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Introduction Programmed death-ligand 1 (PD-L1) expression is not a completely reliable predictive marker of the efficacy of anti–programmed cell death protein-1 (PD-1)/PD-L1 therapy in patients with advanced NSCLC. Immune-related tumor microenvironment (TME) is classified into four different types based on the tumor-infiltrating lymphocyte (TIL) status and PD-L1 expression. Methods We retrospectively reviewed patients with advanced NSCLC treated with anti–PD-1/PD-L1 therapy between 2015 and 2019. We investigated the association between the efficacy of anti–PD-1/PD-L1 therapy, the types of TME based on PD-L1 (clone: 22C3) expression, the density of CD8-positive TILs assessed by immunohistochemistry, and mutational profiles by next-generation sequencing. Results Overall, 228 patients were included in the analysis. The patients were classified into the following four groups: type I: PD-L1High (tumor proportion score ≥ 50%)/TILHigh (≥85/mm2; n = 73); type II: PD-L1Low (tumor proportion score Conclusions Differential types of TME, including PD-L1 expression and TIL status, could accurately predict the efficacy of anti–PD-1/PD-L1 therapy.

NTRK fusions in malignant tumors are therapeutic targets of tyrosine kinase inhibitors. Because they occur only in a small subset of mesenchymal tumors, knowledge regarding the corresponding histology is important to effectively identify patients who could benefit from targeted therapy. In this study, using RNA sequencing, we identified novel NTRK3 fusions involving related partner genes in 2 adult bone and soft tissue tumors that met the current histologic criteria of fibrosarcoma. Case 1 involved the left radius of a 38-year-old woman, whereas in case 2, the right thigh of a 26-year-old man was affected. Histologically, both tumors consisted of the long fascicular growth of long spindle cells. The tumor in case 1 additionally showed focal myxoid changes. Tumor cells had nonpleomorphic, atypical nuclei, and lacked evidence of a specific line of differentiation. Both tumors showed widespread CD34 immunoreactivity and very limited expression of actin. RNA sequencing detected in-frame fusion transcripts of STRN (exon 3)-NTRK3 (exon 14) in case 1 and STRN3 (exon 3)-NTRK3 (exon 14) in case 2, which were confirmed by reverse transcription polymerase chain reaction and Sanger sequencing. Pan-TRK immunostaining was diffusely positive in both cases. Fluorescence in situ hybridization showed signal patterns compatible with NTRK3 rearrangements in both cases, with case 2 additionally harboring a CDKN2A homozygous deletion. This study expands the clinicopathologic and genetic spectrum of sarcomas associated with NTRK fusions, and suggests that CD34-positive fibrosarcoma of bone and soft tissue could be a good candidate for NTRK testing.

Granular cell tumors (GCTs) are rare mesenchymal tumors that exhibit a characteristic morphology and a finely granular cytoplasm. The genetic alterations responsible for GCT tumorigenesis had been unknown until recently, when loss-of-function mutations of ATP6AP1 and ATP6AP2 were described. Thus, we performed whole-exome sequencing, RNA sequencing, and targeted sequencing of 51 GCT samples. From these genomic analyses, we identified mutations in genes encoding vacuolar H+ -ATPase (V-ATPase) components, including ATP6AP1 and ATP6AP2, in 33 (65%) GCTs. ATP6AP1 and ATP6AP2 mutations were found in 23 (45%) and 2 (4%) samples, respectively, and all were truncating or splice site mutations. In addition, seven other genes encoding V-ATPase components were also mutated, and three mutations in ATP6V0C occurred on the same amino acid (isoleucine 136). These V-ATPase component gene mutations were mutually exclusive, with one exception. These results suggest that V-ATPase function is impaired in GCTs not only by loss-of-function mutations of ATP6AP1 and ATP6AP2 but also through mutations of other subunits. Our findings provide additional support for the hypothesis that V-ATPase dysfunction promotes GCT tumorigenesis.

