Loss Of 10q Research Articles

Synthetic Lethal Interaction between the ESCRT Paralog Enzymes VPS4A and VPS4B in Cancers Harboring Loss of Chromosome 18q or 16q

SUMMARYFew therapies target the loss of tumor suppressor genes in cancer. We examine CRISPR-SpCas9 and RNA-interference loss-of-function screens to identify new therapeutic targets associated with genomic loss of tumor suppressor genes. The endosomal sorting complexes required for transport (ESCRT) ATPases VPS4A and VPS4B score as strong synthetic lethal dependencies. VPS4A is essential in cancers harboring loss of VPS4B adjacent to SMAD4 on chromosome 18q and VPS4B is required in tumors with co-deletion of VPS4A and CDH1 (E-cadherin) on chromosome 16q. We demonstrate that more than 30% of cancers selectively require VPS4A or VPS4B. VPS4A suppression in VPS4B-deficient cells selectively leads to ESCRT-III filament accumulation, cytokinesis defects, nuclear deformation, G2/M arrest, apoptosis, and potent tumor regression. CRISPR-SpCas9 screening and integrative genomic analysis reveal other ESCRT members, regulators of abscission, and interferon signaling as modifiers of VPS4A dependency. We describe a compendium of synthetic lethal vulnerabilities and nominate VPS4A and VPS4B as high-priority therapeutic targets for cancers with 18q or 16q loss.

Clinical-Pathological, Immunohistochemical, and Genetic Characterization of a Series of Posterior Pituitary Tumors.

Posterior pituitary tumors are supposed to represent the morphological spectrum of a single entity. Herein, we report the clinical-pathological, immunohistochemical, and genetic features of 5 spindle cell oncocytomas (SCOs), 3 pituicytomas, and 1 granular cell tumor (GCT). SCOs had the highest local invasiveness and affected older subjects. The 3 histotypes differed in the content of spindle cells (predominant in pituicytoma and absent in GCT), presence of lymphocytic infiltrate (in SCO and GCT, but not in the pituicytoma) and EMA/GFAP staining (negative in GCT; EMA-positive/GFAP-negative in 4/5 SCO and GFAP-positive in 3/3 pituicytomas). Three SCOs and 1 pituicytoma analyzed with next-generation sequencing had no mutations in 409 genes. However, 1 SCO had previously unreported homozygous deletion of CDKN2A/B and another of SMARCA4, SMARCB1, and NF2. All 3 SCOs had loss of heterozygosity of chromosome 1p, while the pituicytoma had chromosome 19 homozygous loss and chromosomes 10, 13q, and 18q loss of heterozygosity. Since 1p and 13q losses were previously reported in 1 pituicytoma and 1 SCO, respectively, our data demonstrate that posterior pituitary tumors share common genetic alterations. The possibility that posterior pituitary tumors are SMARCA4/SMARCB1-deficient should be kept in mind in the differential diagnosis toward other entities.

Accessing Genomic Alternations in Chronic Lymphocytic Leukemia Using an NGS-Based Comprehensive Genomic Profiling Assay

Background: Next generation sequencing (NGS) is an integral component in the characterization of hematologic malignancies, including chronic lymphocytic leukemia (CLL). Fluorescence in situ hybridization (FISH) and conventional cytogenetics (CC) are cost effective and are currently the gold standard for detecting copy number abnormalities (CNAs) in hematologic malignancies. NGS is emerging as a comprehensive assay that can detect CNAs while surveying the whole genome for single nucleotide variants and loss of heterozygosity (CN-LOH). Identifying CNA events in addition to mutations and RNA fusions may help identify and characterize the highly complex genetic landscape of hematologic malignancies. Methods: A custom total nucleic acid (TNA) NGS panel was designed which consists of mutation profiles of 297 genes, transcriptome profile of 213 genes, and genomic backbones of 14 chromosomes to identify unbalanced abnormalities. Two-hundred seventy CLL patients were included in the study (abnormalities detected in 236 cases in total: 61 cases by CC; 230 cases by FISH; and 53 cases by both CC and FISH, and no abnormalities detected in 34 cases by both FISH and CC). Mutation profiles including SNVs, indels, and structural changes were interrogated with a custom bioinformatic pipeline which utilized PureCN and CNVkit algorithms to identify structural changes. NGS results were compared to results of CC and FISH. CNA detection of sex chromosome and balanced rearrangement including translocation and inversion was excluded from the analysis Results: CNAs were detected by NGS in 56 of 61 cases (91%) reported by CC and in 178 of 230 cases (77%) detected by FISH. Seventy-seven CNAs detected by CC and 202 CNAs detected by FISH were identified by NGS. NGS failed to detect 13q deletion, detected by FISH in 48 cases. Abnormalities not detected by neither cytogenetics nor FISH were detected by NGS in 108 (gain) and 32 (loss) cases. In addition, we observed abnormalities in 9 of 34 cases by NGS reported as normal by both FISH and cytogenetics. CN-LOH was detected in 9% of cases predominantly on 13q, 17p and 22q. In addition to trisomy 12, gains of 20p and 20q were observed in each 72 (30%) and 43 (18%) cases. CN gains of 7p, 8q, and 17q were also observed in 12%, 12%, and 7% of cases, respectively. Oncogenic driver mutations in KRAS (p.G12D) and (p.G13D) were observed in four and five cases with CN gains, respectively. IKZF3, a recurrent hotspot pathogenic mutation in CLL and a potential prognostic marker that may positively regulate MYC, was detected in five patients with CN gains. CN loss of 11q, 2q, 13q, 3p, 17p, 21q, and 6q were among the most common chromosomes with CN loss (Figure 1). Notably, LOH of RB1, DLEU7, COG3, and FOX1 genes on 13q, of TP53, WRAP53, SLC52A1, CTC1, and ABR genes on 17p and of PRDM1, EPHA7, and CASP8AP2 genes on 6q were observed. Identifying cases with 13q14 deletions that include RB1 could change the CLL patient management due to the aggressive clinical course. Recurrent loss of function mutations in KMT2C (p.E2798Gfs*11), NOTCH1 (p.P2514Rfs*4), and TP53 (p.H179R) in 7q, 9q, and 17p were observed. Identifying both CN loss combined with loss of function mutations in tumor suppressors could help improve patient care. Conclusions: Abnormalities detected by cytogenetics were mostly detected by NGS, but NGS offers a higher resolution including CN events of various length, LOH events, and single gene mutations. CNAs detected at higher resolution is useful in identifying patients with 13q14 loss that include/exclude RB1 which may affect patient management. However, an accurate detection of the CNA could be affected in part by a baseline established by a panel of normal and the depth of coverage. Differences in sensitivity of methodologies can also be attributed to in vitro proliferation and tissue culture conditions utilized for CC analysis. CC and FISH can identify clones with multiple abnormalities as well as clonal evolution. Comprehensive genomic profile including high resolution copy number changes and mutational profiles, detectable by NGS, may provide better profiling for a patient for clinical management. Disclosures Jung: NeoGenomics: Current Employment. Thangavelu:NeoGenomics: Current Employment. Nam:NeoGenomics: Current Employment. Bender:NeoGenomics: Current Employment. Agersborg:NeoGenomics: Current Employment. Weiss:Bayer: Other: speaker; Genentech: Other: Speaker; Merck: Other: Speaker; NeoGenomics: Current Employment. Funari:NeoGenomics: Current Employment.

