Loss Of 10q Research Articles

Abstract 554: Aqueous humor as a liquid biopsy for uveal melanoma

Abstract Purpose: Metastatic Uveal Melanoma (UM) is usually fatal within one year of symptom onset. There are a variety of biomarkers that can stratify patients' metastatic risk, but identifying them requires intraocular tumor biopsy. Liquid biopsy offers a less invasive alterative without the risks of retinal detachment and intraocular hemorrhage. We investigated whether the aqueous humor (AH) has sufficient tumor-derived cell-free DNA (cfDNA) to function as a liquid biopsy for UM. Methods: This case series includes 12 AH samples from 7 eyes of 7 UM patients from whom AH was sampled during routine brachytherapy plaque placement and/or removal. Double stranded DNA (dsDNA), single stranded DNA (ssDNA), RNA, and microRNA (miRNA) were quantified with respective Qubit High Sensitivity Assay Kits (Thermo Fischer) for each sample. The lower limit of detection was 0.01 ng/µL for dsDNA, 0.05 ng/µL for ssDNA, 0.25 ng/µL for RNA, and 0.05 ng/µL for miRNA. CfDNA was then isolated and sequenced on an Illumina platform to assess genome-wide somatic copy number alterations (SCNAs). Results: Aqueous humor derived cfDNA was detected in all samples, ranging from 0.085-28.8 ng/µL (mean = 2.7 ng/µL). RNA was detected in 2 of 12 samples (mean = 1.6 ng/µL, range = 0.7-2.5 ng/µL), and miRNA was detected in 11 of 12 samples (mean = 1.7 ng/µL, range = 0.094-16.2 ng/µL). There was a significantly higher cfDNA concentration in post-radiation samples than pre-radiation samples, (median = 0.082 ng/µL in pre-radiation, 0.21 ng/µL in post-radiation, W = 1, z = -2.55, p < 0.05, Wilcoxon Signed-rank test). This finding was similar when comparing all nucleic acid concentrations (dsDNA, ssDNA, RNA, and miRNA) between pre- and post-radiation (median = 0.085 ng/µL in pre-radiation, 0.333 ng/µL in post-radiation, W = 1, z = -3.46, p = 0.00027). Confirmed UM genomic alterations of 6p gain, 6q loss, and 8q gain were identified in one post-radiation AH sample from an eye with an iridociliary body tumor. These SCNAs are characteristic of UM. No SCNAs were found in the AH from the pre-radiation sample in the same eye, or any of the other AH samples sequenced to date. Conclusions: The AH is a source of cfDNA in eyes with UM, with a higher yield of all nucleic acids detected after brachytherapy radiation treatment. UM associated SCNAs were identified in the AH of one eye with an iridociliary tumor after radiation only. We hypothesize this may be due to intratumoral necrosis from the radiation as well as the proximity of the primary tumor to the AH in the case where SCNAs were identified. These results represent the first time that UM SCNAs have been identified in cfDNA isolated from the AH, and this provides preliminary evidence that the AH can serve as a liquid biopsy for UM. Citation Format: Mary E. Kim, Liya Xu, Chen-Ching Peng, Peter Kuhn, James Hicks, Jesse L. Berry. Aqueous humor as a liquid biopsy for uveal melanoma [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 554.

Abstract 865: Genomic and evolutionary classification of lung cancer in never smokers from the Sherlock-Lung study

Abstract Lung cancer in never smokers (LCINS) is a common cause of cancer mortality, but is poorly characterized. Here we show, through high coverage whole genome sequencing of 232 tumors/blood pairs, that LCINS can be separated into three subtypes, defined by somatic copy number aberrations. While the dominant subtype (‘piano'), rarely found in lung cancer from smokers, is characterized by quiet copy number profiles, the other subtypes are associated with specific arm-level amplifications and EGFR mutations (‘mezzo-forte'), and whole genome doubling (‘forte'). Piano tumors feature somatic UBA1 mutations, germline AR variants, and stem cell-like properties, including low mutational burden, infrequent TP53 alterations, high intra-tumor heterogeneity, long telomeres, and slow growth as suggested by the occurrence of cancer driver genes decades prior to tumor diagnosis. In contrast, driver mutations in mezzo-forte and forte tumors are generally late clonal events acquired close to tumor diagnosis, thus potentially facilitating target identification with a single biopsy. The mutational signature associated with direct exposure to tobacco smoking (SBS4) was not observed in our study, even in cases with reported exposure to second-hand tobacco smoke. No significant differences of mutational spectra were found between passive and non-passive smokers. While genes within the RTK-RAS pathway have distinct impacts on survival, mutations in TP53, CHEK2, EGFR, or loss of 15q or 22q independently doubled mortality. These genomic alterations in forte and mezzo-forte subtypes, and stem cell-like features in piano tumors create avenues for personalized therapeutic strategies in LCINS. Citation Format: Tongwu Zhang, Ludmil B. Alexandrov, Wei Zhao, Jian Sang, Jianxin Shi, Bin Zhu, Dmitry A. Gordenin, Montserrat Garcia-Closas, Qing Lan, Nathaniel Rothman, Stephen J. Chanock, David C. Wedge, Maria Teresa Landi. Genomic and evolutionary classification of lung cancer in never smokers from the Sherlock-Lung study [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 865.

