Additional Genomic Aberrations Research Articles

Conventional Cytogenetic Analysis and Array CGH+SNP Identify Essential Thrombocythemia (ET)/Prefibrotic Primary Myelofibrosis (pre-PMF) Patients Who Are at Risk for Disease Progression

ET and pre-PMF are myeloproliferative neoplasms (MPNs) that are each characterized by thrombocytosis but are difficult to distinguish by clinical and histopathological criteria. Moreover, each has the propensity to progress at variable rates to more advanced forms of the disease. The cause of progression to more overt forms of myelofibrosis (MF) or accelerated /blast phase (AP/BP) has yet to be fully understood, making prognostication difficult. To identify patients (pts) who are at risk for disease progression, we retrospectively reviewed the karyotypes from conventional cytogenetics (CC) and array Comparative Genomic Hybridization + Single Nucleotide Polymorphism (aCGH+SNP) of 158 pts with a diagnosis of ET or pre-PMF from 2017 to 2022 at our institution. Among these pts, 58% were female (92 pts) and 42% were male (66 pts), and the median age was 61 years (range 13-92). Mutational analysis was performed in 155 pts, and JAK2 mutations were present in 63% of cases, CALR in 19%, and MPL in 7% of cases. The remaining 11% of pts were triple negative. An abnormal karyotype was observed in 15% (24/158), and the remaining 85% (134/158) were either normal or had non-diagnostic results due to failed or limited peripheral blood chromosome analysis. Of the 111 pts that had aCGH+SNP testing, 6% (7/111) had prior abnormalities as detected by conventional cytogenetic analyses, while abnormalities were found in 30% (33/111) with aCGH+SNP exclusively. In this cohort, 29% (46/158) of pts underwent disease progression. Among these cases, 65% (30/46) had progression to either MF or chronic myeloid leukemia or myelodysplasia, and 22% (10/46) progressed to AP/BP. In addition, 13% (6/46) evolved to polycythemia vera. Disease progression, irrespective of its form, was associated with genomic alterations detected by aCGH+SNP, cytogenetic analysis, or both at the time of first encounter. Of the 46 pts undergoing progression during their clinical course, 65% (30/46) had an abnormal genomic finding by either technology compared to 35% (16/46) who were normal (p<0.001). Strikingly, among 24 pts who were abnormal by classical cytogenetics, 79% (19/24) progressed to either AP/BP or MF compared to only 20% (26/125) who were cytogenetically normal (p<0.001). As shown in Figure 1, there was a shorter progression-free survival among pts who were cytogenetically abnormal as compared to normal pts (p<0.001). Among 134 pts with ET/pre-PMF who were normal or had non-diagnostic results by classical cytogenetic studies, aCGH+SNP testing was performed in 104 cases and additional genomic aberrations were detected in 32% (33/104). Within this group, 30% (10/33) progressed to MF with a trend towards a shorter progression-free survival. In those patients that progressed to MF, the most frequent abnormality was 9p CNLOH occurring in 6 pts. In the remaining 71 pts that were cytogenetically and aCGH+SNP normal, only 11% (8/71) had disease progression. Overall, the combination of both conventional cytogenetics and aCGH+SNP increased the sensitivity of detecting genomic abnormalities from 15% (cytogenetics alone) to 37% when incorporating aCGH+SNP. These data indicate that genomic alterations detected by conventional cytogenetic analyses or CGH+SNP array identify a subset of ET/pre-PMF pts that are at a higher risk of disease progression.

Polytypic B-Cells, B-Cell Lymphoproliferative Disorders/Lymphomas, and Neoplastic T-Cells Divergently Differentiate from TET2-/ DNMT3A-Mutated Clonal Hematopoiesis in Patients with Follicular Helper T-Cell Lymphomas/Lymphoproliferative Disorders