Genetic and Non-Genetic Mechanisms of Resistance to BCR Signaling Inhibitors in B Cell Malignancies.

The approval of BTK and PI3K inhibitors (ibrutinib, idelalisib) represents a revolution in the therapy of B cell malignancies such as chronic lymphocytic leukemia (CLL), mantle-cell lymphoma (MCL), diffuse large B cell lymphoma (DLBCL), follicular lymphoma (FL), or Waldenström’s macroglobulinemia (WM). However, these “BCR inhibitors” function by interfering with B cell pathophysiology in a more complex way than anticipated, and resistance develops through multiple mechanisms. In ibrutinib treated patients, the most commonly described resistance-mechanism is a mutation in BTK itself, which prevents the covalent binding of ibrutinib, or a mutation in PLCG2, which acts to bypass the dependency on BTK at the BCR signalosome. However, additional genetic aberrations leading to resistance are being described (such as mutations in the CARD11, CCND1, BIRC3, TRAF2, TRAF3, TNFAIP3, loss of chromosomal region 6q or 8p, a gain of Toll-like receptor (TLR)/MYD88 signaling or gain of 2p chromosomal region). Furthermore, relative resistance to BTK inhibitors can be caused by non-genetic adaptive mechanisms leading to compensatory pro-survival pathway activation. For instance, PI3K/mTOR/Akt, NFkB and MAPK activation, BCL2, MYC, and XPO1 upregulation or PTEN downregulation lead to B cell survival despite BTK inhibition. Resistance could also arise from activating microenvironmental pathways such as chemokine or integrin signaling via CXCR4 or VLA4 upregulation, respectively. Defining these compensatory pro-survival mechanisms can help to develop novel therapeutic combinations of BTK inhibitors with other inhibitors (such as BH3-mimetic venetoclax, XPO1 inhibitor selinexor, mTOR, or MEK inhibitors). The mechanisms of resistance to PI3K inhibitors remain relatively unclear, but some studies point to MAPK signaling upregulation via both genetic and non-genetic changes, which could be co-targeted therapeutically. Alternatively, drugs mimicking the BTK/PI3K inhibition effect can be used to prevent adhesion and/or malignant B cell migration (chemokine and integrin inhibitors) or to block the pro-proliferative T cell signals in the microenvironment (such as IL4/STAT signaling inhibitors). Here we review the genetic and non-genetic mechanisms of resistance and adaptation to the first generation of BTK and PI3K inhibitors (ibrutinib and idelalisib, respectively), and discuss possible combinatorial therapeutic strategies to overcome resistance or to increase clinical efficacy.

