1p Loss Research Articles

RBM10‐TFE3 fusions: A FISH‐concealed anomaly in adult renal cell carcinomas displaying a variety of morphological and genomic features: Comprehensive study of six novel cases

The accurate diagnosis of Xp11-translocation renal cell carcinoma (RCC) in adults is challenging. TFE3 (located on chromosome X) fuses with a partner gene generally located on another chromosome. In rare cases TFE3 may fuse with a neighboring gene: RBM10. Because TFE3 false-positive immunostaining is a common pitfall in many laboratories, demonstration of the chromosomal rearrangement is required in order to ascertain the diagnosis. Fluorescence in situ hybridization (FISH)-that has been considered as the gold standard method-reaches its limits for detecting small Xp11 paracentric inversions. We performed a comprehensive clinical, histological and genomic study of six novel cases of RCC with RBM10-TFE3 fusion. Using FISH, TFE3 rearrangement was equivocal in one case and negative in others. RBM10-TFE3 fusion was discovered using targeted RNA sequencing (RNASeq). As all the previously reported cases (mean age: 50), the six patients were adults (mean age: 42), suggesting an epidemiologic difference between RBM10-TFE3 RCC and tumors harboring some other partner genes, such as ASPSCR1 that rather occur in children. Array-comparative genomic hybridization showed several alterations, notably a gain of 17q in four cases with papillary features and loss of 3p in one case with clear cells. Our study demonstrates that, though rare among adult cases of RCC, RBM10-TFE3 fusion is not exceptional and warrants appropriate molecular detection. Notably, it would be worthy to systemically investigate by RNASeq challenging RCC with type-2 papillary features and 17q gain.

IDH2 R172 Mutations Across Poorly Differentiated Sinonasal Tract Malignancies: Forty Molecularly Homogenous and Histologically Variable Cases With Favorable Outcome.

IDH2 R172 mutations occur in sinonasal undifferentiated carcinoma (SNUC), large-cell neuroendocrine carcinoma (LCNEC), sinonasal adenocarcinomas, and olfactory neuroblastoma (ONB). We performed a clinical, pathologic, and genetic/epigenetic analysis of a large IDH2-mutated sinonasal tumor cohort to explore their distinct features. A total 165 sinonasal/skull base tumors included 40 IDH2 mutants studied by light microscopy, immunohistochemistry, and genome-wide DNA methylation, and 125 IDH2 wild-type tumors used for comparison. Methylation profiles were analyzed by unsupervised hierarchical clustering, t-distributed stochastic neighbor embedding dimensionality reduction and assessed for copy number alterations (CNA). Thirty-nine histologically assessable cases included 25 (64.1%) SNUC, 8 (20.5%) LCNEC, 2 (5.1%) poorly differentiated adenocarcinomas, 1 (2.7%) ONB, and 3 (7.7%) IDH2-mutated tumors with ONB features. All cases were high-grade showing necrosis (82.4%), prominent nucleoli (88.9%), and median 21 mitoses/10 HPFs. AE1/AE3 and/or CAM 5.2 were positive in all and insulinoma-associated protein 1 (INSM1) in 80% cases. All IDH2 mutants formed one distinct group by t-distributed stochastic neighbor embedding dimensionality reduction separating from all IDH2 wild-type tumors. There was no correlation between methylation clusters and histopathologic diagnoses. Recurrent CNA included 1q gain (79.3%), 17p loss (75.9%), and 17q gain (58.6%). No CNA differences were observed between SNUC and LCNEC. IDH2 mutants showed better disease-specific survival than SMARCB1-deficient (P=0.027) and IDH2 wild-type carcinomas overall (P=0.042). IDH2-mutated sinonasal tumors are remarkably homogeneous at the molecular level and distinct from IDH2 wild-type sinonasal malignancies. Biology of IDH2-mutated sinonasal tumors might be primarily defined by their unique molecular fingerprint rather than by their respective histopathologic diagnoses.

ECOA-9. Integration of molecular and methylation classifications yields three meningioma groups and suggests chromosome 1p loss may be critical to the aggressive group

Next-generation sequencing has deepened our understanding of meningiomas, particularly those that behave aggressively. Classifications using either DNA methylation profiling or RNA-sequencing predict tumor behavior more reliably than WHO grade, and segregate common meningioma features similarly, implying possible overlap between classifications. In this study, we performed DNA methylation profiling, RNA-sequencing, and whole-exome sequencing on 110 primary meningiomas (90 WHO I, 20 WHO II). Unsupervised non-negative matrix factorization demonstrated three epigenetic types which were highly concordant with our published transcriptional types (87.3% concordance). Two additional classifications (one using 1p/22 loss, the other merlin expression/chromosomal instability) were also highly concordant and an overall meningioma group (MenG) classification was assigned integrating all four together. MenG A and B rarely recur, while MenG C behave aggressively (median recurrence free survival (RFS) of 3.1 years), even after gross total resection (median RFS 4.2 years). MenG A tumors retain Merlin expression (no chromosome loss or NF2 mutation) and harbor mutations in TRAF7, AKT1, or KLF4. Both MenG B and C are merlin-deficient, but MenG B demonstrate low rates of CNV and MenG C high rates of CNV, particularly loss of chromosome 1p. Using partial least squares regression (PLS), we explored how gene expression correlated with promoter methylation and CNV, thereby classifying genes which correlated closely as ‘methylation-driven’ or ‘CNV-driven’. Overall, there were more methylation-driven (5.7%) than CNV-driven (2.9%) genes. Differentially expressed genes (DEGs) were enriched for both methylation- and CNV-driven genes at similar proportions (10.6% and 5.8%), but DEGs unique to MenG C were significantly enriched for CNV-driven (23.7%), but not methylation-driven (4.7%) genes, primarily due many MenG C DEGs on chromosome 1p. Overall, this work suggests three underlying meningioma groups which are identifiable through methylation, transcriptional, or genetic/cytogenetic profiling and warrants exploration of the role of chromosome 1p in group that behaves aggressively.