Introduction: Patients with follicular helper T-cell lymphomas/lymphoproliferative disorders (TFH-LPDs) commonly develop B-cell lymphoproliferative disorders and lymphomas (B-LPDs), but the pathophysiology is incompletely understood. TFH-LPDs often arise from TET2-/ DNMT3A-mutated clonal hematopoiesis (CH). Here we show TET2/ DNMT3A mutations are commonly shared by polytypic B-cells (PBCs), B-LPDs, and TFH-LPDs, with the latter two often carrying additional unique mutations. Methods: Pathology database search at Memorial Sloan Kettering identified patients with TFH-LPDs and PBCs or B-LPDs available for genotyping. If available, myeloid cells were also genotyped. PBCs lacked aberrancies by morphology and flow cytometry (FC) immunophenotyping (including polytypic light chain expression). TFH-LPDs and B-LPDs were defined using International Consensus Classification criteria. Genotyping assays included targeted next-generation sequencing (NGS) with (n=13) or without (n=8; cases 1, 4, 9, 10, 15, 16, 17, 22) a matched germline control and droplet digital PCR (n=4; cases 2, 3, 5, 6). Genotyping was performed on bulk samples or FC-sorted cells, including T-, B-, and myeloid as available. In bulk samples, mutations with allele frequencies (VAFs) >2x the percentage of neoplastic T- and myeloid cells combined in B-LPD samples and of neoplastic T- and total B-cells in bulk blood (PB)/bone marrow (BM) samples were considered present in B-LPD or myeloid compartments, respectively. A >2% VAF cutoff was utilized. EBV status was assessed using EBER in situ hybridization on tissues, serum EBV DNA by quantitative PCR, and/or evaluation of off target EBV reads by NGS. Statistical analyses utilized Mann-Whitney U tests. Results: In total, 25 patients (10 female, 15 male; median age 70 [range 38-82] years) with TFH-LPDs (19 TFH lymphoma, angioimmunoblastic type, 4 TFH lymphoma, NOS, 2 T-LPD with TFH phenotype [defined by morphologically and/or immunophenotypically atypical T-cells with both TFH-like phenotypes and genomic alterations that did not fulfill morphologic criteria for diagnosis of lymphoma]) were identified. The TFH-LPDs harbored TET2 (25/25, 100%), DNMT3A (12/25, 48%), RHOA p.G17 (15/25, 60%), and IDH2 p.R172 (9/25, 36%) mutations (Figure 1A). PBCs from 11 patients were evaluated (7 EBV+, 3 EBV-, 1 EBV unknown; 5 from lymph node, 4 PB, 2 BM). PBCs from 7/11 (64%) shared identical TET2 (n=6; median VAF 0.11 [range 0.02 - 0.27]) and/or DNMT3A (n=3; median VAF 0.05 [range 0.03-0.49]) mutations with corresponding TFH-LPDs. PBCs in 1 patient harbored a unique mutation ( TET2). Four of 8 (50%) patients in which myeloid cells were assessed harbored identical TET2 and/or DNMT3A mutations in the myeloid, PBC, and TFH-LPD compartments. B-LPDs were evaluated from 14 patients (5 diffuse large B-cell lymphoma, 5 polymorphic B-LPD, 2 follicular lymphoma, 1 B-LPD, NOS, 1 plasma cell myeloma; 11 EBV+, 3 EBV-; 9 from tissue, 4 PB, 1 BM). Polymorphic B-LPDs showed atypical polymorphic morphology and an abnormal B-cell immunophenotype by FC and/or a clonal IgH gene rearrangement. The B-LPD, NOS showed an abnormal CD5-/CD10- immunophenotype with light chain restriction by FC without available tissue for morphologic evaluation. Nine of 14 (64%) B-LPDs shared identical mutations with corresponding TFH-LPDs ( TET2 [n=9; median VAF 0.42 (range 0.10- 0.56)], DNMT3A [n=5; median VAF 0.27 (range 0.05-0.50)], TET3 [n=1; VAF 0.32]). Myeloid compartments in 9/12 (75%) patients shared identical TET2 and/or DNMT3A mutations with B- and/or T-LPDs. B-LPDs more commonly harbored additional mutations absent in corresponding TFH-LPDs than PBCs (12/14 [86%] vs 1/8 [13%] evaluated by NGS; p=0.001). Unique mutations in B-LPDs involved epigenetic/transcriptional regulation (n=14, e.g. TET2, KMT2D, SETD5), signaling pathways (n=22, e.g. DTX1, KRAS, EPHA5), and DNA damage response (n=3, e.g. ATM, CHEK2, BRCA2). Incidences of shared and unique mutations did not significantly differ among EBV+ and EBV- cases. Conclusions: Both PBCs and B-LPDs in TFH-LPD patients are commonly EBV+ and share TET2/ DNMT3A mutations with TFH-LPDs, consistent with common origin from CH. B-LPDs more often harbor additional unique mutations than PBCs. Thus, CH mutations and EBV activation in B-cells may predispose TFH-LPD patients to B-LPDs, which may develop with additional private genomic aberrations aiding transformation (Figure 1B).

Loss of Function of SETD2 Tumor Suppressor in Chronic Myeloid Leukemia (CML) Progenitors Fosters Genomic Instability and Enhances Clonogenic Potential