Identifying High-Risk Phenotypes Using Three-Way Feature Interactions in Multiple Myeloma: An Analysis of Publically Available Mmrf Commpass Study

The introduction of next generation sequencing has exponentially increased the number of potential disease targets from a limited panel of probes to markers across the entire genome. Previous studies of NGS data in multiple myeloma have identified key features associated with outcome in additive models, typically showing involvement of TP53, gain of 1q, MYC translocations, and others.We modified the multi-factor dimensionality reduction method (MDR) method, a computationally efficient method for detecting non-linear patterns of gene-gene interactions in genome-wide association studies (GWAS) first proposed by Richie et al, to identify three-way interactions significantly associated with outcome in MM. Specifically, we altered the ranking strategy of the survival adapted Cox-MDR method of Lee et al from balanced accuracy (average of sensitivity and specificity) to the absolute sum of deviance residuals. Due to the high level of censoring (94 events in 559 cases), balanced accuracy ranking favored interactions that identified few high risk cases and failed to validate well on external data.The publically available MMRF CoMMpass trial database (release version IA10) was utilized as primary training set for our MDR method. We included in our comparisons 164 RNA-SEQ genes with significant association with overall survival, the 20 most abundant non-synonymous, non-IG variants, and 24 estimated whole chromosome arm copy number gains or loss calls from the long insert WGS data of 559 baseline cases with outcome and clinical data (median follow-up 1.8 years). We searched for all three-way interactions across 210 total features resulting in the ranking of over 1.5 million unique three-way interactions, adjusted for age, ISS, creatinine level, and treatment by modified MDR. The top three-way RNA-expression interactions from our discovery phase were validated on 540 cases with GEP microarray and clinical data from UAMS Total Therapy 2 and 3 with median follow-up of 4.66 years (publically available data from GSE2658, GSE31161, and GSE24080).Of the top 100 three-way gene expression interactions discovered that we could validate on Affymetrix GEP data, 84 were significant in the UAMS TT2 and TT3 data set (log rank p-value < 0.05). HMGB3, a proposed inhibitor of myeloid differentiation, and DDX51, known to be differentially methylated in ALL, were the most commonly involved genes in the top significant interactions across both data sets. Pathway analysis of the 150 most frequently observed genes across the top 5,000 interactions in CoMMpass revealed an enrichment of DNA replication and cell cycle associated genes: RFC5, MCM2, MCM3, MCM4, MCM6, CCNA2, CDK2, etc.Within the top interactions that included whole arm copy number variants, gain of 1q and loss of 13q were the most frequently observed. We note that the top three-way interactions with gain of 1q paired exclusively with ALYREF, a transcriptional promoter, and one additional gene. Of the top interactions that included a SNV, mutations of DIS3 and DNAH5 were the most frequently observed. Many of the top interactions including DIS3 mutations paired with HMGB3 or TRIB3, a negative regulator of NF-kB and sensitizer to apoptosis, and one additional gene.MDR models are capable of identifying complex interactions of features that are not strictly additive. We observed a variety of interaction models, and present below an example where presence of 2 or more features resulted in overall negative outcome while presence of 1 or fewer did not. This situation was observed for the interaction of high expression of HMGB3, DDX51, and PSMB2, a bortezomib sensitizer in MM cell lines, where high expression in 2 or more of these genes imparted a negative outcome in CoMMpass and TT2/TT3 data sets. In the TT2/TT3 data set, cases identified as high risk from this interaction were enriched for the PR subtype and GEP70 HR.This modified MDR method is an effective tool at mining large genomic data sets for all k -way interactions associated with outcome among binary or multi-level features, adjusted for clinical covariates. Our modifications to MDR method allow us to identify more relevant interactions and increase reliability in validation data. This method could potentially further knowledge of the complex relationships between key features in genomic landscape of MM by discovering novel interactions that identify high-risk phenotypes. [Display omitted] DisclosuresBergsagel:Phosplatin Therapeutics: Research Funding.

Chromosomal complexity as a biomarker to de-escalate adjuvant imatinib treatment in high-risk gastrointestinal stromal tumor.