Abstract 3132: Tumorigenesis of MEN2 pheochromocytoma

Abstract Backgrounds: Pheochromocytomas are catecholamine-secreting neuroendocrine tumors, which occur sporadically or as part of inherited syndromes, such as multiple endocrine neoplasia type2 (MEN2), caused by deleterious mutations within the RET protooncogene. Although the RET mutation is implicated in the development of MEN2-associated pheochromocytoma, there is a long (typically 40 years) latency period before the disease onset, suggesting the need of the acquisition of secondary hits. However, little is known about such secondary alterations involved in the pathogenesis of MEN2. In this study, we compared genetic profiles of MEN2 pheochromocytoma and its precursor lesion, adrenal medullary hyperplasia (AMH). Materials and methods: Samples were collected from surgical specimens in 6 MEN2 pheochromocytoma cases, including a MEN2B case with RET p.M918T and 5 MEN2A cases with RET p.C634F, p.C634F, p.C634Y, p.E768D and p.C620S, and 1 AMH case with MEN2A carrying RET p.C634Y. Spatially separated samples were collected from a tumor slice (“tumor parts”). Multiple microscale sampling was also performed from apparently normal adrenal medulla (“non-tumor parts”). Somatic mutations and copy number alterations (CNAs) were then evaluated by whole exome sequencing. Result: Regarding “tumor parts”, a total of 20 samples from 6 MEN2 pheochromocytomas was analyzed. The number of somatic mutations per sample was ranging from 2 to 10. No somatic mutations commonly implicated in sporadic pheochromocytomas were detected. In a case with MEN2B, 1p, 3q and 21q losses were shared across all 3 samples, while 11p and 11q losses and 5p, 5q, 12p, 12q and 15q gains were private, suggesting that 1p, 3q and 21q losses occurred early in the tumorigenesis. In 5 MEN2A cases, 1p (80%), 3q (100%), 21q (60%) and 22q (60%) losses were frequently shared across all samples in each case. As for “non-tumor parts”, a total of 12 samples from 2 MEN2 pheochromocytoma cases and 1 AMH case were analyzed. The number of somatic mutations per sample ranged from 0 to 15. In 2 MEN2 pheochromocytoma cases, no somatic mutations were shared across the samples or no CNAs were detected, while 12 mutations were shared in two samples derived from an AMH case. Interestingly, these samples harbored 17p loss alone, while 1p, 3q, 21q or 22q losses were not found. Conclusion and perspective: MEN2 pheochromocytoma and AMH had different genetic characteristics, where 1p, 3q, 21q and 22q losses could have a role in early tumorigenesis. Further studies involving an increasing number of patients with MEN2 pheochromocytoma and AMH are warranted. Citation Format: Tatsuki Ogasawara, Yoichi Fujii, Nobuyuki Kakiuchi, Yusuke Shiozawa, Hiromichi Suzuki, Ryuichi Sakamoto, Yusaku Yoshida, Yuichi Shiraishi, Satoru Miyano, Seishi Ogawa. Tumorigenesis of MEN2 pheochromocytoma [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 3132.

The Genetics of Uveal Melanoma: Overview and Clinical Relevance.

Over the last ten years, much has been learnt about the genetic characteristics and genetic evolution of uveal melanoma. It has been shown that uveal melanoma differs fundamentally from non-uveal melanoma and is an independent genetic subtype. Compared to other tumours, uveal melanoma has a low mutational burden. There are recurring chromosomal aberrations with losses of 1p, 6q, 8p and 16q, gains of 6p and 8q, and the presence of monosomy 3. GNAQ, GNA11, PLCB4, CYSLTR2, MAPKAPK5, as well as mutations in BAP1, SF3B1, SRSF2 and EIF1AX, the latter being linked to a higher risk of metastasis, have been identified as significantly mutated genes. In rare cases, a BAP1 germline mutation may also be present. In addition to higher risk of uveal melanoma, this variant is also linked with other tumours. In this case, additional work-up, genetic counselling and screening of family members should be offered. While the knowledge about the genetic characteristics of uveal melanoma is already routinely used for diagnostic and prognostic purposes, targeted genotype-dependent therapy of uveal melanoma is currently still missing.

Abstract 2210: Chromosome arm aneuploidy landscape in lung adenocarcinoma

Abstract Background: Lung adenocarcinoma (LUAD) is the most common subtype of non-small cell lung cancer. Overall aneuploidy frequency in LUAD have been previously documented. We aimed to assess whether specific chromosome arm aneuploidy (CAA) patterns can be found in primary tumor and different metastatic sites, and across patients with different driver mutations. We also investigated CAA and driver mutation co-occurrence patterns in LUAD, as well as probable order of CAA acquisition. Methods: We performed targeted panel sequencing (425 cancer-related genes) on 3533 tissue samples from LUAD patients. CAA status (arm gain or loss) was derived from segmental copy number analysis using Sequenza, accounting for purity and ploidy. 4 types of CAA analysis were conducted: total CAA burden, gain-loss pattern, co-occurrence/exclusivity, and population level CAA progression assessed by Tronco on network-based stratified (NBS) subtypes. Results: Use of panel sequencing data for analysis of CAA status was validated against WES results, and achieved high concordance (Kappa: 0.75, Pearson: 0.85). Primary tumor and different metastatic sites had dramatic differences in CAA burdens (p=3.4e-06). Liver and brain had more arm losses than gains compared to primary (q=0.047 and 0.049 respectively). Different driver mutation groups also had significant difference in CAA burden (p<2.2e-16). BRAF group had more arm gains than losses; whereas PIK3CA had more arm losses than gains. TP53 mutation status had a moderating effect on both CAA burden and gain-loss pattern across metastatic sites and driver mutation groups. EGFR 19del and L858R mutations showed significant differences in CAA co-occurrence/exclusion. EGFR 19del tend to co-occur with 4q-, 5p-, 5q-, 8q+, 9p+, 16q+, 17p+, 17q-, 22q-, and was in exclusion with 14q-. EGFR L858R co-occurred with 4q+, but was in exclusion with 1p-, 8p+ and 16p-. MET significantly co-occurred with 1q-, and PIK3CA with 2p+. Clustering of LUAD patients using NBS gave us 2 tight clusters with high silhouette score (0.86). These two LUAD subtypes showed distinct orders of CAA acquisition. Both clustered subtypes began with acquisition of 7p gain. Subtype 1 is dominated by initial arm gains, followed by few arm losses, seen in previously published literature; whereas subtype 2 began with a simultaneous acquisition of 19p loss, with further additions of many arm losses, before eventual accumulation of arm gains. Proportion of these 2 subtypes varied across primary and metastatic sites, and driver mutant groups. Conclusions: There are significant differences in arm aneuploidy patterns across metastatic sites and mutation groups. Overall, LUAD patients exhibit a unique pattern of co-occurrence and exclusivity between CAA and driver mutations, as well as two subtypes with distinct CAA evolution trajectories. These CAA patterns may hold biological meaning and may be useful predictors of clinical outcomes and warrants further investigation. Citation Format: Hua Bao, Ming Han, Yi Shen, Xue Wu, Yang Shao. Chromosome arm aneuploidy landscape in lung adenocarcinoma [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 2210.