The genomic characteristics of dedifferentiated liposarcoma (DDLPS) that are associated with clinical features remain to be identified. Here, we conduct integrated whole exome and RNA sequencing analysis in 115 DDLPS tumors and perform comparative genomic analysis of well-differentiated and dedifferentiated components from eight DDLPS samples. Several somatic copy-number alterations (SCNAs), including the gain of 12q15, are identified as frequent genomic alterations. CTDSP1/2-DNM3OS fusion genes are identified in a subset of DDLPS tumors. Based on the association of SCNAs with clinical features, the DDLPS tumors are clustered into three groups. This clustering can predict the clinical outcome independently. The comparative analysis between well-differentiated and dedifferentiated components identify two categories of genomic alterations: shared alterations, associated with tumorigenesis, and dedifferentiated-specific alterations, associated with malignant transformation. This large-scale genomic analysis reveals the mechanisms underlying the development and progression of DDLPS and provides insights that could contribute to the refinement of DDLPS management., Understanding the genomic features of dedifferentiated liposarcoma (DDLPS) is likely to uncover new options for management. Here, the authors reveal three prognostic groups, and highlight molecular markers associated with malignant transformation.

// Naofumi Asano 1, 2 , Akihiko Yoshida 3 , Sachiyo Mitani 4 , Eisuke Kobayashi 5 , Bunsyo Shiotani 6 , Motokiyo Komiyama 7 , Hiroyuki Fujimoto 7 , Hirokazu Chuman 5 , Hideo Morioka 2 , Morio Matsumoto 2 , Masaya Nakamura 2 , Takashi Kubo 8 , Mamoru Kato 9 , Takashi Kohno 8, 10 , Akira Kawai 5 , Tadashi Kondo 1 , Hitoshi Ichikawa 4, 8 1 Division of Rare Cancer Research, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan 2 Department of Orthopaedic Surgery, Keio University School of Medicine, Shinjuku-ku, Tokyo 160-8582, Japan 3 Department of Pathology and Clinical Laboratory, National Cancer Center Hospital, Chuo-ku, Tokyo 104-0045, Japan 4 Department of Clinical Genomics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan 5 Department of Musculoskeletal Oncology, National Cancer Center Hospital, Chuo-ku, Tokyo 104-0045, Japan 6 Division of Genetics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan 7 Department of Urology, National Cancer Center Hospital, Chuo-ku, Tokyo 104-0045, Japan 8 Division of Translational Genomics, National Cancer Center-Exploratory Oncology Research and Clinical Trial Center, Chuo-ku, Tokyo 104-0045, Japan 9 Department of Bioinformatics, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan 10 Division of Genome Biology, National Cancer Center Research Institute, Chuo-ku, Tokyo 104-0045, Japan Correspondence to: Hitoshi Ichikawa, email: hichikaw@ncc.go.jp Keywords: liposarcoma, well-differentiated liposarcoma, dedifferentiated liposarcoma, next-generation sequencing, receptor tyrosine kinase Received: September 26, 2016 Accepted: January 08, 2017 Published: January 14, 2017 ABSTRACT Well-differentiated liposarcoma (WDLPS) and dedifferentiated liposarcoma (DDLPS) are closely related tumors commonly characterized by MDM2/CDK4 gene amplification, and lack clinically effective treatment options when inoperable. To identify novel therapeutic targets, we performed targeted genomic sequencing analysis of 19 WDLPS and 37 DDLPS tumor samples using a panel of 104 cancer-related genes (NCC oncopanel v3) developed specifically for genomic testing to select suitable molecular targeted therapies. The results of this analysis indicated that these sarcomas had very few gene mutations and a high frequency of amplifications of not only MDM2 and CDK4 but also other genes. Potential driver mutations were found in only six (11%) samples; however, gene amplification events (other than MDM2 and CDK4 amplification) were identified in 30 (54%) samples. Receptor tyrosine kinase (RTK) genes in particular were amplified in 18 (32%) samples. In addition, growth of a WDLPS cell line with IGF1R amplification was suppressed by simultaneous inhibition of CDK4 and IGF1R, using palbociclib and NVP-AEW541, respectively. Combination therapy with CDK4 and RTK inhibitors may be an effective therapeutic option for WDLPS/DDLPS patients with RTK gene amplification.