11535 Background: Gastrointestinal stromal tumors (GISTs) are characterized molecularly by oncogenic KIT or platelet-derived growth factor alpha ( PDGFRA) mutations. Malignant progression of primary GISTs occurs through stepwise accumulation of additional chromosomal aberrations, such as losses of chromosome arms 14q, 22q, 1p, 15q and Xp. After surgical resection of primary GIST, three years of adjuvant imatinib treatment is recommended for patients with an estimated high risk of recurrence. Still, nearly half of high-risk patients are cured by surgery alone, indicating that selection of patients could be improved. We hypothesized that high-risk GISTs with few chromosomal aberrations had a favorable outcome, and might not benefit from adjuvant therapy. The aim of the study was to investigate if chromosomal complexity could be used as a biomarker in de-escalation of adjuvant imatinib treatment. Methods: GIST patients undergoing surgical resection of their primary tumor between 1998 and 2020 were identified in the sarcoma database at Oslo University Hospital. All samples with available karyotype analysis made on fresh tumor tissue were included. Karyotypes were categorized as simple if they had ≤5 chromosomal changes, and complex if there were &gt; 5 chromosomal aberrations. Results: Chromosomal aberrations were detected in 226 tumors, of which 181 (80.1 %) were gastric. The most frequent resulting imbalances were loss of 14q (75.9 %), 22q (43.5 %), 1p (36.6 %), and 15q (29.6 %). One-hundred and thirty-six tumors (60.2 %) had simple karyotypes whereas 90 (39.8 %) were complex. Cytogenetically complex tumors were larger ( P&lt; 0.001), had a higher mitotic count ( P= 0.009), and were more often non-gastric ( P&lt; 0.001). There was a strong association between chromosomal complexity and risk classification according to the modified NIH criteria ( P&lt; 0.001). Thirty-eight of 58 (65.5 %) high-risk tumors were karyotypically complex compared to 37 of 144 (25.7 %) tumors that were not high-risk. In the high-risk group, 17 patients experienced disease recurrence, of whom one had a simple and 16 had a complex tumor karyotype. Estimated 5-year recurrence-free survival (RFS) for patients with simple tumor karyotypes was 94 % compared to 51 % for patients with cytogenetically complex tumors ( P= 0.004). Adjuvant and/or neoadjuvant imatinib treatment was administered to 40 high-risk patients with a median treatment duration of 33 months (range 2-60 months). A complex karyotype was associated with poor RFS both in patients with ( P= 0.016) and without ( P= 0.046) adjuvant imatinib. Conclusions: Chromosomal complexity was strongly associated with poor RFS in localized, high-risk GIST. Recurrences were infrequent for tumors with simple karyotypes, indicating that de-escalation of adjuvant imatinib treatment should be further explored in patients with cytogenetically simple GISTs.

Glioblastoma Primary Cells Retain the Most Copy Number Alterations That Predict Poor Survival in Glioma Patients.

Gliomas are solid tumors that originate from glial cells in the brain or spine and account for 74.6% of malignant primary central nervous system tumors worldwide. As patient-derived primary cells are important tools for drug screening and new therapy development in glioma, we aim to understand the genomic similarity of the primary cells to their parental tumors by comparing their whole-genome copy number variations and expression profile of glioma clinicopathologic factors. We found that the primary cells from grade II/III gliomas lost most of the gene copy number alterations (CNAs), which were mainly located on chromosome 1p and 19q in their parental tumors. The glioblastoma (GBM) primary cells preserved 83.7% of the gene CNAs in the parental GBM tumors, including chromosome 7 gain and 10q loss. The CNA gains of LINC00226 and ADAM6 and the chromosome 16p11 loss were reconstituted in primary cells from both grade II/III gliomas and GBMs. Interestingly, we found these CNAs were correlated to overall survival (OS) in glioma patients using the Merged Cohort LGG and GBM dataset from cBioPortal. The gene CNAs preserved in glioma primary cells often predicted poor survival, whereas the gene CNAs lost in grade II/III primary cells were mainly associated to better prognosis in glioma patients. Glioma prognostic factors that predict better survival, such as IDH mutations and 1p/19q codeletion in grade II/III gliomas, were lost in their primary cells, whereas methylated MGMT promoters as well as TERT promoter mutations were preserved in GBM primary cells while lost in grade II/III primary cells. Our results suggest that GBM primary cells tend to preserve CNAs in their parental tumors, and these CNAs are correlated to poor OS and predict worse prognosis in glioma patients.

Genomic heterogeneity and copy number variant burden are associated with poor recurrence-free survival and 11q loss in human papillomavirus-positive squamous cell carcinoma of the oropharynx.