Abstract 2367: Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma

Abstract MicroRNAs (miR) are evolutionarily highly conserved non-coding RNAs that play roles in fundamental cellular functions by post-translational gene regulation. In cancer pathogenesis, miRs exert both anti-tumorigenic and pro-tumorigenic effects by virtue of miR-specific and context-dependent mechanisms. The role of chromosomal changes and mutation status on miR expression in cancer remains unknown. Here we provide evidence that chromosome arm 1p loss and P53 mutations are associated with lower levels of miR-34a, particularly in squamous cancers such as head and neck squamous cell carcinoma (HNSCC). miR-34a acts as a tumor suppressor and physically interacts with and functionally targets the proto-oncogene MET, and modulates cell proliferation. miR-34a expression is inversely correlated with MET in tumor tissue. We found that miR-34a suppresses HNSCC carcinogenesis, at least in part, by downregulating MET, consequently inhibiting HNSCC proliferation. Moreover, ectopic expression of miR-34a reduces HNSCC cell proliferation and tumor burden in vitro and in vivo, represses expression of genes and proteins involved in epithelial-mesenchymal transition, and negates the oncogenic effect of MET in mouse tumors. Our work demonstrates a role of miR-34a in maintaining tumor immunity, as overexpression of miR-34a resulted in a decease in percentage of anti-inflammatory (M2-like) TAMs and PDL1+TAMs, which are reported to have pro-tumorigenic activity. In HNSCC patient samples, higher levels of miR-34a are significantly associated with a higher frequency of Th1 cells, myeloid cells, and regulatory T cells. Our results demonstrate that miR-34a directly targets MET and maintains anti-tumor immune activity, and could potentially represent a new therapeutic approach for HNSCC. Citation Format: Xun Wu, Yi-Shing Lisa Cheng, Methew Matthen, Angela Yoon, Gary Schwartz, Shashi Bala, Alison M. Taylor, Fatemeh Momen-Heravi. Role of miR-34a-MET axis in head and neck cancer squamous cell carcinoma [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 2367.

Chromosome 1q12 Instability Drives Both 1q21 Amplification and Arm-Length Deletions in Multiple Myeloma

IntroductionChromosome instability is a driver of copy number aberrations (CNAs) in cancer, and is a factor leading to tumor heterogeneity and resistance to therapy. In multiple myeloma (MM) CNAs involving 1q21 and p53 are high-risk secondary aberrations identified in increasing frequency in relapsed and refractory patients. The 1q21 region contains a large number of possible candidate genes which show amplification (amp) and/or deregulated expression important in myeloma pathogenesis; these genes include MCL1, MUC1, IL6R, BCL9, CKS1B, ANP32E, PSMD4 and ILF2, among others. Given that 1q21 copy number gains are one of the most frequent CNAs in all cancers, the mechanisms for the accumulation of CNAs of 1q21 in MM have important implications in cancer genomics. The most common mechanisms for CNAs of 1q21 in MM are direct and inverted duplications of 1q21, focal amplification of 1q12-23 by breakage-fusion-bridge (BFB) cycles, and unbalanced jumping translocations of 1q12 (JT1q12s) pericentromeric heterochromatin. JT1q12s translocate the entire 1q to the pericentromeric or telomeric regions of non-homologous receptor chromosomes (RCs). Unfortunately, in addition to an increase of 1q21 CNAs, collateral damage involving concomitant losses of arm-length segments of the RCs occurs in the unbalanced pericentromeric translocations. This type of loss may be an underappreciated mechanism for the accumulation of haploinsufficiency and loss of tumor suppressor genes in MM, which have been reported in other malignancies. In our cytogenetic database the most frequent losses of RCs associated with JT1q12 gains are losses of 16q, 19q, 1p, 6q, and 17p, respectively.MethodsTo investigate the cytogenetic impact and progression of these arm-level deletions in RCs we performed a comprehensive metaphase analysis of 50 patients showing segmental aneuploidies with 4 or more copies of 1q21. G-banding, metaphase FISH, and spectral karyotyping were used to identify the key transient and clonal structural aberrations of 1q12 resulting in concomitant segmental aneuploidies in RCs.ResultsThe losses of 16q (8 patients), 19q (7 patients), and 17p (3 patients), were associated with whole-arm JT1q12s to the pericentromeric regions of these RCs. Losses of 6q (6 patients) were associated with a spectrum of JT1q12s aberrations which included the entire 6q from 6q10 through 6q27. Strikingly, isochromosome 1q (i1q) formation, which results in the deletion of 1p and gain of 1q (14 patients) showed the highest range CNAs for 1q21 (4-10). The higher copy number of 1q21 in the i1q patients resulted from BFB cycles of the 1q12-23 amplicon involving sister chromatid fusions (SCFs) at 1q12 breakpoints in these patients. Segmental amplifications of regions harboring MYC and MET were identified hitchhiking on JT1q12s, resulting in cryptic insertions in non-homologous RCs. Unexpectedly, BFB cycle amp of MYC involving SCFs at 1q12 breakpoints identified a novel mechanism for the amp of MYC in MM.ConclusionIn this study the unbalanced pericentromeric translocation of 1q12 involving RCs 16q, 19q, 6q, and 17p provides evidence for recurring arm-length deletions in the cytogenetic progression of MM. Importantly, loss of 17p is currently recognized as a high risk-stratification marker by interphase FISH (iFISH), and has previously been shown to result from JT1q12s in a subset of patients. We suggest that the accumulation of other concomitant copy number gains of 1q21 and losses of RCs such as 16q, 19q, and 6q may provide new risk stratification markers in myeloma. DisclosuresDavies:Seattle Genetics: Consultancy, Honoraria; Amgen: Consultancy, Honoraria; Bristol-Myers: Consultancy, Honoraria; Takeda: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. Morgan:Takeda: Consultancy, Honoraria; Bristol Myers: Consultancy, Honoraria; Celgene: Consultancy, Honoraria, Research Funding.