9044 Background: Programmed death ligand-1 (PD-L1) expression is not a completely reliable predictive marker of the efficacy of anti-programmed cell death protein-1 (PD-1)/anti-PD-L1 therapy in advanced non-small cell lung cancer patients (NSCLC). Immune-related tumor microenvironment (TME) is classified into four different types based on the status of tumor-infiltrating lymphocytes (TILs) and PD-L1 expression. Methods: We retrospectively reviewed advanced NSCLC patients treated with anti-PD-1/anti-PD-L1 therapy between 2015 and 2019, and investigated the association between the efficacy of anti-PD-1/anti-PD-L1 therapy, the types of TME based on PD-L1 (clone: 22C3) expression, and the density of CD8-positive TILs by immunohistochemistry (/mm2), and mutational profiles assessed by next-generation sequencing. Results: Overall, 228 patients without driver mutation ( EGFR, ALK, ROS1, and RET) were included in the analysis. The patients were classified into four following groups: Type I: PD-L1High (tumor proportion score [TPS]≥50%)/TILHigh (≥85/mm2; n = 73), Type II: PD-L1Low (TPS < 50%)/TILLow ( < 85/mm2; n = 70), Type III: PD-L1High/TILLow (n = 37), and Type IV: PD-L1Low/TILHigh (n = 48). The progression-free survival (PFS) and objective response rate (ORR) of anti-PD-1/anti-PD-L1 therapy clearly differed according to the different tumor microenvironment (TME) types (ORR and median PFS; Type I: 64%, 14.5 months, Type II: 12%, 2.1 months, Type III: 24%, 3.6 months, Type IV: 41%, 10.8 months). In patients with PD-L1High tumors, Type I tumors had significantly better ORR and PFS than Type III(ORR: p < 0.001, and PFS: p < 0.001) tumors. Regarding the association between mutational profiles, histology and the TME types, the presence of TP53 mutation and KRAS mutation significantly related to TILHigh (Type I and IV) and PD-L1High tumors (Type I and III), respectively. Pleomorphic and NSCLC- not otherwise specified histology were associated with Type I tumors, while LCNEC was associated with PD-L1 low tumors (Type II and IV). Conclusions: Various factors (mutational profile and histology) are related to TME classification based on the status of TILs and PD-L1 expression. Differential types of TME, including PD-L1 expression and TILs status, can accurately predict the efficacy of anti-PD-1/anti-PD-L1 therapy.[Table: see text]

Granular cell tumors (GCTs) are rare mesenchymal tumors that exhibit a characteristic morphology and a finely granular cytoplasm. The genetic alterations responsible for GCT tumorigenesis had been unknown until recently, when loss-of-function mutations of ATP6AP1 and ATP6AP2 were described. Thus, we performed whole-exome sequencing, RNA sequencing, and targeted sequencing of 51 GCT samples. From these genomic analyses, we identified mutations in genes encoding vacuolar H

EVI1 and MEL1 are homolog genes whose transcriptional activations by chromosomal translocations are known in small subsets of leukemia. From gene expression profiling data of 130 Japanese pediatric acute myeloid leukemia (AML) patients, we found that EVI1 and MEL1 were overexpressed in ~30% of patients without obvious translocations of these gene loci, and that their high expression was significantly associated with inferior survival. High EVI1 expression was detected mainly in myelomonocytic-lineage (designated as e-M4/M5 subtype) leukemia with MLL rearrangements and in megakaryocytic-lineage (designated as e-M7 subtype) leukemia, and its prognostic association was observed in the e-M4/M5 subtype but not in the e-M7 subtype. On the other hand, high MEL1 expression was detected in myelocytic-lineage (designated as e-M0/M1/M2 subtype) and e-M4/M5 subtype leukemia without MLL rearrangements, and its prognostic association was independent from the subtypes. Because of their subtype-dependent and mutually exclusive expression, a combined evaluation of their high expression enabled a clear distinction of patients with inferior survival (P