Human papillomavirus-positive (HPV+) squamous cell carcinoma of the oropharynx (OPSCC) is the most prevalent HPV-associated malignancy in the United States. Favorable treatment outcomes have led to increased interest in treatment de-escalation to reduce treatment morbidity as well as the development of prognostic markers to identify appropriately low-risk patients. Intratumoral genomic heterogeneity and copy number alteration burden have been demonstrated to be predictive of poor outcomes in many other cancers; therefore, we sought to determine whether intratumor heterogeneity and genomic instability are associated with poor outcomes in HPV+ OPSCC. Tumor heterogeneity estimates were made based on targeted exome sequencing of 45 patients with HPV+ OPSCC tumors. Analysis of an additional cohort of HPV+ OPSCC tumors lacking matched normal sequencing allowed copy number analysis of 99 patient tumors. High intratumorally genomic heterogeneity and high numbers of copy number alterations were strongly associated with worse recurrence-free survival. Tumors with higher heterogeneity and frequent copy number alterations were associated with loss of distal 11q, which encodes key genes related to double-strand break repair, including ATM and MRE11A. Both intratumor genomic heterogeneity and high-burden copy number alterations are strongly associated with poor recurrence-free survival in patients with HPV+ OPSCC. The drivers of genomic instability and heterogeneity in these tumors remains to be elucidated. However, 11q loss and defective DNA double-strand break repair have been associated with genomic instability in other solid tumors. Copy number alteration burden and intratumoral heterogeneity represent promising avenues for risk stratification of patients with HPV+OPSCC.

50. Personalized medicine in practice: Comprehensive cytogenomic profiling of a lung adenocarcinoma leads to re-classification of a concurrent lymphoma

Through the use of comprehensive genomic characterization, a personalized medicine approach has the potential to improve diagnosis, ameliorate prognostication and identify actionable molecular alterations. Herein, we describe a 77-year old patient under surveillance for untreated CLL, diagnosed via flow cytometry and further characterized by cytogenetic and molecular studies. In a peripheral blood specimen, FISH analyses identified trisomy 12 in 77% of nuclei and loss of TP53 in 9% of nuclei and was negative for all other markers tested including: loss of 13q14 and/or ATM and IGH-CCND1 rearrangement. Concurrent molecular studies detected a TP53 p.V157F mutation. PET-CT scan for surveillance of patient's CLL incidentally identified multiple bilateral pulmonary nodules, concerning for primary lung cancer. A pulmonary wedge resection confirmed the presence of a lung adenocarcinoma which was profiled using a targeted Next-Generation Sequencing (NGS) panel. This analysis revealed an actionable EGFR mutation (in frame exon 19 deletion), two TP53 mutations (p.P152Q and p.V157F) as well as an IGH-CCND1 rearrangement. Follow-up immunohistochemistry (IHC) studies performed on the intrapulmonary nodules showed positive staining for cyclin D1 (CCND1) and negative staining for SOX11. Together, the presence of the TP53 p.V157F in the lung resection, IGH-CCND1 rearrangement and IHC positivity for CCND1 were most suggestive of an infiltration of an extra-nodal mantle cell lymphoma (MCL) within the lung adenocarcinoma. Genomic characterization of this tumor led to the identification of a CCND1-IGH rearrangement, pathognomonic of MCL, which was misdiagnosed at the onset of the disease due to a cytogenetically cryptic event. This led to the re-evaluation of the patient's heme disorder which was reclassified as a CCND1-positive, SOX11-negative indolent MCL. This further highlights the importance of holistic genomic characterization for accurate classification of neoplasms and disease management.

New developments in existing WHO entities and evolving molecular concepts: The Genitourinary Pathology Society (GUPS) update on renal neoplasia.