Conditioning Chemotherapy with BOR-MEL in Autologous Stem Cell Transplant in Multiple Myeloma

INTRODUCTIONIn multiple myeloma (MM) the intensive chemotherapy strategy followed by autologous stem cell transplantation (ASCT) has improved the overall median survival of patients with MM from 36 months to more than 5 years in patients at standard risk. Despite modern therapy, most patients with MM will have disease progression. Several prognostic factors have been identified for the progression of the disease, such as beta-2-microglobulin, albumin (international staging system score), elevated serum lactate dehydrogenase and certain chromosomal abnormalities such as t (14; 16), 17 , 8q21 and the loss of 1p or gains of 1q. Among the main strategies to achieve the best response in the transplanted MM, is the improvement of the conditioning regimen.In some Latin American cities, access to melphalan IV is limited, the use of oral melphalan has been previously reported. The addition of bortezomib to high doses of melphalan has been reported by several groups and has shown that the toxicity profile Is comparable to the high doses of Melphalan (mel200).OBJECTIVESTo assess the impact of bortezomib in combination with oral doses of oral melphalan (BorMel) on progression-free survival and overall survival of patients with multiple myeloma. We will also assess the toxicity and complications that occur during and after transplantation as well as time of grafting of both neutrophils and platelets.METHODS AND PATIENTSIt is a unilateral, observational, descriptive study that included patients 18 to 70 years of age with diagnosis of multiple myeloma included in the program of autologous stem cell transplantation at the National Medical Center “20 de Noviembre” in the Hematology Service since 2010 to 2017. All of them received oral melphalan 200mg / m2SC conditioning from day -5 to -2, in addition to Bortezomib 1.3mg / m2SC on days -11, -8, -4, -1.RESULTSWe included 47 patients, of whom 31 (65.9%) were men and 16 (34.1%) were women. The median age was 52 years (31-67 years).Regarding the immunoglobulin involved, 55% were IgG (26 patients), 12% IgA, non-secretors 8%. The Durie-Salmon stage I, II and III were 15, 14 and 71% respectively, 100% received first line bortezomib, the combination with Cyclophosphamide in 25%, Thalidomide 36% and Doxorubicin 29% mainly. To be admitted to ASCT, they had to have at least MBRP, 73% of the patients were transplanted after the first line of treatment, 17% required a second line, of these 4 (8.5%) by primary progression. The mobilization was based on Cyclophosphamide in 76.6%, the median of mononuclear cells obtained was 3.19 x 10(8) CMN / kg. There was no difference in the myeloid and platelet graft with mean of 13 days with a range of 9 to 24 days . During transplantation, 10% of the patients presented gastrointestinal toxicity, an infectious process in 40%, and the upper airway infection in 8%, the mortality during transplantation was 0%. 94% of patients entered maintenance treatment. At present, 63% of the patients continue to be maintained, progression-associated death in 6%, 17% in follow-up in other units, 25% progressed with an average of 18 months posttransplant.CONCLUSIONSThe addition of bortezomib to oral melphalan proved to be safe, as it did not increase toxicity or transplant-related mortality, however, if there was an improvement in event- free survival when compared with previous studies of oral melphalan, where the mean was 14 months. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Complex Karyotype Predicts the Prognostics Impact of WBC Count in Untreated Mantle Cell Lymphoma and Is Associated with Shortened Survival Independent of Specific Cytogenetic Abnormalities

Complex Karyotype Predicts the Prognostics Impact of WBC Count in Untreated Mantle Cell Lymphoma and Is Associated with Shortened Survival Independent of Specific Cytogenetic Abnormalities

A Retrospective Analysis of 450 TP53 Mutations in a Real Life Cohort of CLL from the French Innovative Leukemia Organization (FILO) Group

A Retrospective Analysis of 450 TP53 Mutations in a Real Life Cohort of CLL from the French Innovative Leukemia Organization (FILO) Group

Preferential In Vitro and In Vivo Induction of Endogenous Retrovirus Transcription from a Monoallelic Chromosome 7q in Decitabine (DAC) Treated AML Blasts