Thus far, only 23 cases of the ectopic production of parathyroid hormone (PTH) have been reported. We have characterized the genome-wide transcription profile of an ectopic PTH-producing tumor originating from a retroperitoneal histiocytoma. We found that the calcium-sensing receptor (CaSR) was barely expressed in the tumor. Lack of CaSR, a crucial braking apparatus in the presence of both intraparathyroid and, probably, serendipitous PTH expression, might contribute strongly to the establishment and maintenance of the ectopic transcriptional activation of the PTH gene in nonparathyroid cells. Along with candidate drivers with a crucial frameshift mutation or copy number variation at specific chromosomal areas obtained from whole exome sequencing, we identified robust tumor-specific cytochrome P450 family 24 subfamily A member 1 (CYP24A1) overproduction, which was not observed in other non–PTH-expressing retroperitoneal histiocytoma and parathyroid adenoma samples. We then found a 2.5-kb noncoding RNA in the PTH 3′-downstream region that was exclusively present in the parathyroid adenoma and our tumor. Such a co-occurrence might act as another driver of ectopic PTH-producing tumorigenesis; both might release the control of PTH gene expression by shutting down the other branches of the safety system (e.g., CaSR and the vitamin D3–vitamin D receptor axis)., We examined a genome-wide transcription profile of an ectopic PTH-producing tumor. Scarcity of CaSR, CYP24A1 overproduction, and an lncRNA near the PTH gene are candidate culprits of PTH ectopy.

Acute myeloid leukaemia, French-American-British M4 and M5 subtypes (AML-M4/M5) is frequently associated with MLL gene rearrangement and its incidence is relatively high among infants. Clinically, paediatric AML-M4/M5 has been considered as an intermediate or undefined prognostic group. In this study, we analysed gene expression of 40 paediatric AML-M4/M5 patients excluding inv(16) and t(8;21) patients, and found striking differences among the patients in an age-associated manner. In particular, most of the infants displayed very distinct gene expression. On the basis of this difference, we divided paediatric patients into three subgroups (A, B and C) with the average age of 0.3, 3.1 and 6.6 years old respectively. All subgroups included patients with MLL gene rearrangement as well as normal and other karyotypes. Surprisingly, gene expression signatures of MLL gene rearrangement differed substantially among these subgroups. In addition, subgroup C presented extremely poor outcome (3-year event-free survival 28%) whilst eight patients with MLL gene rearrangement in subgroup C had all relapsed within 18 months. These results suggest that age is an important factor contributing to the biology of AML-M4/M5 and the sub-grouping procedures developed in this study could be a powerful tool to identify unfavourable risk patients within paediatric AML-M4/M5.

AML1-MTG8 fusion protein, which is produced from the rearranged gene formed between AML1 and MTG8 in myeloid leukemia with t(8;21) chromosomal translocation, plays an important role in the pathogenesis of leukemia. We previously showed that ectopically expressed AML1-MTG8 fusion protein is associated with an MTG8-like protein in the mouse myeloid precursor cell line L-G, and this association seemed to be required for AML1-MTG8 to stimulate proliferation. As a candidate cDNA for this MTG8-like protein, a 6.4 kb MTGR1 cDNA encoding human MTGR1b protein of 604 amino acids was isolated. Since this cDNA was shorter than the main mRNA (about 7.5 kb), the 5'-end of the MTGR1 cDNA was extended using Marathon Ready cDNA. When the newly obtained 5'-sequence was combined with the previous cDNA, the resultant MTGR1 cDNA (6995 bp), including exon 3 that the previous cDNA lacked, could encode MTGR1a protein of 575 amino acids. Transcripts of the MTGR1 gene were expressed ubiquitously in the human tissues and cell lines examined. PCR analyses of the cDNAs from human tissues showed the presence of various splicing variants with regard to the 5'-region including exons 1, 2 and 3. The MTGR1 gene consists of 14 exons and spans about 68 kb. The genomic structure of MTGR1 is highly similar to those of other MTG 8-family genes, MTG8 and MTG16. MTG16 was recently cloned from the translocation breakpoint of myeloid malignancies with t(16;21) chromosomal translocation.