The Genitourinary Pathology Society (GUPS) reviewed recent advances in renal neoplasia, particularly post-2016 World Health Organization (WHO) classification, to provide an update on existing entities, including diagnostic criteria, molecular correlates, and updated nomenclature. Key prognostic features for clear cell renal cell carcinoma (RCC) remain WHO/ISUP grade, AJCC/pTNM stage, coagulative necrosis, and rhabdoid and sarcomatoid differentiation. Accrual of subclonal genetic alterations in clear cell RCC including SETD2, PBRM1, BAP1, loss of chromosome 14q and 9p are associated with variable prognosis, patterns of metastasis, and vulnerability to therapies. Recent National Comprehensive Cancer Network (NCCN) guidelines increasingly adopt immunotherapeutic agents in advanced RCC, including RCC with rhabdoid and sarcomatoid changes. Papillary RCC subtyping is no longer recommended, as WHO/ISUP grade and tumor architecture better predict outcome. New papillary RCC variants/patterns include biphasic, solid, Warthin-like, and papillary renal neoplasm with reverse polarity. For tumors with 'borderline' features between oncocytoma and chromophobe RCC, a term "oncocytic renal neoplasm of low malignant potential, not further classified" is proposed. Clear cell papillary RCC may warrant reclassification as a tumor of low malignant potential. Tubulocystic RCC should only be diagnosed when morphologically pure. MiTF family translocation RCCs exhibit varied morphologic patterns and fusion partners. TFEB-amplified RCC occurs in older patients and is associated with more aggressive behavior. Acquired cystic disease (ACD) RCC-like cysts are likely precursors of ACD-RCC. The diagnosis of renal medullary carcinoma requires a negative SMARCB1 (INI-1) expression and sickle cell trait/disease. Mucinous tubular and spindle cell carcinoma (MTSCC) can be distinguished from papillary RCC with overlapping morphology by losses of chromosomes 1, 4, 6, 8, 9, 13, 14, 15, and 22. MTSCC with adverse histologic features shows frequent CDKN2A/2B (9p) deletions. BRAF mutations unify the metanephric family of tumors. The term "fumarate hydratase deficient RCC" ("FH-deficient RCC") is preferred over "hereditary leiomyomatosis and RCC syndrome-associated RCC". A low threshold for FH, 2SC, and SDHB immunohistochemistry is recommended in difficult to classify RCCs, particularly those with eosinophilic morphology, occurring in younger patients. Current evidence does not support existence of a unique tumor subtype occurring after chemotherapy/radiation in early childhood.

Somatic Copy Number Alterations and Associated Genes in Clear-Cell Renal-Cell Carcinoma in Brazilian Patients.

Somatic copy number aberrations (CNAs) have been associated with clear-cell renal carcinoma (ccRCC) pathogenesis and are a potential source of new diagnostic, prognostic and therapeutic biomarkers. Recurrent CNAs include loss of chromosome arms 3p, 14q, 9p, and gains of 5q and 8q. Some of these regional CNAs are suspected of altering gene expression and could influence clinical outcomes. Despite many studies of CNAs in RCC, there are currently no descriptions of genomic copy number alterations in a Brazilian ccRCC cohort. This study was designed to evaluate the chromosomal profile of CNAs in Brazilian ccRCC tumors and explore clinical associations. A total of 92 ccRCC Brazilian patients that underwent nephrectomy at Barretos Cancer Hospital were analyzed for CNAs by array comparative genomic hybridization. Most patients in the cohort had early-stage localized disease. The most significant alterations were loss of 3p (87.3%), 14q (35.8%), 6q (29.3%), 9p (28.6%) and 10q (25.0%), and gains of 5q (59.7%), 7p (29.3%) and 16q (20.6%). Bioinformatics analysis revealed 19 genes mapping to CNA significant regions, including SETD2, BAP1, FLT4, PTEN, FGFR4 and NSD1. Moreover, gain of 5q34-q35.3 (FLT4 and NSD1) and loss of 6q23.2-q23.3 (MYB) and 9p21.3 (MLLT3) had gene expression levels that correlated with TCGA data and was also associated with advanced disease features, such as larger tumors, Fuhrman 3, metastasis at diagnosis and death. The loss of region 14q22.1 which encompasses the NIN gene was associated with poor overall survival. Overall, this study provides the first CNA landscape of Brazilian patients and pinpoints genomic regions and specific genes worthy of more detailed investigations. Our results highlight important genes that are associated with copy number changes involving large chromosomal regions that are potentially related to ccRCC tumorigenesis and disease biology for future clinical investigations.

Integrated molecular analysis of adult sonic hedgehog (SHH)-activated medulloblastomas reveals two clinically relevant tumor subsets with VEGFA as potent prognostic indicator.

Up to now, adult medulloblastoma (MB) patients are treated according to the protocols elaborated for pediatric MB although these tumors are different in terms of clinical outcomes and biology. Approximately 70% of adult MB disclose a sonic hedgehog (SHH) molecular signature in contrast to about 30% in pediatric cohorts. In addition, adult SHH-MB (aSHH-MB) are clinically heterogeneous but there is consensus neither on their optimal treatment nor on risk stratification. Thus, the identification of clinically relevant molecular subsets of aSHH-MB and identification of potential treatment targets remains inconclusive. We analyzed 96 samples of institutionally diagnosed aSHH-MB through genome-wide DNA methylation profiling, targeted DNA sequencing, and RNA sequencing to identify molecular subcategories of these tumors and assess their prognostic significance. We defined two aSHH-MB numerically comparable epigenetic subsets with clinical and molecular variability. The subset "aSHH-MBI" (46%/48%) was associated with PTCH1/SMO (54%/46%) mutations, "neuronal" transcriptional signatures, and favorable outcomes after combined radio-chemotherapy (5-year PFS = 80% and OS = 92%). The clinically unfavorable "aSHH-MBII" subset (50%/52%; 5-year PFS = 24% and OS = 45%) disclosed GLI2 amplifications (8%), loss of 10q (22%), and gene expression signatures associated with angiogenesis and embryonal development. aSHH-MBII tumors revealed strong and ubiquitous expression of VEGFA both at transcript and protein levels that was correlated with unfavorable outcome. (1) The histologically uniform aSHH-MB cohort exhibits clear molecular heterogeneity separating these tumors into two molecular subsets (aSHH-MBI and aSHH-MBII), which are associated with different cytogenetics, mutational landscapes, gene expression signatures, and clinical course. (2) VEGFA appears to be a promising biomarker to predict clinical course, which needs further prospective validation as its potential role in the pathogenesis of this subset.