BACKGROUND AND AIMS - Genomic instability is a hallmark of chronic myeloid leukemia (CML) cells since the chronic phase (CP) of the disease, and results in BCR::ABL1 mutations and/or additional genetic and genomic aberrations that may drive resistance to tyrosine kinase inhibitors (TKIs) and progression to blast crisis (BC). Genomic instability is also a feature of CML stem and progenitor cells and may contribute to their persistence. The SETD2 tumor suppressor codes for a protein that trimethylates histone H3 at lysine 36 (H3K36me3). In solid tumors, SETD2 loss of function has been shown to impair H3K36me3-mediated recruitment of DNA damage response components. We have recently reported that non genomic loss of function of SETD2 is a feature of BC CML and results from premature proteasome-mediated degradation of the SETD2 protein triggered by Aurora kinase A phosphorylation and MDM2 ubiquitination. In the present study, we aimed to assess SETD2/H3K36me3 status in CD34+ progenitors of CP CML patients (pts) and whether SETD2/H3K36me3 deficiency may play a role in genomic instability in CML models. METHODS - Western blotting (WB) was used to assess SETD2 protein expression and H3K36me3 as a surrogate marker of SETD2 function in the CD34+ cell fraction isolated from the bone marrow of 20 newly diagnosed CP CML pts and from a pool of healthy donors (HD). SETD2 forced expression in CD34+ progenitors from newly diagnosed CP CML pts and in the SETD2-deficient KCL22 cell line was performed by nucleofection. SETD2 knock-down in the SETD2-proficient LAMA84 cell line was performed by RNAi. Clonogenic capacity was evaluated by clonogenic assays. Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K36me3 was performed on an Illumina HiSeq2000 with a min 50 million 150-bp single-end reads per replicate. High resolution karyotyping wias perfomed with Cytoscan HD arrays. DNA damage and DNA repair activation were assessed in primary samples and cell lines by WB and immunofluorescence (IF) using antibodies specific for phospho-H2AX, mismatch repair (MMR) and homologous recombination repair (HR) proteins. RESULTS - WB demonstrated a marked down-modulation of SETD2 expression, paralleled by H3K36me3 deficiency, in the CD34+ cells of all newly diagnosed CP CML pts as compared to the total mononuclear fraction and to CD34+ cells from HDs. To investigate whether SETD2 loss affects the activation and proficiency of HR and MMR, we used chronic exposure to UV rays or a single exposure to hydrogen peroxide (1mM for 30 and 60 min). Both induced DNA damage in SETD2 siRNA-depleted LAMA84 cells. Compared to parental cells, cells silenced for SETD2 failed to activate the ATM-dependent repair pathway. Moreover, we found that ATM inactivation prevents H2AX foci formation, associated with a loss of RAD51 and RAD54 (HR) and MSH6 (MMR) repair foci. To confirm the hypothesis that SETD2 is a tumor suppressor implicated in maintaining genomic stability in CML, we transfected SETD2-deficient KCL22 cells with an ectopic SETD2 plasmid. SETD2 forced expression was able to restore DNA damage response, as demonstrated by WB and IF detection of ATM, p95 and H2AX phosphorylation, BRCA1, BRCA2 and CtIP expression and finally RAD51 and RAD54 localization on HR repair foci observed after UV or hydrogen peroxide exposure. High-resolution karyotyping after DNA damage showed increased rate of DNA breakpoints in SETD2-deficient cells, preferentially occuring at loci where H3K36me3 marks were lost as assessed by ChiP-seq. In line with the effects observed in cell line models, forced expression of SETD2 in CD34+ cells from 5 CP CML pts was found to restore proliferation control, since a >50% reduction in clonogenic potential was observed after nucleofection. CONCLUSIONS - Our findings demonstrate that SETD2 is a bona fide tumor suppressor in CML progenitors and establish a functional link between SETD2 loss of function and genomic instability. SETD2 inactivation may thus play a pivotal role in the harmful cascade of events that may foster drug resistance and ultimately lead to disease progression. Since SETD2 loss of function in CML is mediated by post-translation mechanisms, hence is reversible, therapeutic strategies aimed at interfering with these mechanisms may restore proliferation control and interrupt this cascade. Supported by AIRC IG 2019 (23001) and by Italian Ministry of Health, “Bando Ricerca Finalizzata 2016”, project GR-2016-02364880.

Loss of the KN Motif and AnKyrin Repeat Domain 1 (KANK1) Leads to Lymphoid Compartment Dysregulation in Murine Model.

The KN Motif and AnKyrin Repeat Domain 1 (KANK1) is proposed as a tumour suppressor gene, as its expression is reduced or absent in several types of tumour tissue, and over-expressing the protein inhibited the proliferation of tumour cells in solid cancer models. We report a novel germline loss of heterozygosity mutation encompassing the KANK1 gene in a young patient diagnosed with myelodysplastic neoplasm (MDS) with no additional disease-related genomic aberrations. To study the potential role of KANK1 in haematopoiesis, we generated a new transgenic mouse model with a confirmed loss of KANK1 expression. KANK1 knockout mice did not develop any haematological abnormalities; however, the loss of its expression led to alteration in the colony forming and proliferative potential of bone marrow (BM) cells and a decrease in hematopoietic stem and progenitor cells (HSPCs) population frequency. A comprehensive marker expression analysis of lineage cell populations indicated a role for Kank1 in lymphoid cell development, and total protein analysis suggests the involvement of Kank1 in BM cells' cytoskeleton formation and mobility.

The oncogenic driving force of CD30 signaling-induced chromosomal instability in adult T-cell leukemia/lymphoma.

Adult T-cell leukemia/lymphoma (ATL) develops via stepwise accumulation of gene mutations and chromosome aberrations. However, the molecular mechanisms underlying this tumorigenic process are poorly understood. We previously reported the presence of a biological link between the expression of CD30, which serves as a marker for ATL progression, and the actively proliferating fraction of human T-cell leukemia virus type 1 (HTLV-1)-infected cells that display polylobulation. Here, we demonstrated that CD30 signaling induced chromosomal instability with clonal expansion through DNA double-strand breaks (DSBs) via an increase of intracellular reactive oxygen species. CD30+ ATL cells were composed of subclones with additional genomic aberrations compared with CD30- ATL cells in ATL patients. Furthermore, we found an accumulation of copy number loss of DSB repair-related genes as the disease progressed. Taken together, CD30 expression on ATL cells appears to be correlated with genomic instability, suggesting that CD30 signaling is one of the oncogenic factors of ATL progression with clonal evolution. This study provides new insight into the biological roles of CD30 signaling and could improve our understanding of tumorigenic processes of HTLV-1-infected cells.

