 years) and were negative for Philadelphia chromosome (BCR-ABL) translocation and negative for the recurrent known molecular rearrangements (E2A-PBX, TEL, AML1-MLL-AF4). Peripheral blood and/or bone marrow samples were collected from adult B-ALL at the time of diagnosis and/or at the time of relapse. Matched samples of primary tumour (peripheral blood or bone marrow) and germline DNA from buccal swab or peripheral blood at the remission time were analyzed. MuTect and GATK tools to call mutations (Single Nucleotide Variants=SNVs and/or INDELs) were used and we selected variants with a minor allele frequency (MAF) lower than 0.05 and filtered using dbSNP142. Results: The WES analysis of the 41 Ph negative cases identified 735 point mutations and 25 mutations that occur in splicing sites in 651 genes. The average number of somatic coding mutations was 17 per case (range 1-47). 38 genes were recurrently mutated with 11 genes mutated in at least 3 cases: PAX5, PRDM12, JAK2, TTN, TP53, PTPN11, PKHD1L1, CUL3, PIEZO2, TACC2, RBBP6. The first two genes present more point mutations, in 5 pts and in 4 pts respectively. Some mutations in genes like PAX5, JAK2, TP53, PTPN11 were deeply described in acute lymphoblastic leukemic; PKHD1L1 was described mutated in one case of T-cell large granular lymphocyte leukemia; PRDM12 disruption was described in an aggressive CML case;TACC2 expression in infant ALL was described as predictor of outcome andtranscription factor and RBBP6 expression was differentially expressed in leukemic cells that overexpressed Gfi-1B gene. The alterations in the remaining genes were not previously described in ALL and/or leukemia. Using KEGG database we mapped the 651 mutated genes to detect the mostly represented pathways. The following resulted significantly enriched (p=0.0004 to p=0.006): Jak-STAT signaling pathway (11 genes), Cell Cycle (13), Dilated Cardiomyophaty (9), Hypertrophic cardiomyophaty (8), Axon Guidance (9), Calcium Signaling pathways (10), Huntington's disease (10), Wnt signaling pathway (9), Metabolic pathways (30), Pacreat Secretion (7). Preliminar analysis lead considering both SNVs and INDELs, detected totally 956 gene variations. Again the pathways mainly significantly (p=8.05e-05 to p=0.0076) affected are the Jak-STAT signaling pathway (14) and the Cell Cycle (13). Also Huntington's disease (14), Dilated Cardiomyophaty (10), Hypertrophic cardiomyophaty (9), Wnt signaling pathway (12), Metabolic pathways (40 genes), Calcium Signaling pathways (12), Metabolic pathways (40), TGF-Beta signaling pathway (8), Pacreat Secretion (8) were alterated. Prediction of protein interactions, using STRING database, generated a network with the genes mutated in more than 5 patients. Then, the nodes were clustered with K-means identifying 4 groups that contain several of our analysis variations (Fig.1). Conclusions: Point mutations are the prevalent mechanism identified in our pts cohort (75.5%). INDELs are less represented (21.5%). Altogether the identified mutations may help cluster Ph- ALL pts. Analysis of SNVs confirmed mutations in important genes known to be involved in leukemogenesis. Relevant alterations affect crucial pathways as cell cycle and Jak-STAT signaling which may be effectively targeted by currently available JAK inhibitors. Supported by: ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:AMGEN: Consultancy; Ariad: Consultancy; Pfizer: Consultancy; ROCHE: Consultancy; MSD: Consultancy; BMS: Consultancy, Speakers Bureau; Novartis: Consultancy, Speakers Bureau.