Background: Hypomethylating agents (HMAs) show an encouraging but not yet well-understood activity in AML/MDS patients (pts) with adverse cytogenetics, such as -7/7q-, often embedded in a complex, monosomal karyotype (Lübbert et al., Haematologica 2012). Integrative methylome/transcriptome studies have provided evidence for aberrant hypermethylation/silencing on monoallelic gene loci, including tumor suppressor genes (TSGs, Kotini et al., Nat. Biotechnol. 2015). Recently, an alternative mechanism of HMA action was described (Roulois et al., Chiappinelli et. al, Cell 2015): induction of silenced endogenous retroviral (ERV) dsRNA transcription, resulting in activation of an interferon-mediated, antitumor immune response (“viral mimicry”). To unravel in vivo HMA activity in different (cyto)genetic backgrounds, we hypothesized that in AML haploinsufficient for 7q, transcriptional repression of monoallelic genes in this region may be preferentially reversed by HMA treatment.Materials and Methods: For identification of genes induced by HMAs, two AML cell lines were selected, ELF-153 (ELF) and UCSD-AML1 (AML1): copy number variations were analyzed by Genome-Wide Human SNP Array 6.0 (Affymetrix), and revealed no monosomies but loss of 7p; in AML1 loss of one entire chromosome 7 was the only numerical aberration. Cells were treated with DAC at equitoxic and -effective concentrations for 4 days, harvested on day 5. Total RNA was depleted of ribosomal RNA (Ribo-Zero rRNA Removal Kit, Illumina) and cDNA libraries (strand-specific, 50 bp) were sequenced with ≥33 million paired-end reads, read quality checked with FastQC (≥95% >Q30). Alignment to the reference genome hg19 was performed with TopHat2, read-counting with HTseq, differential expression testing with DESeq2 (protein-coding transcripts only). Selected target genes were validated by RT-qPCR. Peripheral blood mononuclear cells (PBMC) were collected from 9 AML pts (5 pts with 7q-, 4 cytogenetically normal [CN] pts) treated with DAC (i.v. 20 mg/m2 for 5 days) within the DECIDER trial (NCT00867672). Leukemic blasts before treatment and at day 8 were isolated using automatic magnetic sorting of cells labeled with anti-human CD34 and CD117 MACS microbeads.Results: DAC treatment of AML1 and ELF cells resulted in massive transcriptome changes in both cell lines, with 1,704 genes induced in AML1 (total of 3,583) and 1,206 in ELF (total of 1,867; adjusted FDR <0.01). Comparing DAC-induced expression changes only of genes on 7q, we identified 43 genes significantly differentially expressed only in AML1 cells (up: 15), 24 only in ELF (up: 21), and 19 in both (up: 12). The 15 genes on 7q that were selectively upregulated in AML1 included 3 with TSG features: zyxin (ZYX; up 6.0-fold), homeodomain interacting protein kinase 2 (HIPK2; up 3.8-fold), and high mobility group box transcription factor 1 (HBP1, up 3.1-fold). However, the most heavily upregulated transcripts in AML1 were the endogenous retrovirus 3 group member 1 (ERV3-1 ; up 86.4-fold in AML1 vs 14.3-fold in ELF) and the neighboring zinc finger 117 (ZNF117; up 119.4-fold in AML1vs. 6.5-fold in ELF), both transcribed from the ERV3-1 promoter. ERV3-1 induction was accompanied by induction of the interferon type I response genes retinoic acid inducible gene I (RIG-I) and interferon regulatory factor 7 (IRF7) in both cell lines. Therefore, we next interrogated induction of these genes by DAC in vivo, utilizing serially sorted, matched primary AML blasts (purified before and 3 days after DAC administration). Stronger induction of ERV3-1 and RIG-I mRNA was seen in the 7q- pts compared to the CN pts: By qRT-PCR, median ERV3-1 and RIG-I expression was induced 1.5- and 1.4-fold, respectively, in the pts with 7q- (range: 1.1-2.1 and 1.0-3.2), 1.1- and 0.7-fold, respectively, in the CN pts (range: 0.8-1.7 and 0.3-0.9).Conclusions: We successfully developed an unbiased RNA-seq approach of AML cell lines either mono- or bi-allelic for 7q, demonstrating that DAC treatment preferential upregulates several monoallelic TSGs, and massively activates the ERV3-1 gene (implicated in “viral mimicry”). Induction of ERV3-1 and the dsRNA sensor RIG-I was validated in vivo, and preferentially seen in 7q- AML purified primary blasts. Thus, under clinically established treatment conditions, both the ERV and an interferon response gene can be activated by DAC, supporting a combination with immunotherapy. DisclosuresLübbert:Janssen-Cilag: Other: Travel Funding, Research Funding; Ratiopharm: Other: Study drug valproic acid; Celgene: Other: Travel Funding.

The Contribution of Gene Mutations to Long-Term Clinical Outcomes: Data from the Randomised UK LRF CLL4 Trial

Despite the excitement of targeted therapies such asibrutinib and idelalisib, the longer-term follow-up offered by early immuno/-chemotherapy trials presents opportunities to explore the disease features that contribute most to long-term outcomes.We employed next generation sequencing (NGS) to identify somatic mutations targeting 22 genes in DNA from 499 LRF CLL4 cases, a trial with >12 years follow-up on all surviving cases. The analysis was performed using a bespoke TruSeq panel, with confirmation of key sub-clonal mutations using orthogonal approaches (Ion Torrent [n = 19], SureSeq [n = 27], and ddPCR [n = 30]). Germ-line DNA was unavailable, so only COSMIC annotated mutations were included in downstream analysis. Variant allele frequencies were adjusted for tumor purity using CD19+CD5+% to define the clonal cell fraction (CCF). Significant associations between gene mutations and a comprehensive panel of clinical and biological features reported in previous LRF CLL4 papers were identified by Fisher's Exact test. Univariate Kaplan-Meier (KM) and multivariate Cox regression analyses were used to assess the impact of gene mutation status on progression-free (PFS) and overall survival (OS).We identified 608 mutations (mean= 1.22, min/max = 0/7 per patient), with 93% of cases harbouring >=1 mutation or copy number alteration (CNA) (Fig1A). After correction for background mutation rate (0.5/Mb, >=3% recurrence), 11/22 genes were recurrently mutated at frequencies between 3% (NFKBIE) and 25% (SF3B1), (Fig1A). SAMHD1 and KRAS mutations were principally clonal and subclonal, respectively, while ATM, MYD88, NOTCH1, SF3B1, TP53, and XPO1 occurred at mostly clonal CCFs(two-way binomial test , False Discovery Rate [FDR], Q >P [ P <0.05]) (Fig1B). Co-occurrence analysis of these genes with available clinical/biological/genetic features (Fisher's Exact, n = 1799 tests), identified 31 associations (FDR, Q >P [ P <0.05]), including those with other mutated genes (e.g. SF3B1 and POT1, OR = 3.4, P <0.01), CNAs (e.g. TP53 and del(17p), OR = 111.2, P <0.001), biological features (e.g. NOTCH1 and prolymphocytes, OR = 3.7, P <0.001), and clinical outcome (e.g. KRAS and 10-year survival, OR<0.1, P <0.001).Univariate survival analysis identified mutations of two genes associated with short PFS (TP53, EGR2) and nine with inferior OS (TP53, SF3B1, NOTCH1 +3'UTR , EGR2, RPS15, NFKBIE, BRAF, KRAS, NRAS). TP53 (median PFS = 7 months (mutated) vs. 26 (wild-type), HR = 1.95, P <0.001) and EGR2 (median = 13 vs. 25, HR = 2, P= 0.01) mutationswere associated with reduced PFS. In addition to the expected impact of TP53 (median OS = 31 vs. 73, HR = 2, P <0.001), NOTCH1 +3'UTR (median = 51 vs. 72, HR = 2, P= 0.001) and SF3B1 (median = 53 vs. 75, HR = 2, P <0.001) mutations, RPS15 (median = 34 vs. 70, HR = 2, P= 0.02) and NFKBIE (median= 43 vs. 70, HR = 2, P <0.01) also associated with reduced OS. Importantly, mutations of the MAPK-ERK genes, BRAF, KRAS, and NRAS, were principally mutually exclusive (87%), and all associated with reduced OS (BRAF : median = 46.5 vs. 71, HR = 1.69, P <0.01, KRAS : median = 46 vs. 70, HR = 1.87, P <0.01, NRAS : median = 50.5 vs. 70, HR = 2.22, P= 0.01). Only mutations in MYD88 associated with superior OS (median = 121 vs. 68, HR = 0.41, P= 0.01), but not in an analysis of only IGHV-mutated cases (median = 105 vs. 103, HR = 0.57, P= 0.27). Multivariate Cox models identified TP53, SF3B1, NOTCH1 +3'UTR, EGR2, as well as NRAS mutations (HR = 4, P <0.01) to predict for reduced OS.Using Ion Torrent to confirm subclonal TP53 mutations (all IARC annotated) , we assessed the clinical impact of TP53 clonality in the context of del(17p) (Fig1C).Contrary to previous studies, whilst clonal TP53 lesions, and subclonal mutations with concomitant 17p loss were associated with reduced PFS and OS, solely subclonal TP53 mutations did not impact on outcome (OS: median = 59 vs. 73, P=0.74, PFS: median = 34 vs. 26, P=0.94).Through resequencing analysis of the LRF CLL4 clinical trial, we identified several novel associations between gene mutations and clinic-biological disease features. We confirm the impact of several gene mutations on PFS and OS, and show that MAPK-ERK pathway mutations were associated with reduced survival. Furthermore, we provide evidence that subclonal TP53 mutations without detectable 17p loss do not exhibit reduced outcome, at least after initial treatment with chemotherapy. [Display omitted] DisclosuresSteele:Gilead: Consultancy, Honoraria; Portola Pharmaceuticals: Consultancy, Honoraria, Research Funding. Schuh:Abbvie: Honoraria; Janssen: Honoraria; Roche: Honoraria; Novartis: Honoraria; Gilead: Consultancy; Janssen: Honoraria; Gilead: Honoraria, Research Funding; Abbvie: Honoraria; Novartis: Honoraria; Celgene: Honoraria; Roche: Honoraria; Celgene: Honoraria. Strefford:Roche: Research Funding.