Targeted genomic profiling revealed a unique clinical phenotype in intrahepatic cholangiocarcinoma with fibroblast growth factor receptor rearrangement

Genomic aberrations (GAs) in fibroblast growth factor receptors (FGFRs) are involved in the pathogenesis of intrahepatic cholangiocarcinoma (ICC), and clinical trials have shown efficacy of FGFR inhibitors in treating ICC patients with FGFR GAs such as FGFR2 rearrangement. To clarify the FGFRs GA profile and corresponding clinicopathological features in Chinese patients with ICC, a total of 257 cases were identified. Fourteen cases (5.45%) were positive for FGFR2 rearrangement. Further analysis on the 110 FGFR2 rearrangement negative cases showed that 13 patients present additional FGFRs GAs, including FGFR3 rearrangement (2.73%), and FGFRs mutations. When compared with patients without FGFRs GAs, those with FGFR2 or FGFR3 rearrangement presented more under the age of 58 years, female sex, HBsAb positivity, CD10 expression, and PD-L1 expression. The clinical characteristics between patients with FGFRs mutation and those without FGFRs GAs were similar, with the exception that cases with FGFRs mutation have more hepatolithiasis. We concluded that FGFR rearrangement is associated with unique clinical phenotypes in ICC.

Characterization of RB1 in Pediatric TCF3-PBX1+ acute Lymphoblastic Leukemia

Introduction. Genomic alterations are frequently associated with acute lymphoblastic leukemia (ALL) prognosis. TCF3-PBX1 fusion is one of these alterations associated with B-cell precursor ALL (BCP-ALL) subtype. Additional genomic aberrations, observed in most pre-leukemic clones, are crucial in both BCP-ALL leukaemogenesis and treatment management. Aims. To explore the role of deletions in genes involved in lymphoid differentiation, cell cycle regulation and cytokine receptors on BCP-ALL TCF3-PBX1 + patients; To evaluate the difference in the gene expression profile of genes involved in cell cycle regulation when comparing patients with and without RB1 deletions (RB1del); To correlate the alterations found with clinical-laboratorial characteristics and with risk stratification variables. Methods. BCP-ALL patients younger than 18 years-old and without previous leukemic treatment were included in the present study. Submicroscopic deletions were identified by multiplex ligation-dependent probe amplification (MLPA) using SALSA MLPA P335 A4/B1 kit. RB1delwill be confirmed by FISH using in-house probes targeting the regions of interest in RB1 . Gene expression of genes involved in cell cycle regulation was assessed using the TaqMan Array 96-well plate - Human cyclins & Cell Cycle Regulation. Univariable analyses of characteristics were performed using the Fisher's exact test or χ2 test for qualitative variables using GraphPad Prism software. Results. Fifty cases harboring the TCF3-PBX1 (6.9% of the overall BCP-ALL series) were identified in the present study. They were predominantly male, aged 1-10 years-old, with WBC count ≤50x109/L and classified as pre-B ALL. Additional alterations in those TCF3-PBX1 + cases were identified by MLPA, with the most frequent alterations affecting genes involved in cell cycle regulation and lymphoid differentiation: CDKN2A/Bdel (40.0%) and PAX5del (37.7%), followed by RB1del (31.1%) and BTG1del (17.7%). The frequency of RB1del in TCF3-PBX1 +patients was markedly different from those with other cytogenetic subgroups of BCP-ALL (P <0.05). RB1del cases were mainly female (68.7% vs 31.3%) and molecular characterized by 9p21 deletions (37.5% CDKN2A/Bdel plus PAX5del). FISH analyses revealed heterogeneity of nuclei harbouring RB1del, varying from 12% to 97% of the interphase nucleus evaluated. Regarding the expression profile, we observed that the presence of RB1del is associated with significantly increased expression of CCND2 (P =0.032) , while the expression of CDKN2D was reduced compared to cases without RB1del (P =0.043). Additionally, we observed that the complete deletion of RB1 (involving all exons) was significantly associated with increased expression of CCNA2, CCNB1 , CDC2 and E2F3 , as a consequence of the loss of critical phosphorylation sites that regulate pRB interactions (P <0.05). On the other hand, RB1 partial deletion (involving exons 17 to 26) was associated with the reduction of CDKN2D expression. Conclusions.RB1 deletions were frequently found as an additional aberration particularly in TCF3-PBX1 + patients. Deletions in 9p21 locus were also frequently found in this cytogenetic subgroup. The loss of critical RB1 phosphorylation sites, as a consequence of RB1del affecting all exons, deregulate the expression of E2F3 , an important transcript factor that interacts directly with pRB and regulate the expression of other genes involved in the cell cycle, such as cyclins A2 and B1 , and CDC2 , essentials for G1/S and G2/M phase transitions. DisclosuresNo relevant conflicts of interest to declare.

P76.43 Co-occurring genomic alterations and treatment outcomes in patients with EGFR exon 20 insertion positive NSCLC