Aneuploidy causes a proliferative disadvantage, mitotic and proteotoxic stress in non-malignant cells ( Torres et al. Science 2007). Chromosome gain or loss, which is the hallmark of aneuploidy, is a relatively common event in Acute Myeloid Leukemia (AML). About 10% of adult AML display isolated trisomy 8, 11, 13, 21 (Farag et al. IJO 2002), or either an isolated autosomal monosomy or monosomal karyotype (Breems et al. JCO 2008). This evidence suggests that tumor-specific mechanisms cooperate to overcome the unfitness barrier and maintain aneuploidy. However, the molecular bases of aneuploid AML are incompletely understood. We analyzed a cohort of 166 cytogenetically-characterized AML patients (80 aneuploid (A-) and 86 euploid (E-)) treated at Seràgnoli Institute (Bologna). Aneuploidy was significantly associated with poor overall survival (median survival: 13 and 26 months in A-AML and E-AML respectively; p=.006, Fig.1). To identify AML-specific alterations having a causative and/or tolerogenic role towards aneuploidy, we integrated high-throughput genomic and transcriptomic analyses. We performed 100 bp paired-end whole exome sequencing (WES, Illumina Hiseq2000) of 70 samples from our A-AML and E-AML cohort of 166 patients. Variants where called with MuTect or GATK for single nucleotide variant and indels detection, respectively. AML samples were genotyped by CytoScan HD Array (Affymetrix). Gene expression profiling (GEP) was also conducted on bone marrow cells from 24 A-AML, 33 E-AML (≥80% blasts) and 7 healthy controls (HTA 2.0, Affymetrix). We detected a significantly higher mutation load in A-AML compared with E-AML (median number of variants: 31 and 15, p=.04) which was interestingly unrelated to patients' age (median age: 63.5 years in A-AML and 62 years in E-AML, Xie et al, Nat. Med. 2014). C>A and C>T substitutions, which are likely mediated by endogenous 5mCdeamination, were the most frequent alterations (Alexandrov et al. Nat. 2013). However, aneuploidy associated with an increased variability in terms of mutational signatures, with the majority of A-AML displaying 3 or more signatures compared to few E-AML cases (p=.04). WES analysis also revealed a specific pattern of somatic mutations in A-AML. A-AML had a lower number of mutations in signaling genes (p=.04), while being enriched for alterations in cell cycle genes (p=.01) compared with E-AML. The mutated genes were involved in different cell cycle phases, including DNA replication (MCM6, PURB, SSRP1), centrosome dynamics (CEP250, SAC3D1, HEPACAM2, CCP110), chromosome segregation (NUSAP1, ESPL1, TRIOBP), mitotic checkpoint (ANAPC7, FAM64A) and regulation (CDK9, MELK, ZBTB17, FOXN3, PPM1D, USP2). Moreover, genomic deletion of cell cycle-related genes was frequently detected in A-AML. Notably, ESPL1 which associated with aneuploidy, chromosome instability and DNA damage in mammary tumors (Mukherjee et al. Oncogene 2014) was mutated and also upregulated in A-AML compared with E-AML (p=.01), the latter showing expression levels comparable to controls. Among the top-ranked genes differentially expressed between A-AML and E-AML, we identified a specific signature characterized by increased CDC20 and UBE2C and reduced RAD50 and ATR in A-AML (p Our data show a link between aneuploidy and genomic instability in AML. Deregulation of the cell cycle machinery, DNA damage and repair checkpoints either through mutations, copy number and transcriptomic alterations is a hallmark of A-AML. The results define specific genomic and transcriptomic signatures that cooperate with leukemogenic pathways, as KRAS signaling, to the development of the aggressive phenotype of A-AML and suggest that a number of A-AML patients may benefit frompharmacological reactivation of TP53pathway (e.g. MDM2 inhibitor, clinical trial NP28679). Supported by: FP7 NGS-PTL project, ELN, AIL, AIRC, PRIN, progetto Regione-Università 2010-12 GS & AP: equal contribution Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Martinelli:MSD: Consultancy; BMS: Speakers Bureau; Roche: Consultancy; ARIAD: Consultancy; Novartis: Speakers Bureau; Pfizer: Consultancy.