Ultra high-risk PFA ependymoma is characterized by loss of chromosome 6q.

Within PF-EPN-A, 1q gain is a marker of poor prognosis, however, it is unclear if within PF-EPN-A additional cytogenetic events exist which can refine risk stratification. Five independent non-overlapping cohorts of PF-EPN-A were analyzed applying genome-wide methylation arrays for chromosomal and clinical variables predictive of survival. Across all cohorts, 663 PF-EPN-A were identified. The most common broad copy number event was 1q gain (18.9%), followed by 6q loss (8.6%), 9p gain (6.5%), and 22q loss (6.8%). Within 1q gain tumors, there was significant enrichment for 6q loss (17.7%), 10q loss (16.9%), and 16q loss (15.3%). The 5-year progression-free survival (PFS) was strikingly worse in those patients with 6q loss, with a 5-year PFS of 50% (95% CI 45%-55%) for balanced tumors, compared with 32% (95% CI 24%-44%) for 1q gain only, 7.3% (95% CI 2.0%-27%) for 6q loss only and 0 for both 1q gain and 6q loss (P = 1.65 × 10-13). After accounting for treatment, 6q loss remained the most significant independent predictor of survival in PF-EPN-A but is not in PF-EPN-B. Distant relapses were more common in 1q gain irrespective of 6q loss. RNA sequencing comparing 6q loss to 6q balanced PF-EPN-A suggests that 6q loss forms a biologically distinct group. We have identified an ultra high-risk PF-EPN-A ependymoma subgroup, which can be reliably ascertained using cytogenetic markers in routine clinical use. A change in treatment paradigm is urgently needed for this particular subset of PF-EPN-A where novel therapies should be prioritized for upfront therapy.

Recurrent Somatic Chromosomal Abnormalities in Relapsed Extraocular Retinoblastoma.

Simple SummaryRelapse outside the eye of retinoblastoma (the most common eye cancer in children) is an uncommon event in developed countries, however it is the main cause of death in patients with retinoblastoma worldwide. The genomic features of this population are not known. We studied 23 cases from four countries and found a characteristic pattern in chromosomal copy number alterations that could help guide future clinical management of these patients.Most reports about copy number alterations (CNA) in retinoblastoma relate to patients with intraocular disease and features of children with extraocular relapse remain unknown, so we aimed to describe the CNA in this population. We evaluated 23 patients and 27 specimens from 4 centers. Seventeen cases had extraocular relapse after initial enucleation and six cases after an initial preservation attempt. We performed an analysis of CNA and BCOR gene alteration by SNP array (Single Nucleotide Polymorfism array), whole-exome sequencing, IMPACT panel and CGH array (Array-based comparative genomic hybridization). All cases presented CNA at a higher prevalence than those reported in previously published studies for intraocular cases. CNA previously reported for intraocular retinoblastoma were found at a high frequency in our cohort: gains in 1q (69.5%), 2p (60.9%) and 6p (86.9%), and 16q loss (78.2%). Other, previously less-recognized, CNA were found including loss of 11q (34.8%), gain of 17q (56.5%), loss of 19q (30.4%) and BCOR alterations were present in 72.7% of our cases. A high number of CNA including 11q deletions, 17q gains, 19q loss, and BCOR alterations, are more common in extraocular retinoblastoma. Identification of these features may be correlated with a more aggressive tumor warranting consideration for patient management.

Prognostic value of integrated cytogenetic, somatic variation, and copy number variation analyses in Korean patients with newly diagnosed multiple myeloma.