Linear and Branching Progression of Chronic Lymphocytic Leukemia (CLL) Under PI3K Inhibition (PI3Ki)

Introduction: B-cell receptor (BCR) signaling is a critical pathway in chronic lymphocytic leukemia (CLL). BTK or PI3K antagonists can abolish BCR-derived signals. Idelalisib inhibits the delta isoform of PI3K and has been already approved for patients with CLL, whereas duvelisib inhibits both the delta and gamma isoforms of PI3K and is currently being evaluated in clinical trials. Despite their remarkable efficacy, a proportion of patients develop progressive disease after initial response to the drug, sometimes in the form of transformation to high-grade lymphoma. Several mechanisms of resistance have been described in patients taking the BTK inhibitor ibrutinib, including BTK or phospholipase C gamma-2 mutations. There is, however, little information regarding the mechanisms of resistance to PI3Ki.In our institution, out of 12 patients who received PI3Ki as salvage therapy, 6 patients had their medication stopped due to progressive disease, 3 with transformed disease and 1 with accelerated CLL. In this study, we comprehensively evaluated the genomic aberrations of these last 4 patients with the aim of unraveling the molecular mechanisms of disease progression in this patient population.Patients and methods: DNA (tumor and germline) was extracted from peripheral blood/lymphoid tissue samples drawn before and after progression under PI3Ki. One case showed a CLL with increased proliferation (accelerated CLL), two cases showed transformation to a diffuse large B-cell lymphoma (DLBCL), and one to a plasmablastic lymphoma (PBL). Somatic copy number alterations (CNA) were identified using genome-wide copy number arrays. Whole-exome sequencing (WES) or whole-genome sequencing (WGS) was performed as previously described (Puente et al. Nature 2015).Results: All patients had relapsed after rituximab, fludarabine and cyclophosphamide (RFC) first-line therapy, and rituximab plus bendamustine second-line therapy with the exception of one patient, who received RFC twice. All had significant lymphadenopathy before PI3Ki initiation, initially responded to PI3Ki, and then experienced progression 103, 151, 329 and 752 days after treatment initiation. IGH rearrangement analysis revealed that the relapsed tumor was clonally related to the original CLL in all cases. Pre-PI3Ki samples from all patients harbored high-risk genomic aberrations by FISH/SNP arrays, including del(11q) in 3 patients and del(17p) in 1 patient. In 3 patients, all abnormalities identified before PI3Ki were maintained at progression, but several other alterations (1, 14, and 2 lesions, respectively) were acquired, consistent with a linear pattern of evolution. In the PBL transformation, some initial alterations were maintained (8q gain, 17p and 19p losses), some were not found (4q, and 19q gains, 2q loss), and 54 new alterations emerged at relapse, suggesting that the resistant clone arose from an early branch during CLL evolution. Together, 3 out of 4 patients (all except the patient with accelerated CLL) have alterations involving ARID1A (1 patient with a point mutation, and 2 patients with selective losses) . Other acquired aberrations in these patients were CDKN2A losses (2 patients), TP53 disruption (2 patients), MYC activating mutation (1 patient), 1 frameshift mutation in USP28 (1 patients), 2p16 gain (1 patient) and NFKBIE mutation (1 patient).Conclusion: Overall, CLL progression after PI3Ki may follow different pathways including linear progression with increasing proliferation and transformation to DLBCL through different mechanisms (TP53, CDKN2A, NFKBIE inactivation, MYC activation or ARID1A inactivation among others) and branching transformation to PBL that may represent an alternative pathway to escape from BCR-dependent activation. DisclosuresDelgado:Abbvie, Jansen, Gilead, Roche: Consultancy, Speakers Bureau. Lopez-Guillermo:Roche: Consultancy, Other: Research grant; Celgene: Consultancy; Gilead: Consultancy; Janssen: Consultancy; Novartis: Consultancy.