Impact of Molecular Subgroups on Outcomes Following Radiation Treatment Randomizations for Average Risk Medulloblastoma: A Planned Analysis of Children’s Oncology Group (COG) ACNS0331

The COG conducted a randomized trial for average-risk medulloblastoma (AR-MB). Patients age 3-21 years were randomized to a radiation boost to the whole posterior fossa (PFRT) or an involved field volume (IFRT) after receiving CSI. Patients age 3-7 years were also randomized to standard dose CSI (23.4Gy, SDCSI) or low dose CSI (18Gy, LDCSI). A total of 464 evaluable patients were available to compare PFRT vs. IFRT and 226 for SDCSI vs. LDCSI. 380 cases had sufficient tissue for DNA methylation-based molecular classification: 362 confirmed medulloblastoma; 6 non-medulloblastoma; and 12 inconclusive. Molecular subgrouping confirmed the following representation amongst the evaluable cohort: 156 Group 4 (43.1%), 76 Group 3 (21.0%), 66 SHH (18.2%), 64 WNT (17.7%). Five-year event-free survival (EFS) estimates were 82.5±2.7% and 80.5±2.7% for IFRT and PFRT, respectively (p = 0.44). Five-year overall survival (OS) estimates were 84.6±2.5% and 85.2±2.4% for the IFRT and PFRT regimen, respectively (p = 0.44). Five-year EFS estimates were 71.4±4.4% and 82.9±3.7% for LDCSI and SDCSI, respectively (p = 0.028). Five-year estimates of OS were 77.5±4.0% and 85.6±3.5% for the LDCSI and SDCSI regimens, respectively (p = 0.049). EFS distributions differed significantly by molecular subgroup (p<0.0001). Group 3 had the worst outcome, while WNT had the best outcome. There was a significant difference in EFS by RT group among SHH patients; SHH patients receiving IFRT arm had better EFS compared to PFRT (p = 0.018). There was a significant difference in EFS distributions by CSI group in Group 4 patients; young Group 4 patients treated with SDCSI had better EFS compared to LDCSI (p = 0.047). There were differences in EFS by the presence of specific mutations and/or copy number variations (CNVs). Among SHH patients, chromosome 14q loss (p<0.001) and chromosome 10q loss (p = 0.012) were associated with worse EFS. SHH patients with GLI2 amplification had inferior outcome, though not statistically significant (p = 0.056); there were only 3 SHH patients with amplified GLI2 however. Group 3 patients with MYC amplification and/or isochromosome 17 had inferior EFS. For group 4 patients none of the mutations or CNVs showed statistically significant differences in EFS at the nominal 0.05 level. These included presence of isochromosome 17 (p = 0.48), chromosome 11 loss (p = 0.056), chromosome 17 gain (p = 0.84), and chromosome 11 loss and/or chromosome 17 gain (p = 0.32). As previously reported, IFRT is noninferior to PFRT in all patients with AR-MB but LDCSI is worse than SDCSI in younger children. Significant differences in outcome by study randomization and molecular subgroup are observed.

Association between copy-number alteration of +20q, \u221214q and \u221218p and cross-sensitivity to tyrosine kinase inhibitors in clear-cell renal cell carcinoma

BackgroundWe aim to explore association between copy number alteration (CNA) and sensitivity to common tyrosine kinase inhibitors (TKIs) used in clear-cell renal cell carcinoma (ccRCC) treatment.MethodsCNA with related sensitivity profiles were extracted from the Genomics of Drug Sensitivity in Cancer (GDSC) dataset and was cross-referenced with common CNA in ccRCC in the Cancer Genome Atlas (TCGA) dataset. Functional annotation was profiled using GSEA and NET-GE. Target genes within cytobands of interest were screened in silico and validated in vitro using proliferation assays in A498 and 786-O ccRCC cells.ResultsFour TKIs (Sunitinib, Cabozantinib, Axitinib and Sorafenib) that were clinically used in ccRCC were selected. In silico analysis showed gain of 20q (+20q) occurred in ~ 23% of cases and was associated with resistance to all four TKIs; loss of 14q (−14q) occurred in ~ 39% of cases and was associated with resistance to Sunitinib and Sorafenib; loss of 18p (−18p) occurred in ~ 39% of cases and was associated with sensitivity to Sunitinib and Sorafenib. All 3 CNAs were associated with worsened prognosis, respectively. Candidate target genes included of RBL1 on 20q, KLHL33 on 14q and ARHGAP28 on18q. In vitro validation showed RBL1 overexpression induced resistance to Sunitinib and Cabozantinib; KLHL33 silencing induced resistance to Sunitinib; ARHGAP28 silencing induced sensitivity to Cabozantinib. Functional annotation indicated FoxO signaling, hypoxic response and Wnt pathway, and Rho-related cellular adhesion were mechanistically associated with +20q, −14q and −18p, respectively.ConclusionCommon CNAs in ccRCC are associated with cancer-intrinsic cross-sensitivity to common TKIs. Further validation and functional analyses are therefore needed.

Identifying bifurcated paths with differential function impact in glioblastomas evolution.