NSCLC harboring an EGFR exon 20 insertion (ex20ins) often has additional genomic aberrations, which may influence the underlying biology and predict outcomes. Clinical outcomes data overall remain limited in patients (pts) with ex20ins and comprehensive characterization of these tumors is lacking. NSCLC samples were profiled by targeted NGS (Caris Life Sciences, Phoenix, AZ). Mutations and copy number variations (CNV) were assessed. Gene amplification was defined as gene copy number ≥ 6 and copy number loss as gene copy number < -1.4. Clinical outcomes data were obtained through an IRB-approved retrospective chart review. Among 3,359 pts with NSCLC harboring an EGFR mutation, 250 (7.4%) had EGFR ex20ins. 170 (68%) were females with a mean age 65 (range 29-90). 60 different ex20ins variants were identified with the most common being A767_V769dup (25%), followed by S768_D770dup (18%), N771_H773dup (7%), H773_dup (6%) and A763_Y764insFQEA (3%). 4% had compound ex20ins mutations. The most common co-mutations included TP53 (56.6%), followed by CTNNB1 (6.6%), RB1 (5.1%), PIK3CA (4.7%) and CDKN2A (4.3%). Commonly amplified genes included EGFR (9.6%), FOXA1 (4%), MDM2 (3.6%) and CDK4 (3.5%). Copy number loss was observed in CDKN2A (4.6%), BRCA2 (1.1%), FANCL (1.1%), CTNNB1 (0.5%), and ATR (0.5%). PD-L1 expression by 22C3 was high (TPS ≥ 50%) in 16%, low (1-49%) in 27%, and negative in 57%. Clinical data were obtained from 26 pts with advanced NSCLC with ex20ins mutations from four institutions. In the first-line setting, 12 (46%) received chemotherapy, 10 (39%) received an EGFR-TKI and 4 (15%) received chemo-IO. Mean PFS was 9.3 months with chemotherapy-based regimens and 6 months with TKI therapy. One patient with P772_H773dup treated with TKI achieved a partial response. Across all treatments, mean PFS was 8 months and mean OS was 28.3 months. The diverse spectrum of EGFR ex20ins mutations demonstrates notable molecular heterogeneity within this uncommon EGFR mutation. PFS and OS in this population remain poor. Understanding the role of co-occurring genomic alterations and their impact on outcomes may have therapeutic implications on clinical trial development.

Impact of Autologous Transplantation in Patients with Multiple Myeloma with t(11;14): A Propensity-Score Matched Analysis.

Patients with multiple myeloma with t(11;14) have been considered to have standard-risk disease. However, several recent reports have shown contradictory results. We identified 95 patients with multiple myeloma with t(11;14) on FISH studies, who underwent upfront autologous hematopoietic stem cell transplant (auto-HCT) at our center. We compared their outcome with a group of standard-risk patients with multiple myeloma who had diploid cytogenetics by both conventional cytogenetics (CC) and FISH (n = 287). To reduce the bias between the groups, we performed a 1:1 propensity score matching technique for analysis. A total of 160 patients, 80 in each group, were identified. Patients in the 2 groups were matched for age, International staging system stage at diagnosis, serum creatinine at presentation, disease status at auto-HCT, type of preparative regimens, dose of melphalan used for conditioning, and induction and maintenance regimens. Patients in t(11;14) group had a post auto-HCT overall response rate (ORR) of 97.5% (78/80), compared with 100% (80/80) in the standard-risk control group (P = 0.50). Complete response rate in the t(11;14) group was 35% (28/80), compared with 45% (36/80) in the standard-risk control group (P = 0.26). The 4-year PFS rates were 40.8% (95% CI, 29.6%-56.1%) and 51.1% (95% CI, 39.4%-66.3%) in the t(11;14) and standard-risk control groups, respectively (P = 0.14). The 4-year OS rates were 74.9% (95% CI, 63.3%-88.7%) and 88.3% (95% CI, 80.4%-97.0%) in the t(11;14) and standard-risk control groups, respectively (P = 0.17). Also, patients with t(11;14) with concurrent cytogenetics had significantly poor PFS and OS compared with a propensity matched standard-risk control group. Our study confirms that t(11;14) multiple myeloma undergoing upfront autologous transplantation had similar outcomes as patients with multiple myeloma with normal cytogenetic and FISH studies. Existence of additional genomic aberrations by CC or FISH was associated with a worse outcome.

SeqPlus sequencing methodology enables robust whole-genome sequencing, true variant detection, and novel genomic insights from archival esophageal carcinoma FFPE samples.

e13016 Background: Whole-genome sequencing (WGS) of formalin-fixed, paraffin-embedded (FFPE) samples could enable novel insights from archival sample collections, yet robust FFPE WGS is challenged by fragmented DNA, uneven genomic coverage &amp; sequencing artifacts attributed to FFPE fixation. We report our proprietary extraction &amp; library preparation methodology (SeqPlus) with high quality, uniform WGS sequencing performance comparable to that from fresh-frozen samples. Methods: We analyzed 20 paired esophageal carcinoma (EC) samples i.e., primary tumors &amp; matched germline samples to assess SeqPlus performance on 10-15-year-old FFPE tissues, measure variant concordance between WGS and a high-depth sequencing panel (269 genes, 400x coverage) &amp; identify novel genomic features. Results: At a targeted 70x WGS tumor sequencing depth, 93% of the genome was covered by ³ 20 reads, 99% of bases had 10x coverage &amp; average duplicate reads were 31%. We noted similar transition/transversion ratios &amp; mutational spectra as from fresh-frozen EC specimens, suggesting that extraction &amp; library preparation contributes to prior FFPE artifacts. Concordance of tumor-specific SNVs &amp; indels derived from WGS &amp; targeted panel was high at 86%. All 76 targeted panel-detected variants above the WGS limit of detection (mutant allele frequency [MAF] &gt; 10%) were detected by WGS, 2 variants (2 tumors) were detected only by WGS, and 12 variants at MAF ≤ 6% (9 tumors) were only detected by the targeted panel. Tumor WGS yielded SNV, indels &amp; CNV findings beyond variants detected by targeted sequencing. WGS enabled detection of 10.4 putative cancer variants per tumor compared to 12 variants per patient from frozen specimens and a median of 7 (up to 16) cancer-associated variants in genes outside the targeted panel. WGS copy number analysis revealed CCND1, EGFR, TP63, and SOX2amplification, CDKN2A/B deletion and additional unrecognized genomic aberrations. Conclusions: Our study reinforces the utility of high-quality, uniform WGS sequencing of archival FFPE cancer samples with SeqPlus and unlocks the potential for massive-scale retrospective genomic analysis of archived pathology samples with associated clinical &amp; outcomes data.