Systemic mastocytosis (SM) includes a heterogeneous group of disorders ranging from indolent SM to the rare and aggressive mast cell leukemia (MCL). Somatic mutations in the KIT receptor kinase (most frequently, D816V) can be detected in >90% of patients and are thought to play an important pathogenetic role. Nevertheless, morphological and clinical diversity, as well as the fact that some patients are negative for KIT mutations, suggest that the underlying molecular picture is far from being fully elucidated. To shed further light on this issue, we undertook an integrated molecular genetic study of a KIT gene mutation-negative MCL case who came to our attention in 2012. After having obtained written informed consent, we extracted genomic DNA and total RNA from purified mast cells (MCs) isolated from bone marrow at diagnosis and at progression, as well as DNA from saliva, and performed whole exome sequencing (WES) and RNA-seq on an HiSeq1000 (Illumina, San Diego CA). High resolution karyotyping was also performed with Cytoscan HD arrays (Affymetrix, Santa Clara CA). Among the mutated genes detected in MCs but not in saliva by WES, SETD2 stood out among others because two loss-of-function mutations (a nonsense and a frameshift mutation) inactivating both alleles of the gene were identified. Western Blotting (WB) confirmed the expression of the truncated SETD2 isoform resulting from the nonsense mutation. The SETD2 gene encodes a histone methyltransferase responsible for trimethylation of Lysine 36 of histone H3 (H3K36Me3), a key hystone mark associated not only with active chromatin but also with transcriptional elongation, alternative splicing, DNA replication and repair. Loss of the highly conserved WW and SRI domains was predicted to impair SETD2 binding with RNA polymeraseII and hNRNP L, as confirmed by co-immunoprecipitation. Accordingly, RNA-seq showed evidence of spurious transcripts initiated from cryptic promoter-like sequences within genes as well as non-canonical splice isoforms. More importantly, WB confirmed that H3K36Me3 was completely abrogated. In line with the role of SETD2-dependent H3K36Me3 in DNA repair and genome stability, Cytoscan HD arrays and WES showed that several losses at many chromosomal loci, together with more than 70 additional point mutations, undetectable at diagnosis, were acquired at the time of progression. Absence of SETD2 protein expression and/or reduced H3K36Me3 were detected in 3/3 additional MCLs and in 6/8 aggressive SMs so far screened. Our data point to epigenetic regulation and/or DNA repair as candidate pathways deserving further investigation in SM - in an attempt to elucidate the mechanisms underlying enhanced clinical aggressiveness and to identify more effective treatment modalities. Molecular characterization of 10 additional MCLs and aggressive SMs is ongoing. Supported by FP7 NGS-PTL project and Progetto Regione-Università 2010-12(L. Bolondi) Citation Format: Simona Soverini, Caterina De Benedittis, Michela Rondoni, Manuela Mancini, Cristina Papayannidis, Luca Zazzeroni, Viviana Guadagnuolo, Elisa Zago, Francesca Griggio, Alberto Ferrarini, Marianna Garonzi, Massimo Delledonne, Giorgina Specchia, Roberta Zanotti, Omar Perbellini, Livio Pagano, Michele Cavo, Peter Valent, Giovanni Martinelli. Integrated molecular characterization of mast cell leukemia reveals recurrent inactivation of the SETD2 tumor suppressor gene. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3957. doi:10.1158/1538-7445.AM2015-3957