Ywhae Located on Chromosome 17p13 Promotes Myeloma Cells Proliferation Via Induction of Anti-Apoptosis and Survival Signaling Pathways

Patients with multiple myeloma (MM) carrying 17p deletion (del17p) have poor outcome. Around a third of MM patients with del(17p) are also reported to have a TP53 mutation. The acquisition of TP53 mutations seems to occur after allelic loss of 17p, and confers worse clinical outcomes. However, a recent publication has raised possibility that other additional genes resident at 17p13 may play a role in the clinical outcome of this subgroup of MM patients.The YWHAE gene is located at 17p13.3 and encodes the 14-3-3ε protein, one of the mammalian 14-3-3 protein family members that are highly conserved in eukaryotes. Altered 14-3-3ε expression is associated with development and progression of cancer. It has a context dependent pleotropic effect both as oncogene or tumor suppressor gene. However, its precise role in tumorigenesis has yet to be fully discovered.Using a large cohort of MM patients (n=369) RNA sequencing and SNP array data with complete clinical annotation, we observed a significant positive correlation (r=0.7) between YWHAE and TP53 at gene and copy number levels. In MM patients with del17p, YWHAE expression was significantly lower compared to normal plasma cells. Importantly, low expression of YWHAE correlated with poor clinical outcome (both EFS and OS), suggesting that YWHAE may be important in this specific myeloma clinical setting. However, in 2 independent MM datasets, we observed that overall YWHAE expression is higher in CD138+ MM cells compared to normal plasma cells. Moreover, we confirmed high expression of 14-3-3ε at the protein level in a large panel of MM cell lines and primary MM cells.To clarify this apparent paradox and shed light on the role of 14-3-3ε in MM, we used gene-specific CRISPR screen as well as shRNA knock down (KD) to study the functional impact of perturbation of YWHAE in MM. For the loss-of-function studies, we selected a large panel of MM cell lines based on their p53 status (WT, HD, Mu). We found a significant inhibitory effect of YWHAE KD on MM cell growth and survival over time, and induction of apoptosis via PARP cleavage and BAX increase. The antiproliferative effects were even more pronounced in p53 WT myeloma cell lines, such as MM1S and H929.Gene expression profiling (GEP) analysis showed a significant number of differentially expressed genes in YWHAE KD cells compared to control cells. Gene enrichment analysis revealed a major impact of YWHAE KD on a large set of genes involved in the mTORC1 signaling pathway. WB analysis confirmed a reduction of p-mTOR along with a decreased of p-p70 S6 kinase and p-4E BP1 after YWHAE KD. Considering the crucial role of mTOR pathway in the regulation of autophagy, we are now in the process of analyzing the effect of YWHAE on the autophagic response. Moreover, we found that YWHAE KD exerted a significant suppression of known E2F1 gene targets. Notably, a significant decrease in the expression of genes involved in the unfolded protein response was observed upon silencing/knock-out of YWHAE. As a consequence, myeloma cell sensitivity to bortezomib was increased after 14-3-3ε KD.In conclusion, YWHAE and its coding protein 14-3-3ε are constitutively expressed in MM cells. Perturbation of 14-3-3ε expression impacts MM cell growth via induction of apoptosis and inhibition of mTOR pathway. 14-3-3ε KD increase cell sensitivity to bortezomib. Ongoing efforts will further elucidate the potential molecular mechanism of YWHAE/14-3-3ε action in MM. DisclosuresAnderson:Oncopep: Other: scientific founder; Bristol-Myers Squibb: Membership on an entity's Board of Directors or advisory committees; Gilead Sciences: Membership on an entity's Board of Directors or advisory committees; MedImmune: Membership on an entity's Board of Directors or advisory committees; C4 Therapeutics: Other: scientific founder; Millenium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees.

Genomic profile of columnar cell variant of papillary thyroid carcinoma

Columnar cell variant (CCV) is a rare papillary thyroid carcinoma subtype. The majority of CCV occur in older patients and are large, invasive tumours that pursue an aggressive clinical course. Rare well-circumscribed CCV occur in younger female patients and are comparatively indolent. We retrospectively identified CCV with material available to perform targeted next-generation sequencing and correlated molecular results with clinicopathological features and outcome. Our cohort was comprised of nine CCV. Nearly all were aggressive tumours; however, one was predominantly well-circumscribed and arose in a thyroglossal duct cyst of a 26-year-old woman who had no evidence of disease at last follow-up. Seven (78%) cases demonstrated activating oncogenic driver alterations in BRAF, including BRAF V600E, an activating N486_P490del deletion, and BRAF-AGK fusions. Activating RAS mutations were seen in two (22%) cases. Additionally, three (33%) cases had TERT promoter mutations, four (44%) had loss of the tumour suppressor CDKN2A and one (11%) case had a loss of function TP53 mutation. Most cases (89%) also demonstrated copy number alterations, including recurrent gain of chromosome 1q (five cases) and losses of chromosome 9p (three cases) and 22q (four cases). The one case without secondary pathogenic mutations or copy number alterations was the tumour in the 26-year-old woman. We found that CCV is primarily a BRAF-driven tumour, with most also harbouring secondary oncogenic mutations and multiple chromosomal gains and losses. Moreover, our findings suggest that molecular analysis could potentially be used to help risk stratify CCV.

Correlating genomic copy number alterations with clinicopathologic findings in 75 cases of hepatocellular carcinoma

BackgroundOligonucleotide array comparative genomic hybridization (aCGH) analysis has been used for detecting somatic copy number alterations (CNAs) in various types of tumors. This study aimed to assess the clinical utility of aCGH for cases of hepatocellular carcinoma (HCC) and to evaluate the correlation between CNAs and clinicopathologic findings.MethodsaCGH was performed on 75 HCC cases with paired DNA samples from tumor and adjacent nontumor tissues. Survival outcomes from these cases were analyzed based on Barcelona-Clinic Liver Cancer Stage (BCLC), Edmondson-Steiner grade (E-S), and recurrence status. Correlation of CNAs with clinicopathologic findings was analyzed by Wilcoxon rank test and clustering vs. K means.ResultsThe survival outcomes indicated that BCLC stages and recurrence status could be predictors and E-S grades could be a modifier for HCC. The most common CNAs involved gains of 1q and 8q and a loss of 16q (50%), losses of 4q and 17p and a gain of 5p (40%), and losses of 8p and 13q (30%). Analyses of genomic profiles and clusters identified that losses of 4q13.2q35.2 and 10q22.3q26.13 seen in cases of stage A, grade III and nonrecurrence were likely correlated with good survival, while loss of 1p36.31p22.1 and gains of 2q11.2q21.2 and 20p13p11.1 seen in cases of stage C, grade III and recurrence were possibly correlated with worst prognosis.ConclusionsThese results indicated that aCGH analysis could be used to detect recurrent CNAs and involved key genes and pathways in patients with HCC. Further analysis on a large case series to validate the correlation of CNAs with clinicopathologic findings of HCC could provide information to interpret CNAs and predict prognosis.