BackgroundTo investigate the prognostic value of gene variants and copy number variations (CNVs) in patients with newly diagnosed multiple myeloma (NDMM), an integrative genomic analysis was performed.MethodsSixty-seven patients with NDMM exhibiting more than 60% plasma cells in the bone marrow aspirate were enrolled in the study. Whole-exome sequencing was conducted on bone marrow nucleated cells. Mutation and CNV analyses were performed using the CNVkit and Nexus Copy Number software. In addition, karyotype and fluorescent in situ hybridization were utilized for the integrated analysis.ResultsEighty-three driver gene mutations were detected in 63 patients with NDMM. The median number of mutations per patient was 2.0 (95% confidence interval [CI] = 2.0–3.0, range = 0–8). MAML2 and BHLHE41 mutations were associated with decreased survival. CNVs were detected in 56 patients (72.7%; 56/67). The median number of CNVs per patient was 6.0 (95% CI = 5.7–7.0; range = 0–16). Among the CNVs, 1q gain, 6p gain, 6q loss, 8p loss, and 13q loss were associated with decreased survival. Additionally, 1q gain and 6p gain were independent adverse prognostic factors. Increased numbers of CNVs and driver gene mutations were associated with poor clinical outcomes. Cluster analysis revealed that patients with the highest number of driver mutations along with 1q gain, 6p gain, and 13q loss exhibited the poorest prognosis.ConclusionsIn addition to the known prognostic factors, the integrated analysis of genetic variations and CNVs could contribute to prognostic stratification of patients with NDMM.

A Distinctive Adnexal (Usually Paratubal) Neoplasm Often Associated With Peutz-Jeghers Syndrome and Characterized by STK11 Alterations (STK11 Adnexal Tumor): A Report of 22 Cases.

We describe 22 examples of a novel, usually paratubal, adnexal tumor associated with Peutz-Jeghers syndrome in nearly 50% of cases that harbored STK11 alterations in all tested (n=21). The patients ranged from 17 to 66 years (median=39 y) and the tumors from 4.5 to 25.5 cm (median=11 cm). Most (n=18) were paratubal, with metastases noted in 11/22 (50%) and recurrences in 12/15 (80%). Morphologically, they were characterized by interanastomosing cords and trabeculae of predominantly epithelioid cells, set in a variably prominent myxoid to focally edematous stroma, that often merged to form tubular, cystic, cribriform, and microacinar formations, reminiscent of salivary gland-type tumors. The tumor cells were uniformly atypical, often with prominent nucleoli and a variable mitotic index (median=9/10 HPFs). The tumors were usually positive to a variable extent for epithelial (CAM5.2, AE1/AE3, cytokeratin 7), sex cord (calretinin, inhibin, WT1), and mesothelial (calretinin, D2-40) markers, as well as hormone receptors. PAX8, SF1, and GATA-3 were rarely positive, while claudin-4, FOXL2, and TTF-1 were consistently negative. All sequenced tumors (n=21) harbored alterations in STK11, often with a loss of heterozygosity event. There were no other recurrently mutated genes. Recurrent copy number alterations included loss of 1p and 11q, and gain of 1q, 15q, and 15p. Despite an extensive morphologic, immunohistochemical, and molecular evaluation, we are unable to determine with certainty the histogenesis of this unique tumor. Wolffian, sex cord stromal, epithelial, and mesothelial origins were considered. We propose the term STK11 adnexal tumor to describe this novel entity and emphasize the importance of genetic counseling in these patients as a significant number of neoplasms occur in association with Peutz-Jeghers syndrome.

SMAD4 haploinsufficiency in small intestinal neuroendocrine tumors

BackgroundPatients with small intestinal neuroendocrine tumors (SINETs) frequently present with lymph node and liver metastases at the time of diagnosis, but the molecular changes that lead to the progression of these tumors are largely unknown. Sequencing studies have only identified recurrent point mutations at low frequencies with CDKN1B being the most common harboring heterozygous mutations in less than 10% of all tumors. Although SINETs are genetically stable tumors with a low frequency of point mutations and indels, they often harbor recurrent hemizygous copy number alterations (CNAs) yet the functional implications of these CNA are unclear.MethodsUtilizing comparative genomic hybridization (CGH) arrays we analyzed the CNA profile of 131 SINETs from 117 patients. Two tumor suppressor genes and corresponding proteins i.e. SMAD4, and CDKN1B, were further characterized using a tissue microarray (TMA) with 846 SINETs. Immunohistochemistry (IHC) was used to quantify protein expression in TMA samples and this was correlated with chromosome number evaluated with fluorescent in-situ hybridization (FISH). Intestinal tissue from a Smad4+/− mouse model was used to detect entero-endocrine cell hyperplasia with IHC.ResultsAnalyzing the CGH arrays we found loss of chromosome 18q and SMAD4 in 71% of SINETs and that focal loss of chromosome 12 affecting the CDKN1B was present in 9.4% of SINETs. No homozygous loss of chromosome 18 was detected. Hemizygous loss of SMAD4, but not CDKN1B, significantly correlated with reduced protein levels but hemizygous loss of SMAD4 did not induce entero-endocrine cell hyperplasia in the Smad4+/− mouse model. In addition, patients with low SMAD4 protein expression in primary tumors more often presented with metastatic disease.ConclusionsHemizygous loss of chromosome 18q and the SMAD4 gene is the most common genetic event in SINETs and our results suggests that this could influence SMAD4 protein expression and spread of metastases. Although SMAD4 haploinsufficiency alone did not induce tumor initiation, loss of chromosome 18 could represent an evolutionary advantage in SINETs explaining the high prevalence of this aberration. Functional consequences of reduced SMAD4 protein levels could hypothetically be a potential mechanism as to why loss of chromosome 18 appears to be clonally selected in SINETs.