The evolutionary dynamics of human cancers has been investigated popularly and several bifurcated paths in cancer evolutionary trajectories are revealed to be with differential outcomes and phenotypes. However, whether such bifurcated paths exist in glioblastoma (GBM) remains unclear. In 385 GBM samples, through determining the clonal status of cancer driver events and inferring their temporal order, we constructed a temporal map of evolutionary trajectories at the patient population level. By investigating the differential impact on clinical outcome, we identified four key bifurcated paths, namely, "chromosome 10 copy number loss (ie, 10 loss) → chromosome 19 copy number gain (ie, 19 gain): 10 loss → 13q loss"; "10 loss → 19 gain: 10 loss → 15q loss"; "10 loss → 19 gain: 10 loss → 6q loss" and "10 loss → 19 gain: 10 loss → 16q loss". They formed a core multibranches path, with 10 loss being regarded as the common earliest event followed by 19 gain and four other departure events (13q loss, 15q loss, 6q loss and 16q loss), which may account for their difference in genome instability and patient survival time. Compared to "10 loss → 19 gain", the patients with "10 loss → 13q loss" had higher telomerase activity. Notably, there were obvious discrepancies in immune activity and immune cell infiltration level between patients with "10 loss → 13q/16q loss" and "10 loss → 19 gain", highlighting the bifurcated paths' effect on tumor immune microenvironment. In summary, our study identifies four key bifurcated paths in GBM for the first time, suggesting the feasibility of patient stratification and prognosis prediction based on key bifurcated paths.

Age Dependency of the Prognostic Impact of Tumor Genomics in Localized Resectable MYCN-Nonamplified Neuroblastomas. Report From the SIOPEN Biology Group on the LNESG Trials and a COG Validation Group.

PURPOSEFor localized, resectable neuroblastoma without MYCN amplification, surgery only is recommended even if incomplete. However, it is not known whether the genomic background of these tumors may influence outcome.PATIENTS AND METHODSDiagnostic samples were obtained from 317 tumors, International Neuroblastoma Staging System stages 1/2A/2B, from 3 cohorts: Localized Neuroblastoma European Study Group I/II and Children’s Oncology Group. Genomic data were analyzed using multi- and pangenomic techniques and fluorescence in-situ hybridization in 2 age groups (cutoff age, 18 months) and were quality controlled by the International Society of Pediatric Oncology European Neuroblastoma (SIOPEN) Biology Group.RESULTSPatients with stage 1 tumors had an excellent outcome (5-year event-free survival [EFS] ± standard deviation [SD], 95% ± 2%; 5-year overall survival [OS], 99% ± 1%). In contrast, patients with stage 2 tumors had a reduced EFS in both age groups (5-year EFS ± SD, 84% ± 3% in patients < 18 months of age and 75% ± 7% in patients ≥ 18 months of age). However, OS was significantly decreased only in the latter group (5-year OS ± SD in < 18months and ≥ 18months, 96% ± 2% and 81% ± 7%, respectively; P = .001). In < 18months, relapses occurred independent of segmental chromosome aberrations (SCAs); only 1p loss decreased EFS (5-year EFS ± SD in patients 1p loss and no 1p loss, 62% ± 13% and 87% ± 3%, respectively; P = .019) but not OS (5-year OS ± SD, 92% ± 8% and 97% ± 2%, respectively). In patients ≥ 18 months, only SCAs led to relapse and death, with 11q loss as the strongest marker (11q loss and no 11q loss: 5-year EFS ± SD, 48% ± 16% and 85% ± 7%, P = .033; 5-year OS ± SD, 46% ± 22% and 92% ± 6%, P = .038).CONCLUSIONGenomic aberrations of resectable non–MYCN-amplified stage 2 neuroblastomas have a distinct age-dependent prognostic impact. Chromosome 1p loss is a risk factor for relapse but not for diminished OS in patients < 18 months, SCAs (especially 11q loss) are risk factors for reduced EFS and OS in those > 18months. In older patients with SCA, a randomized trial of postoperative chemotherapy compared with observation alone may be indicated.

Abstract 186: Gain of chromosome 6p is a molecular biomarker for prognostication of retinoblastoma ocular survival: The aqueous humor surrogate tumor biopsy

Abstract Purpose: Aqueous humor (AH) is a novel source of tumor-derived cell-free DNA (cfDNA) and can serve as a surrogate tumor biopsy, or liquid biopsy, for retinoblastoma (RB). We previously identified a somatic copy number alteration (SCNA) gain of chromosome 6p as associated with a 10-fold increased risk of enucleation. In this study, we provide a 2-year extended update to further explore 6p gain as a prognostic biomarker for ocular survival. Methods: Patients diagnosed with RB from 12/2014 - 7/2019 from whom AH was sampled were included. AH was extracted via clear corneal paracentesis at diagnosis, during intravitreal injection of chemotherapy or enucleation. CfDNA was extracted; shallow whole genome sequencing was performed to assess for cfDNA tumor fractions and highly recurrent RB SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). Patient demographics, treatment regimen and globe salvage were recorded. Results: 50 eyes from 46 patients were included: 27 eyes were salvaged and 23 were enucleated. Most eyes (36/50,72%) were TNM stage cT2b. 116 samples of AH were analyzed. There were no cases of orbital relapse or metastatic spread. The median follow-up was 30 months (range 6-64 mos). After genomic evaluation, 33 / 50 eyes (66%) demonstrated one or more highly-recurrent RB SCNAs and 39/50 (78%) demonstrated any measurable SCNA. Gain of 6p was the most prevalent RB SCNA among all 50 eyes (50%), followed by 1q gain (38%), 16q loss (30%), 2p gain (including 3 focal MYCN gains; 18%), and 13q loss (16%). RB SCNAs were significantly more prevalent in enucleated eyes (21/23, 91.3%) than in salvaged eyes (12/27, 44.4%; Fisher's exact, P=0.0007). Of these, 6p gain was particularly more prevalent in enucleated eyes (17/23, 73.9%) than in salvaged eyes (8/27, 29.6%; Chi-squared, P=0.002) Of all the RB SCNAs, the presence of 6p gain in the cfDNA in the AH portended nearly 10 times greater odds of being enucleated (OR=9.87,95% CI=1.75-55.65, P=0.009). No single other SCNA had a statistically significant power to predict enucleation although there was a trend towards significance for 2p/focal MYCN gain (P=0.09) limited by the few number of eyes with this SCNA. Conclusions: Although we previously identified chromosomal gain of 6p as a potential indicator of an aggressive phenotype in RB, that study included a smaller cohort of patients and limited follow-up. With extended follow-up and nearly double the number of eyes and AH samples, the data continue to demonstrate that AH is a high-yield surrogate tumor biopsy, and the presence of RB-specific SCNAs–specifically gain of 6p–has the potential to serve as a prognostic biomarker for RB. Citation Format: Jesse L. Berry, Liya Xu, Ashley Polski, Rishvanth Prabakar, Mark Reid, Jonathan Kim, Peeter Kuhn, James Hicks. Gain of chromosome 6p is a molecular biomarker for prognostication of retinoblastoma ocular survival: The aqueous humor surrogate tumor biopsy [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 186.