Chromosome microarray characterisation of chromosome arm 12p loss associated with complex molecular karyotype and recurrent adverse cytogenetic markers in multiple myeloma

Copy number loss within chromosome 12 short arm (12p) has gained attention as an adverse cytogenetic marker in multiple myeloma. The prognostic significance and characterisation of the common minimal deleted region remains controversial between various studies with loss of CD27 proposed as the putative critical gene. We aimed to determine the frequency of 12p loss, its correlation with adverse cytogenetic markers further to define and characterise 12p deletions. Our study included a prospective cohort of 574 multiple myeloma patients referred for cytogenetic testing, including interphase fluorescence in situ hybridisation for IGH (14q32.33) translocations and chromosome microarray. Loss of 12p was detected in 54/574 (9.4%) patients and when compared with the non-12p loss group [520/574 (90.6%)], 12p loss patients demonstrated a statistically significant association with specific recurrent cytogenetic markers: complex molecular karyotypes (98.1% vs 45.2%), 1p loss (50.0% vs 20.2%), t(4;14) (20.4% vs 7.7%), 8p loss (37.0% vs 15.0%), 13/13q loss (70.4% vs 41.7%), and 17p loss (33.3% vs 6.5%). The size and location of 12p losses were heterogeneous with a common 0.88 Mb minimally deleted region that included ~9 genes from ETV6 to CDKN1B in 52/54 (~96.3%) patients but did not include CD27. Our findings support 12p loss being a secondary chromosome abnormality frequently co-occurring with adverse cytogenetic markers and complex molecular karyotypes indicative of chromosome instability.

Integrative structural variant and breakpoint detection using optical genome mapping in a patient with a transformed diffuse large B-cell lymphoma from chronic lymphocytic leukemia.

e19511 Background: The finding of complex karyotypes has been a clinically significant finding in cancers of advanced stage or during cancer progression. Conventional cytogenetic and FISH analyses have been limited by the low-resolution of chromosomes and the number of FISH probes which can be implemented in one assay. Recent study revealed the potential of using optical genome mapping to decipher the architecture of cancer genome at nucleotide level. Methods: Karyotyping, FISH and optical genome mapping of bone marrow specimen. Results: We reported on a 63-year-old female with chronic lymphocytic leukemia for nine years and transformed to diffuse large B-cell lymphoma (DLBCL). The karyotype revealed a hypodiploid chromosomal complement: 42,X,-X,der(2)t(2;?;8)(p25;?;q11.2),t(2;9)(q3?5;p2?2),der(4)t(4;?;8)(p16;?;q11.2)t(4;15)(q35;q15),-8,-15,dic(17;21)(p11.2;p11.2),der(18)t(8;18)(q21.2;p11.32)[12]/46,XX[8]. FISH analysis showed loss of TP53, monosomy 8 with 3-5 copies of the Myc genes. Optical genome mapping analysis revealed 12 insertions, 29 deletions, 8 duplications, 6 intrachromosomal translocations and 12 interchromosomal translocations. Whole genome analysis identified multiple gains of 2p, 8p and 8q, losses of 9p, 15q and 17p. The breakpoints of two unbalanced translocation and one complex chromosomal rearrangement have been narrowed down to nucleotide resolution: ogm[GRCh37] t(2;9)(q34;p21.2) g.[chr2:211,281,003::chr9:26,532,620], ogm t(2;8)(p25.3;p23.3) g.[chr2:15,924::chr8:215,177] and ogm der(4)t(8q24.3→8q21.13→2p24.2→2q34→4p15.2→4q34.3→15q15.1) g.[chr8:80,461,191::chr2:18,079,530→chr2:211,274,379::chr4:26,381,729→chr4:180,989,408::chr15:42,404,514]. Conclusions: Integrative analysis using conventional cytogenetic and molecular cytogenomic methodologies unveil the complex architectural alterations of caner genome. The resolution achieved by optical genome mapping would potentially lead to discovery of carcinogenic mechanisms, new fusion genes, prognostic and therapeutic markers.

POLR2A/TPR mutations as potential biomarker for better efficacy of immunoterpy in non-small cell lung cancer (NSCLC).

e21011 Background: Immunotherapy represents promising treatment in patients with non-small cell lung cancer (NSCLC).However, response rates for immunotherapy remain limited. There is a need to identify potential biomarkers to distinguish patients who respond to immunotherapy. POLR2A has been reported to be a potential target gene for immunotherapy of HER2-low breast cancer with 17p loss, together with TPR, responsible for RNA binding that is essential for cell survival. It is unclear whether POLR2A/TPR mutations could predict the efficacy of immunotherapy in NSCLC. Methods: WES (Whole Exome Sequencing) and survival data of NSCLC patients treated with anti–PD-1/PD-L1 or anti–CTLA-4 antibodies (Rizvi2015; Miao2018) were analyzed. WES, survival data of 1144 patients with NSCLC was obtained from The Cancer Genome Atlas (TCGA). In addition, the tumor-infiltrating immune cells present in NSCLC were analyzed by CIBERSORT, utilizing data from 515 patients by lung adenocarcinoma (LUAD) in TCGA pan-cancer cohort. Results: POLR2A/TPR mutations were significantly associated with better PFS in Rizvi2015 cohort (HR = 0.3; 95% CI, 0.09-1.02; P = 0.041), MIAO2018 cohort (HR = 0; 95% CI, 0-INF; P = 0.01). In TCGA, no association between POLR2A/TPR mutations and OS was observed (P = 0.67), suggesting that POLR2A/TPR mutations were not prognostic factor. POLR2A/TPR mutations were associated with higher TMB and TNB (p = 0.00018 and p = 0.00016 in Rizvi2015 cohort, p = 0.0042 and 0 = 0.0038 in Miao2018 cohort). In addition, POLR2A or TPR mutations was positively correlated with infiltrating levels of T cells CD4+ memory activated( p = 0.011), T cells CD8+(p = 0.017), and Macrophages M1(p = 0.0055). Conclusions: Our finding suggests that POLR2A/TPR mutations were associated with better PFS in NSCLC patients treated with Immunotherapy, with significantly higher TMB and TNB. The role of PTPRD/PTPRT in immunotherapy could be related with better immune cell infiltration.