Metabolic remodeling of cancer is controlled by metabolic enzymes having oncogenic or tumor suppressor functions, along with oncogenes and tumor suppressors, which cooperate with the tissue environment to define specific metabolic profiles (Yuneva et al. Cell met 2012). Dysregulated metabolic pathways contribute to the pathogenesis of Acute Myeloid Leukemia (AML), as demonstrated for IDH1/2 mutations, which force the production of the oncometabolite 2-Hydroxyglutarate (Ward et al. Cancer Cell 2010) and can be selectively targeted (Wang et al. Science 2013). However, the genetic determinants of leukemia metabolic plasticity are largely unexplored. To identify metabolism-related pathogenic mechanisms in AML, we screened 886 AML cases for targeted genomic alterations and performed Whole Exome Sequencing of 143 leukemia samples (100 bp paired-end, HiSeq2000, Illumina), focusing our analysis on 37 AML cases (34 at diagnosis and 3 at relapse). Mutations were called by MuTect and GATK. Moreover, transcriptional analysis was performed on bone marrow cells from 59 AML cases (≥80% blasts) and 7 healthy controls (HTA2.0, Affymetrix). By mapping the mutated genes into functional categories, we identified a previously undescribed class of mutations targeting metabolism-related genes, that we define metabolic acute leukemia genes (MALGs). MALG was the most represented category after signaling pathways (76/915 genomic alterations) and 29/37 patients carried at least one MALG mutation. MALG mutations mostly targeted biosynthesis and catabolism of lipids and of CoA (ACP2, PANK2), bioenergetic pathways, metabolism of amino acids and nucleotides (NUDT18, IMPDH2). Notably, IMPDH2 is a target of MYC, a known regulator of cancer cell metabolism, and balances the nucleotide pool required for DNA replication (Liu et al, Plos one 2008). IMPDH2 was not only mutated but also upregulated at mRNA level in AML compared with controls (p=0.0001), suggesting an oncogenic function of the gene in AML, which is under investigation. Moreover, MYC transcriptional network was affected by additional mutations targeting genes regulating MYC activity (HUWE1, ZBTB17, TRRAP) and degradation (HEPACAM). Mutations in amino acid metabolism affected the synthesis/degradation of serine (PHGDH), glycine (SHMT2), proline (PRODH), tryptophan(CYP1B1) and glutamate (OPLAH), with a glutamate-related metabolic signature being also enriched in AML. These results may be highly relevant to AML therapy, since they may identify patients suitable to glutaminase inhibitor treatment, which is under development by pharmaceutical companies. An additional subset of patients displayed mutations in glucose-dependent bioenergetic pathways: glycolysis (GPI), oxidative phosphorylation (ND1, ND4, ND5, CYTB) and pentose phosphate pathway (H6PD, PGLS). These mutations were mutually exclusive with KRAS/NRAS alterations, which were detected in 8/37 samples. Indeed, oncogenic KRAS stimulates glucose uptake and channeling of glucose intermediates into pentose phosphate pathway (Ying et al. Cell 2012). Mutations in the bioenergetic pathways occurred across different cytogenetic groups and were associated with a poor outcome in terms of overall survival (p=0.016 Fig.1) in our AML cohort. Along with mutations in KRAS- and MYC-oncogenic pathways, which are known to control metabolic processes, we identified a novel functional category of mutated genes involved in metabolism (MALG) in AML. Our results may suggest different types of metabolic remodeling across leukemia subgroups. Mutations targeting a common downstream metabolic pathway are mutually exclusive in our cohort, as shown by KRAS and genes involved in glucose-dependent bioenergetic processes. Glucose metabolism predicts clinical outcome and chemotherapy response in AML (Chen et al. Blood 2014). Our data further suggest that the mutational screening of glucose-related MALGs may define a new subgroup of patients, which could not be identified by cytogenetic analysis. These findings may have implication for AML treatment, since metabolic alterations and genomic determinants of metabolic remodeling are promising targets for tailored therapies, as recently shown for glutaminase and IDH1/2 inhibitors. Acknowledgments: EHA Research Fellowship award, FP7 NGS-PTL project, ELN, AIL, AIRC, progetto Regione-Università 2010-12 Disclosures Soverini: Novartis, Briston-Myers Squibb, ARIAD: Consultancy. Cavo:JANSSEN, CELGENE, AMGEN: Consultancy. Martinelli:Novartis: Consultancy, Speakers Bureau; Pfizer: Consultancy; MSD: Consultancy; Ariad: Consultancy; BMS: Consultancy, Speakers Bureau; ROCHE: Consultancy; AMGEN: Consultancy.