Chromosome 6p Amplification in Aqueous Humor Cell-Free DNA Is a Prognostic Biomarker for Retinoblastoma Ocular Survival.

Aqueous humor contains tumor-derived cell-free DNA (cfDNA) and can serve as a liquid biopsy for retinoblastoma. We previously associated somatic copy-number alteration (SCNA) 6p gain with a 10-fold increased risk of enucleation. Here we provide a 2-year update to further explore 6p gain as a prognostic biomarker for ocular survival. Patients diagnosed with retinoblastoma from December 2014 to July 2019 from whom aqueous humor was sampled were included. cfDNA was extracted and shallow whole-genome sequencing performed to identify highly recurrent retinoblastoma SCNAs (gain of 1q, 2p, 6p, loss of 13q, 16q). 116 aqueous humor samples from 50 eyes of 46 patients were included: 27 eyes were salvaged, 23 were enucleated. Highly recurrent retinoblastoma SCNAs were found in 66% eyes. 6p gain was the most prevalent SCNA (50% eyes). It was particularly more prevalent in enucleated eyes (73.9%) than in salvaged eyes (29.6%; P = 0.004). 6p gain in aqueous humor cfDNA portended nearly 10-fold increased odds of enucleation (OR = 9.87; 95% confidence interval = 1.75-55.65; P = 0.009). In the enucleated eyes, 6p gain was associated with aggressive histopathologic features, including necrosis, higher degrees of anaplasia, and focal invasion of ocular structures. With extended follow-up and nearly double the aqueous humor samples, we continue to demonstrate 6p gain as a potential prognostic biomarker for retinoblastoma. IMPLICATIONS: Aqueous humor is a high-yield source of tumor-derived DNA in retinoblastoma eyes. Detection of 6p gain in the aqueous humor allows for targeted, patient-centered therapies based on this molecular prognostic marker. Prospective, multicenter studies with aqueous humor sampled from all eyes at diagnosis are warranted to validate these findings.

Abstract B07: Prospective germline next-generation sequencing in pediatric patients with neuroblastoma identifies frequent alterations in genes involved in DNA damage repair

Abstract Background: The role of germline ALK and PHOX2B variants in genetic predisposition to neuroblastoma (NB) is well established; however, alterations in genes associated with other syndromes, including RASopathies, Fanconi anemia, and Li-Fraumeni, have also been detected. Emerging data suggest potential roles for DNA damage repair (DDR) pathway genes in NB predisposition. The use of next-generation sequencing (NGS) technologies facilitates the unbiased detection of both known and novel germline variants. Methods: Patients (pts) with newly diagnosed and relapsed malignancies, including 43 NB pts, were referred to our institutional pediatric oncology NGS study to sequence germline DNA, tumor DNA, and RNA. Germline DNA was sequenced using a custom pediatric cancer panel (Agilent Sure Select capture technology) targeting 15,000 exons across 864 genes (1000x coverage). Variant pathogenicity was classified according to ACMG (American College of Medical Genetics and Genomics) criteria and evaluated at multidisciplinary molecular tumor boards. Rare variants of uncertain significance (VUS) with supportive corresponding somatic data, patient phenotypes, literature, and/or in-silico functional analyses were termed “Variants of uncertain significance with limited evidence for pathogenicity” (VUS-LEP). Results: Analyses have been completed for 41/43 enrolled NB pts. Patient history was retrospectively categorized as “high genetic predisposition risk” (HGPR) in 14/41 pts based on ≥1 of the following criteria: (1) family history of NB and/or significant family history of other cancer(s); (2) patient with NB and another metachronous or synchronous malignancy; (3) NB with congenital abnormalities; (4) multifocal NB. Germline pathogenic (P), likely pathogenic (LP) variants, or VUS-LEP in a known cancer predisposition gene were identified in 6/14 HGPR pts (43%), predominantly in DDR-related genes (PALB2, BRCA1/CHEK2, CHEK2/PALB2, NF1, DICER1, MITF). Of note, no ALK or PHOX2B germline variants were identified for the five pts with NB family history. For the 27 non-HGPR pts, germline P, LP variants or VUS-LEP in DDR genes were most prevalent and detected in 8/27 pts (30%), in genes such as BAP1, BARD1 (n=2), BLM, BRCA2, CHEK2, RAD51, RAD51D/NBN. In this non-HGPR population, we also identified a germline P TP53 variant (n=1), a LP FH variant (n=1) as well as additional germline VUS-LEPs in EZH2 (n=1) and ERCC2 (n=1). Conclusion: Prospective sequencing identified frequent germline variants in NB pts, predominantly in genes involved in DDR and homologous recombination, regardless of classification as HGPR. We intend to complete and report somatic characterization of the tumors, with a focus on mutational signatures and 11q loss encompassing ATM. Our findings may have implications for future targeted treatment recommendations (e.g., PARP inhibitors) as well as for appropriate genetic counseling for pts and families. Citation Format: Sarah Cohen-Gogo, Karin Langenberg-Ververgaert, Anita Villani, Winnie Lo, Ted Young, Nisha Kanwar, Scott Davidson, Nathaniel Anderson, Bailey Gallinger, Mehdi Layeghifard, Larissa Waldman, Daniel Morgenstern, Ledia Brunga, Stephen Meyn, David Malkin, Adam Shlien, Meredith Irwin. Prospective germline next-generation sequencing in pediatric patients with neuroblastoma identifies frequent alterations in genes involved in DNA damage repair [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B07.