A novel PAX5 rearrangement in TCF3-PBX1 acute lymphoblastic leukemia: a case report

BackgroundChromosome translocations are a hallmark of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). Additional genomic aberrations are also crucial in both BCP-ALL leukemogenesis and treatment management. Herein, we report the phenotypic and molecular cytogenetic characterization of an extremely rare case of BCP-ALL harboring two concomitant leukemia-associated chromosome translocations: t(1;19)(q23;q13.3) and t(9;17)(p13;q11.2). Of note, we described a new rearrangement between exon 6 of PAX5 and a 17q11.2 region, where intron 3 of SPECC1 is located. This rearrangement seems to disrupt PAX5 similarly to a PAX5 deletion. Furthermore, a distinct karyotype between diagnosis and relapse samples was observed, disclosing a complex clonal evolution during leukemia progression.Case presentationA 16-year-old boy was admitted febrile with abdominal and joint pain. At clinical investigation, he presented with anemia, splenomegaly, low white blood cell count and 92% lymphoblast. He was diagnosed with pre-B ALL and treated according to high risk GBTLI-ALL2009. Twelve months after complete remission, he developed a relapse in consequence of a high central nervous system and bone marrow infiltration, and unfortunately died.ConclusionsTo our knowledge, this is the first report of a rearrangement between PAX5 and SPECC1. The presence of TCF3-PBX1 and PAX5-rearrangement at diagnosis and relapse indicates that both might have participated in the malignant transformation disease maintenance and dismal outcome.

OA10.07 Report on Liquid Biopsies from Advanced Lung Adenocarcinoma Patients and Correlation with Their Tumor Biopsy Profiles

Liquid biopsy (LBx) has emerged as an alternative tool for the management of advanced lung cancer patients (pts) identifying driver mutations, and hence, improving personalized medicine. There are still controversial issues such as standardization, validation of different technologies, concordance with tissue molecular profile results (TMPR), and others. LBx offers many advantages including non-invasive, bypass tumor heterogeneity, an opportunity for serial measurements to evaluate response or early recurrence, and others. Guardant 360 was analyzed in 100 consecutive stage IV or recurrent lung adenocarcinoma (adeno) pts. Guardant 360 is a panel of 70 genes including single nucleotide variations, amplifications, translocations, and short insertions/duplications/deletions in exons 19 and 20 of the EGFR, and others. Cell-free DNA (cfDNA) is extracted from plasma and genomic alterations are analyzed by massively parallel sequencing of amplified target genes. TMPRs from each subject was obtained or recovered for comparison with their LBx counterparts. TMPRs from this cohort was developed in different CLIA laboratories. 69 pts were females; median age 72 (range, 27-99). 84/100 pts had at least 1 genomic alteration by LBx (range, 1-10). Most common abnormalities found in LBx were: TP53 (37 pts), EGFR (35 pts), NF1 (20 pts), KRAS (12 pts), MET (14 pts). From this 84 pts with + LBx results, 67 pts (80%) had TMPRs for comparison. Main reason for lack of TMPRs: insufficient tumor (19/100; 19%). For comparison between the 2 modalities, we considered all pts with available results in both tests; hence, 81 pts were used to compare tumor biopsy (TBx) vs. LBx. 37 pts out of 81 (46%) had at least 1 similar genomic abnormality found in both TBx and LBx, respectively. Most of the concordance was in EGFR alterations (19/28; 68%). LBx caught 16 additional EGFR genomic aberrations not being identified by TBx. A total of 35 EGFR genomics aberrations were identified in LBx; 16/35 EGFR mutations found in LBx were actionable and 5 of these 16 actionable EGFR mutant cases were only found in LBx not in TBx. LBx offers an alternative to identify genomic alterations. Still, insufficient tumor is the major reason for lacking of TMPRs. EGFR mutations are the most common actionable mutations found in LBx; also, it has a high correlation with TBx (68%). LBx identified more gene abnormalities than TBx, and in some cases, the actionable EGFR mutations were found only in LBx sample.

P1.29: “Real World” Use of Liquid Biopsy in Patients With Lung Adenocarcinoma and Correlation With Tumor Tissue Genetic Profile