Differential gain of chromosomal regions 20q or 13q with loss of 8p and 18q differentiates disease-free survival in colorectal cancer.

126 Background: In microsatellite stable (MSS) colorectal cancer (CRC) cases, frequent amplifications in 13q and 20q regions have been reported with limited understanding of their potential role for prognosis and treatment. Based on an internal research molecular tumor board case analysis of a KRAS WT, MSS left-sided (LS), metastatic CRC patient, we noted 50% of the 11 listed variants of unknown significance were on 20q. This study aims to investigate the potential prognostic role for gain of 20q chromosomal regions relative to other chromosomal imbalances using a retrospective cohort. Methods: We performed a genome-wide CN cluster analysis using the TCGA data with resulting patient clusters compared based on Kaplan-Meier estimated disease-free interval (DFI) in months (mos) using a log-rank. We examined associations between CN-derived clusters and CRC phenotypes. Results: Using TCGA data, we identified four CN-derived patient clusters of which two had significantly different DFI’s. One longer DFI cluster (n = 83; median DFI = 28mos) was defined by a signature of copy neutral genes along Chr8p and 18q regions. The shorter DFI cluster (n = 46; median DFI = 20mos) was defined by a signature of Chr8p and 18q regions of loss. The two clusters significantly differed in terms of site (85% right-sided in the shorter vs. 85% LS in the longer DFI cluster) and MS status (81% MSS in the shorter vs. 72% MSI in the longer DFI cluster) with no significant differences in age (mean of 70 years at diagnosis) and KRAS status (around 50% WT) between clusters. The shorter DFI was able to be further differentiated based on gain of chromosomal regions 20q (n = 26; median DFI = 24mos) and 13q (n = 20; median DFI = 18mos). No significant differences in mean age, tumor stage, site, and MS status between the shorter DFI subgroups were noted. Enriched pathways in the Chr20q gain sub-cluster include cell differentiation (Hippo, Rap1, VEGF), whereas metabolic signaling pathways via AGE-RAGE and AMPK, as well as cell growth and malignant transformation via RAS are enriched in the Chr13q gain sub-cluster. Conclusions: Clinical sequencing is able to identify regions of gain in Chr20q and Chr13q as part of VUS’s. Gain of Chr20q has been reported with better survival in mCRC patients. Gain of chromosomal region 20q and loss of 18q has been reported to discriminate between Lynch Syndrome and familial colorectal cancer. Herein, we show that gain of the 20q region with 18q and 8p regions of loss is associated with shorter DFI and gain of 13q region with loss of 8p even worse prognosis.

Defining the molecular features of radiation-induced glioma: A systematic review and meta-analysis.

BackgroundCranial radiation therapy is essential in treating many pediatric cancers, especially brain tumors; however, its use comes with the risk of developing second malignancies. Cranial radiation-induced gliomas (RIGs) are aggressive high-grade tumors with a dismal prognosis, for which no standard therapy exists. A definitive molecular signature for RIGs has not yet been established. We sought to address this gap by performing a systematic review and meta-analysis of the molecular features of cranial RIGs.MethodsA systematic review of the literature was performed according to Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines. Articles and case reports that described molecular analyses of cranial radiation-induced high-grade gliomas were identified and evaluated, and data extracted for collation.ResultsOf 1727 records identified, 31 were eligible, containing 102 unique RIGs with molecular data. The most frequent genetic alterations in RIGs included PDGFRA or TP53 mutations, PDGFRA or CDK4 amplifications, and CDKN2A deletion, along with 1q gain, 1p loss and 13q loss. Of note, mutations in ACVR1, EGFR, H3F3A, HIST1H3B, HIST1H3C, IDH2, SMARCB1 or the TERT promoter were not observed. A comparative analysis revealed that RIGs are molecularly distinct from most other astrocytomas and gliomas and instead align most closely with the pedGBM_RTK1 subgroup of pediatric glioblastoma.ConclusionsThis comprehensive analysis highlights the major molecular features of RIGs, demonstrates their molecular distinction from many other astrocytomas and gliomas, and reveals potential genetic drivers and therapeutic targets for this currently fatal disease.

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.