Dual MGMT inactivation by promoter hypermethylation and loss of the long arm of chromosome 10 in glioblastoma.

BackgroundEpigenetic inactivation of O6‐methylguanine‐methyltransferase (MGMT) gene by methylation of its promoter is predictive of Temozolomid (TMZ) response in glioblastoma (GBM). MGMT is located on chromosome 10q26 and the loss of chromosome 10q is observed in 70% of GBMs. In this study, we assessed the hypothesis that the dual inactivation of MGMT, by hypermethylation of MGMT promoter and by loss the long arm of chromosome 10 (10q), may confer greater sensitivity to TMZ.MethodsA total of 149 tumor samples from patients diagnosed with GBM based on the WHO 2016 classification were included in this retrospective study between November 2016 and December 2018. Methylation status of MGMT promoter was evaluated by pyrosequencing and status of chromosome 10q was assessed by array comparative genomic hybridization.ResultsGlioblastoma patients with chromosome 10q loss associated with hypermethylation of MGMT promoter had significantly longer overall survival (OS) (P = .0024) and progression‐free survival (PFS) (P = .031). Indeed, median OS of patients with dual inactivation of MGMT was 21.5 months compared to 12 months and 8.1 months for groups with single MGMT inactivation by hypermethylation and by 10q loss, respectively. The group with no MGMT inactivation had 9.5 months OS. Moreover, all long‐term survivors with persistent response to TMZ treatment (OS ≥ 30 months) displayed dual inactivation of MGMT.ConclusionsOur data suggest that the molecular subgroup characterized by the dual inactivation of MGMT receives greater benefit from TMZ treatment. The results of our study may be of immediate clinical interest since chromosome 10q status and methylation of MGMT promoter are commonly determined in routine practice.

Narrowing down the Common Cytogenetic Deletion 14q to a 5.6-Mb Critical Region in 1p/19q Codeletion Oligodendroglioma-Relapsed Patients Points to Two Potential Relapse-Related Genes: SEL1L and STON2

Based on a literature review and our database, we report on the smallest 14q deletion identified in a brain tumor characterized by 1p/19q codeletion low-grade oligodendroglioma. In 2013, array-comparative genomic hybridization of the brain tumor revealed 1p/19q codeletion as a sole abnormality. In 2019, the patient relapsed showing additional abnormalities including a 14q deletion of 16.5 Mb at 14q24.2q31.3. This region overlaps with 2 previously identified minimal regions, 14q21.2q24.3 and 14q31.3q32.1, based on 142 cases of glioma. The authors reported no correlation between these 2 regions and survival. By extracting these 2 regions from our patient's deletion and comparing it to 12 other cases of 1p/19q codeletion oligodendrogliomas reported in the literature, we narrowed down the 14q loss possible critical region to 5.6 Mb mapping at 14q31.1q31.2. This region contains 2 potential relapse-related genes: SEL1L and STON2.