Several genomic abnormalities have been discovered in adenocarcinoma of the lung (adenoca) in the last year; however, adequate quantity of tumor tissue for molecular analysis is a major handicap to offer most of the patients (pts) a personalized medicine. Reasons such as difficulty to perform tumor biopsy (TBx) either because of tumor location or patient’s co-morbid conditions are among the common factors for lack of molecular profiling results (MPR). Hence, liquid biopsy (LBx) has emerged as a potential alternative to detect these genomic alterations. We analyzed 81 consecutive pts to whom a LBx using Guardant 360 test was ordered in our thoracic oncology clinics at Lynn Cancer Institute, Boca Raton, Florida and Memorial Cancer Institute, Hollywood, Florida. Results from tissue molecular profiling from each subject was obtained or recovered for comparison. MPRs from this cohort was developed by different CLIA laboratories (e.g. Response Genetics, Caris, Foundation Medicine, BioTheranostics, and Genoptix). For LBx analysis, only Guardant 360 test was considered. The Guardant 360 test assays a panel of 70 genes (see Fig. 1) to identify genomic alterations in cancer-associated somatic variants with high sensitivity. Cell-free DNA (cfDNA) is extracted from plasma and genomic alterations are analyzed by massively parallel sequencing of amplified target genes. The distribution by gender was 56 female patients and 25 males; median age 71 (range, 27-99). 65/81 pts (80%) had at least 1 genomic alteration by LBx (range, 1-10). Most common abnormalities found in LBx were: TP53 (32 pts), EGFR (27 pts), NF1 (16 pts), KRAS (11 pts), MET (10 pts). From this 65 pts with + LBx results, 49 pts (75%) had tumor tissue MPRs for comparison. Major reason for lack of tumor tissue MPRs: insufficient tumor (18/81; 22%). For comparison between the 2 modalities, we considered all pts with available results in both tests; hence, 63 pts were used to compare TBx with LBx. 33 pts out of 63 (52%) had at least 1 similar genomic abnormality or MPRs found in both TBx and LBx. Most of the concordance was in EGFR alterations (17/22; 77%). LBx caught 10 additional EGFR genomic aberrations not being identified by TBx (a total of 27 EGFR genomics aberrations were identified in LBx). 14 EGFR found in LBx were actionable; 3/10 of mutant EGFR found only in LBx were actionable. LBx using Guardant 360 evaluation offers an alternative to identify genomic alterations including actionable mutations; still, insufficient tumor is the major reason for lacking of tumor MPRs and more advances to obtain TBx are needed. Our cohort had 48% concordance between LBx and TBx.

Establishment of SPAST mutant induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia (HSP) patient

Human skin fibroblasts were isolated from a 40-year-old hereditary spastic paraplegia patient carrying an intronic splice site mutation (c.1687+2T>A) in SPAST, leading to hereditary spastic paraplegia type 4 (SPG4). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-SPG4-splice retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG4-splice line might be a useful platform to study the pathomechanism of SPG4.

Generation of induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia patient carrying a homozygous R486C mutation in CYP7B1 (SPG5)

Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation R486C in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-R486C was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-R486C line may be a useful resource for disease modelling of SPG5.

Generation of induced pluripotent stem cells (iPSCs) from a hereditary spastic paraplegia patient carrying a homozygous Y275X mutation in CYP7B1 (SPG5)

Skin fibroblasts were obtained from a 47-year-old hereditary spastic paraplegia patient carrying a homozygous mutation Y275X in CYP7B1 (Cytochrome P450, Family 7, Subfamily B, Polypeptide 1), responsible for causing hereditary spastic paraplegia type 5 (SPG5). Induced pluripotent stem cells (iPSCs) were generated by transfection with episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated line iPS-SPG5-Y275X was transgene-free, retained the specific mutation with no additional genomic aberrations, expressed pluripotency markers and was able to differentiate into cells of all germ layers in vitro. The generated iPS-SPG5-Y275X line may be a useful resource for disease modelling of SPG5.

Generation of optic atrophy 1 patient-derived induced pluripotent stem cells (iPS-OPA1-BEHR) for disease modeling of complex optic atrophy syndromes (Behr syndrome)

Human skin fibroblasts were isolated from a 48-year-old patient carrying compound heterozygous mutations (c.610+364G>A and c.1311A>G) in OPA1, responsible for early onset optic atrophy complicated by ataxia and pyramidal signs (Behr syndrome; OMIM #210000). Fibroblasts were reprogrammed using episomal plasmids carrying hOCT4, hSOX2, hKLF4, hL-MYC and hLIN28. The generated transgene-free line iPS-OPA1-BEHR showed no additional genomic aberrations, maintained the disease-relevant mutations, expressed important pluripotency markers and was capable to differentiate into cells of all three germ layers in vitro. The generated iPS-OPA1-BEHR line might be a useful platform to study the pathomechanism of early onset complicated optic atrophy syndromes.

Genomic aberrations in spitzoid melanocytic tumours and their implications for diagnosis, prognosis and therapy