15. The good, the bad, and the ugly of SNP array testing for cutaneous melanocytic neoplasms

Cutaneous melanocytic neoplasms (CMNs) with ambiguous histologic features are diagnostically challenging for anatomic pathology practices. Ancillary genomic testing by SNP array can identify copy number abnormalities and allelic imbalances potentially associated with malignancy. We tested 445 formalin fixed, paraffin embedded CMNs by SNP array (OncoScan CNV Plus Assay, Thermo Fisher Scientific). Specimens were reviewed by a pathologist for acceptable amount and percentage of tumor cells. The reportable range included deletions > 1 megabase (Mb), duplications > 2 Mb, copy neutral loss of heterozygosity (cnLOH) > 10 Mb, or any detectable alteration affecting gene regions known to be associated with melanoma; benign alterations were excluded. If SNP array testing could not be completed, our laboratory requested reflex testing with a melanoma panel of FISH probes targeting 6p gain, 6q loss, 8q gain, 9p loss, and 11q gain. SNP array yielded interpretable results on 341 out of 426 specimens tested; abnormalities were reported in a total of 162 (47.5%) arrays, including 134 with abnormalities undetectable by FISH. In 97 specimens converted from SNP array to FISH testing, 92 were successfully tested by FISH, and 29 (31.5%) were abnormal. The combination of SNP array and FISH yielded interpretable results on 97.3% of CMNs. FISH interpretation was confounded by technical artifact, cell overlap, and tetraploidy, whereas SNP array had a higher failure rate (20% vs. <5% for FISH), a tendency toward 'noisy' results, and a higher yield of non-specific copy number alterations. Despite the challenges of SNP array, it is more precise and comprehensive.

MECOM rearrangement involving the MYC locus: Two additional patients with the rare translocation, t(3;8)(q26.2;q24), and molecular review

A relatively small subset of myeloid neoplasms involve rearrangements of cytoband 3q26.2. Such rearrangements are often in response to therapy and carry a poor prognosis. The ectopic expression of MECOM is the result of such translocations. To date, thirty-three t(3;8)(q26.2;q24) cases have been reported; we contribute two patients with confirmed MECOM and MYC rearrangements. Both patients presented with pancytopenia and were diagnosed with myelodysplastic/myeloproliferative disorders. In addition to translocation t(3;8), Patient 1 possessed a derivative chromosome 5, while Patient 2 possessed monosomy 7; neither patient’s clonal abnormalities resolved in follow-up studies. Of the previous 33 cases, one exhibited 5q loss, while monosomy 7 was found in fifteen. These findings contribute to the small number of reported cases with t(3;8) translocations. We also speculate about the molecular mechanisms associated with this translocation.

Non-invasive detection of urothelial carcinoma (UC) by cost-effective low-coverage whole genome sequencing from urine exfoliated cells DNA.

1552 Background: Urothelial carcinoma (UC) is a malignancy with frequent chromosomal aberrations. The FISH assays were more sensitive as compared to cytology tests. Here we investigated cost-effective whole genome sequencing technology, which is able to detect all chromosomal aberrations for UC diagnoses. Methods: UC patients and control group are prospectively recruited in trial NCT03998371. First-morning-voided urine were freshly collected before TURBT or cystectomy. Urine Exfoliated Cells DNA was analyzed by illumina HiSeq X10, followed by genotyping by bioinformatics workflow UCAD. Results: 195 individuals were prospectively recruited. 121 UC patients and 67 non-tumor diseases were included in this study. 7 other malignancies as confirmed by pathological testing were excluded. Frequent chromosome copy number changes were found in cancer patients as compared non-tumor controls, including chromosome 3 gain, 17 gain, 7 gain and 9p loss used in FISH assays were found. In addition to that, chr9q loss, 8q gain, 5q loss, 17p loss, 11p loss, 1q gain, 8p loss, 10q loss, 6q loss, 4q loss and 11q loss were also frequent in cancer patients (AUC &gt; 0.65). Metacentric chromosomes showed better AUC compared to acrocentric and telocentric chromosomes (P = 1.7e-03). A novel diagnosis model UCAD was built by incorporating all the chromosomal changes. The model reached performance of AUC = 0.933. At the optimal cutoff |Z| &gt; = 3.16, the sensitivity, specificity and accuracy were 84.7%, 97.9% and 89.0% respectively. The prediction positivity was found correlated with urine microscopy visible epithelial cells (P = 0.00069), tumor invasiveness (Ta/Tis vs the other, P = 0.0048) and tumor grade (P = 0.0030), but not microscopy RBC/WBC findings, urine culture findings, smoke and drinking history. The UCAD model outperformed cytology tests by predicting all 16-cytology positive and 12 cytology negative tumors with comparable specificity. The model found 75.0% more tumors. And UCAD identified more upper urinary tract cancer (P = 0.012) and smaller tumors ( &lt; 3cm, P = 5.9e-04). The adding of cytology to UCAD did not improve diagnosing sensitivity and specificity. UCAD reproduce the diagnoses among morning - void urine, morning, afternoon urine samples with correlation coefficient R2&gt; 0.98. All the urine samples showed high concordances with matched tumor samples (R2&gt; 0.85). Conclusions: UCAD could be a high specific, robust UC diagnoses method with improved sensitivity as compared to cytology tests. Clinical trial information: NCT03998371.

Age-dependent prognostic value of KRAS mutation in metastatic colon cancer.

e16085 Background: KRAS mutation is associated with poor prognosis in metastatic colon cancer (mCC). However, the age-dependent prognostic value of KRAS status is unknown. We aimed to confirm if the prognostic impact of KRAS is dependent on the age of diagnosis in mCC. Methods: We identified adult patients with mCC diagnosed between 2004 and 2016 using the NCDB. We compared patients with KRAS mutation and KRAS wild type, stratified according to age group &lt; 50, 50-70, and ≥70. Categorical variables were compared using the chi-square test. We performed the Kaplan-Meier and Cox regression methods for survival analyses. We adjusted for patient and tumor characteristics (included KRAS status, MSI, 18q loss of heterozygosity, side of the primary tumor). Results: A Total of 19,875 patients had KRAS status reported; 43% had KRAS mutation, and 57% were KRAS wild type. Patients with KRAS mutation were more likely to be female, black, have elevated CEA, and have right-sided tumors (all p &lt; .001). In the overall population, patients with KRAS mutation had significantly worse OS compared to patients with KRAS wild type (20 vs. 22 months, p &lt; .001), and this difference was maintained after multivariable Cox regression analysis ( p &lt; .001) (Table). Young patients ( &lt; 50 years) with KRAS mutation had a worse prognosis than those with KRAS wild type in the tumor (23 vs. 29 months, p &lt; .001). KRAS status did not maintain its prognostic value among older age (≥70 years) patients (14 vs. 14 months, p = NS). Conclusions: In this analysis of a national cancer registry, we confirmed that KRAS mutation was an independent poor prognostic factor for mCC. This effect persisted after adjusting for patient and tumor characteristics, including sidedness and MSI status. However, we identified that this prognostic value of KRAS mutation is significant in young mCC patients &lt; 50 years, as compared to older patients. [Table: see text]

Pancreatic tropism of metastatic renal cell carcinoma.

Renal cell carcinoma (RCC) is characterized by a particularly broad metastatic swath, and, enigmatically, when the pancreas is a destination, the disease is associated with improved survival. Intrigued by this observation, we sought to characterize the clinical behavior, therapeutic implications, and underlying biology. While pancreatic metastases (PM) are infrequent, we identified 31 patients across 2 institutional cohorts and show that improved survival is independent of established prognostic variables, that these tumors are exquisitely sensitive to antiangiogenic agents and resistant to immune checkpoint inhibitors (ICIs), and that they are characterized by a distinctive biology. Primary tumors of patients with PM exhibited frequent PBRM1 mutations, 3p loss, and 5q amplification, along with a lower frequency of aggressive features such as BAP1 mutations and loss of 9p, 14q, and 4q. Gene expression analyses revealed constrained evolution with remarkable uniformity, reduced effector T cell gene signatures, and increased angiogenesis. Similar findings were observed histopathologically. Thus, RCC metastatic to the pancreas is characterized by indolent biology, heightened angiogenesis, and an uninflamed stroma, likely underlying its good prognosis, sensitivity to antiangiogenic therapies, and refractoriness to ICI. These data suggest that metastatic organotropism may be an indicator of a particular biology with prognostic and treatment implications for patients.

EWSR1(22q12) Translocation Positive Pediatric Adrenal Tumor with Loss of 1p, 11q, and Unbalanced Gain of 17q: Neuroblastoma or Ewing Sarcoma?

Background Although neuroblastoma and Ewing sarcoma/Primitive neuroectodermal tumor are different clinical entities, they are both a member of small round blue cell tumors and can mimic each other’s behavior in clinical and molecular aspects. Case report: A 3 year-old girl with an abdominal mass was found to have a small round blue cell tumor originating from the right adrenal gland. High level of neuron specific enolase, initial genetic test results (N-Myc amplification: negative, loss of 1p, 11q, and unbalanced gain of 17q) and characteristic radiological appearance of the tumor suggested a preliminary diagnosis of neuroblastoma but further analysis showed CD99 expression and presence of EWSR1 rearrangement, which are mostly observed in Ewing sarcoma. Conclusion: Adrenal gland tumors of childhood with complex immunophenotypic features requires distinguishing two discrete tumors in the small round blue cell tumor group, neuroblastoma and Ewing sarcoma. Although no exact diagnosis of the tumor was made, we reached a good response with neuroblastoma treatment protocol.

Wait-and-See Treatment Strategy Could be Considered for Lung Adenocarcinoma with Special Pleural Dissemination Lesions, and Low Genomic Instability Correlates with Better Survival.

This study aimed to evaluate the feasibility of a wait-and-see strategy for non-small cell lung cancer (NSCLC) patients with special pleural dissemination lesions (r-pM1a and s-pM1a). Furthermore, the study characterized genomic alternations about disease progression. For this study, 131 NSCLC patients with a diagnosis of pM1a were retrospectively selected. Survival differences were evaluated among patients treated with three different initial postoperative treatments: chemotherapy, targeted therapy, and wait-and-see strategy. Whole-exome sequencing (WES) was performed on primary and metastatic tumors of 10 patients with dramatic progression and 13 patients with gradual progression. The wait-and-see group showed better progression-free survival (PFS) than the chemotherapy group (p < 0.001) but PFS similar to that of targeted group (p = 0.984). This pattern persisted in epidermal growth factor receptor (EGFR)-positive patients. For patients with EGFR-negative/unknown status, PFS was longer in the wait-and-see group than in the two treatment groups. Furthermore, better overall survival (OS) was observed for the patients who received chemotherapy or targeted therapy after the wait-and-see strategy than for those who received chemotherapy or targeted therapy immediately. Lymph node status was an independent prognostic factor for PFS and OS. Finally, WES analysis showed that a high genomic instability index (GIS) and chromosome 18q loss were more common in metastatic tumors, and low GIS was significantly associated with better PFS (p = 0.016). The wait-and-see strategy could be considered for special pM1a patients without lymph nodes metastasis, and patients with a low GIS may be suitable for this strategy.