Histopathological evaluation of melanocytic tumours usually allows reliable distinction of benign melanocytic naevi from melanoma. More difficult is the histopathological classification of Spitz tumours, a heterogeneous group of tumours composed of large epithelioid or spindle-shaped melanocytes. Spitz tumours are biologically distinct from conventional melanocytic naevi and melanoma, as exemplified by their distinct patterns of genetic aberrations. Whereas common acquired naevi and melanoma often harbour BRAF mutations, NRAS mutations, or inactivation of NF1, Spitz tumours show HRAS mutations, inactivation of BAP1 (often combined with BRAF mutations), or genomic rearrangements involving the kinases ALK, ROS1, NTRK1, BRAF, RET, and MET. In Spitz naevi, which lack significant histological atypia, all of these mitogenic driver aberrations trigger rapid cell proliferation, but after an initial growth phase, various tumour suppressive mechanisms stably block further growth. In some tumours, additional genomic aberrations may abrogate various tumour suppressive mechanisms, such as cell-cycle arrest, telomere shortening, or DNA damage response. The melanocytes then start to grow in a less organised fashion and may spread to regional lymph nodes, and are termed atypical Spitz tumours. Upon acquisition of even more aberrations, which often activate additional oncogenic pathways or alter cell differentiation, the neoplastic cells become entirely malignant and may colonise and take over distant organs (spitzoid melanoma). The sequential acquisition of genomic aberrations suggests that Spitz tumours represent a continuous biological spectrum, rather than a dichotomy of benign versus malignant, and that tumours with ambiguous histological features (atypical Spitz tumours) might be best classified as low-grade melanocytic tumours. The number of genetic aberrations usually correlates with the degree of histological atypia and explains why existing ancillary genetic techniques, such as array comparative genomic hybridisation (CGH) or fluorescence in situ hybridisation (FISH), are usually capable of accurately classifying histologically benign and malignant Spitz tumours, but are not very helpful in the diagnosis of ambiguous melanocytic lesions. Nevertheless, we expect that progress in our understanding of tumour progression will refine the classification of spitzoid melanocytic tumours in the near future. By integrating clinical, pathological, and genetic criteria, distinct tumour subsets will be defined within the heterogeneous group of Spitz tumours, which will eventually lead to improvements in diagnosis, prognosis and therapy.

Abstract 3294: Clonal evolution and integral analysis of hepatoblastoma

Abstract [Background] Hepatoblastoma (HB) is a rare and heterogeneous childhood malignancy of liver. It is mainly affecting young children in the first 2 years of life, underscoring the unique sensitivity of the immature liver to tumorigenesis. The unique age range affected by this embryonal tumor suggests that developmental factors contribute to the tumor development. On the other hands it is well recognized activation of Wnt/β-catenin signaling pathway was frequently occurred in HB through somatic mutation of CTNNB1 and germline mutation of APC; however, the exact mechanism that normal hepatic developmental processes are altered by mutational events is an area of current research and the definitive contribution to tumorigenesis is unclear mainly due to its rarity. [Methods] To elucidate the genetic aberration underlying development and progression of HB, we performed whole exome sequencing and RNA sequencing using Illumina HiSeq 2000 on 14 HB specimens. In addition, we employed detailed genomic analysis on 5 multiple samples obtained from primary, post-chemotherapy and relapsed HB with familial adenomatous polyposis (FAP). All the candidates for mutations and fusions were validated by target and Sanger sequencing. Detected genetic alterations were sequenced in additional 53 HB samples. [Results] The somatic mutation rate was 8.1 (range of 1-41) per tumor with median of 0.16 non-silent mutations per Mb. It is statistically significant that number of somatic mutations is correlated with age. Consistent with the previous reports, most frequent mutated gene in our cohort was CTNNB1 with frequency of 71.4%. Overall, HBs have low mutation rates in concordance with other pediatric solid tumors. In our cohort, recurrent somatic mutations with high frequency was not detected, but overlapping germline mutations in Wnt signaling pathway were found, even if they were not affected with FAP. In total, 42 (79.2%) of 53 HB had mutations in genes regulating WNT/β-catenin signaling pathway. In multisampling sequencing analysis based on 5 different sites of HB specimens, progression from normal hepatocyte to HB seemed to be accompanied by few trancal events including only APC mutation in Wnt/β-catenin signaling pathway and gain of 1q and loss of 4q in copy number analysis. The major clone of relapsed tumors derived from minor clone of the primary tumor after chemotherapy getting additional genomic aberrations. [Conclusion] Our results revealed that mutations in Wnt/βcatenin-signaling pathway were truncal and significant mutations. In addition, even if only one cases multisampling sequencing, it is useful for revealing clonal evolution of HB. This study disclosed comprehensive genetic features of HB and identified potentially important therapeutic targets, as well as insight into possible cancer predisposition genes. Citation Format: Noriko Hoshino, Masafumi Seki, Motohiro Kato, Kenichi Yoshida, Yusuke Sato, Atsuko Nakazawa, Satoru Miyano, Akira Oka, Tadashi Iwanaka, Yasuhide Hayashi, Seishi Ogawa, Junko Takita. Clonal evolution and integral analysis of hepatoblastoma. [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 3294. doi:10.1158/1538-7445.AM2015-3294

Large genomic aberrations detected by SNP array are independent prognosticators of a shorter time to first treatment in chronic lymphocytic leukemia patients with normal FISH

BackgroundGenomic complexity can predict the clinical course of patients affected by chronic lymphocytic leukemia (CLL) with a normal FISH. However, large studies are still lacking. Here, we analyzed a large series of CLL patients and also carried out the so far largest comparison of FISH versus single-nucleotide polymorphism (SNP) array in this disease. Patients and methodsSNP-array data were derived from a previously reported dataset. ResultsSeventy-seven of 329 CLL patients (23%) presented with a normal FISH. At least one large (>5 Mb) genomic aberration was detected by SNP array in 17 of 77 patients (22%); this finding significantly affected TTT. There was no correlation with the presence of TP53 mutations. In multivariate analysis, including age, Binet stage, IGHV genes mutational status and large genomic lesion, the latter three factors emerged as independent prognosticators. The concordance between FISH and SNP array varied between 84 and 97%, depending on the specific genomic locus investigated. ConclusionsSNP array detected additional large genomic aberrations not covered by the standard FISH panel predicting the outcome of CLL patients.