We previously isolated RBP56 cDNA by PCR using mixed primers designed from the conserved sequences of the RNA binding domain of FUS/TLS and EWS proteins. RBP56 protein turned out to be hTAFII68 which was isolated as a TATA-binding protein associated factor (TAF) from a sub-population of TFIID complexes (Bertolotti A., Lutz, Y., Heard, D.J., Chambon, P., Tora, L., 1996. hTAFII68, a novel RNA/ssDNA-binding protein with homology to the proto-oncoproteins TLS/FUS and EWS is associated with both TFIID and RNA polymerase II. EMBO J. 15, 5022-5031). The RBP56/hTAFII68, FUS/TLS and EWS proteins comprise a sub-family of RNA binding proteins, which consist of an N-terminal Ser, Gly, Gln and Tyr-rich region, an RNA binding domain, a Cys2/Cys2 zinc finger motif and a C-terminal RGG-containing region. Rearrangement of the FUS/TLS gene and the EWS gene has been found in several types of malignant tumors, and the resultant fusion proteins play an important role in the pathogenesis of these tumors. In the present study, we determined the genomic structure of the RBP56/hTAFII68 gene. The RBP56/hTAFII68 gene spans about 37kb and consists of 16 exons from 33bp to 562bp. The longest exon, exon 15, encodes the C-terminal region containing 19 repeats of a degenerate DR(S)GG(G)YGG sequence. While the structure of the FUS/TLS gene has been reported previously, we determined the total DNA sequence of the FUS/TLS gene, consisting of 12kb. The RBP56/hTAFII68, FUS/TLS and EWS genes consist of similar numbers of exons. Comparison of the structures of these three genes showed that the organization of exons in the central part encoding a homologous RNA binding domain and a cysteine finger motif is highly conserved, and other exon boundaries are also located at similar sites, indicating that these three genes most likely originate from the same ancestor gene.

The cryptic t(5;11)(q35;p15.5) creates a fusion gene between the NUP98 and NSD1 genes. To ascertain the significance of this gene fusion, we explored its frequency, clinical impact, and gene expression pattern using DNA microarray in pediatric acute myeloid leukemia (AML) patients. NUP98-NSD1 fusion transcripts were detected in 6 (4.8%) of 124 pediatric AML patients. Supervised hierarchical clustering analyses using probe sets that were differentially expressed in these patients detected a characteristic gene expression pattern, including 18 NUP98-NSD1-negative patients (NUP98-NSD1-like patients). In total, a NUP98-NSD1-related gene expression signature (NUP98-NSD1 signature) was found in 19% (24/124) and in 58% (15/26) of cytogenetically normal cases. Their 4-year overall survival (OS) and event-free survival (EFS) were poor (33.3% in NUP98-NSD1-positive and 38.9% in NUP98-NSD1-like patients) compared with 100 NUP98-NSD1 signature-negative patients (4-year OS: 86.0%, 4-year EFS: 72.0%). Interestingly, t(7;11)(p15;p15)/NUP98-HOXA13, t(6;11)(q27;q23)/MLL-MLLT4 and t(6;9)(p22;q34)/DEK-NUP214, which are known as poor prognostic markers, were found in NUP98-NSD1-like patients. Furthermore, another type of NUP98-NSD1 fusion transcript was identified by additional RT-PCR analyses using other primers in a NUP98-NSD1-like patient, revealing the significance of this signature to detect NUP98-NSD1 gene fusions and to identify a new poor prognostic subgroup in AML.

Abstract 1391 Background Acute myeloid leukemia (AML) is a complex disease caused by mutations, epigenetic modifications, and deregulated expression of genes leading to increased proliferation and decreased differentiation of hematopoietic progenitor cells. Many important molecular markers have already been discovered in AML that have not only helped to better characterize patients, but also helped to improve risk stratification. However, in a subset of AML patients, no known prognosis-associated cytogenetic aberrations or mutations were detectable. In hematological malignancies, 11p15 translocations involving the nucleoporin 98-kDa (NUP98) protein gene are relatively rare, but more than 20 different chromosomal rearrangements have been identified. The cryptic t(5;11)(q35;p15.5), which is frequently accompanied by deletion of the long arm of chromosome 5, del(5q), creates a fusion gene between NUP98 and the nuclear receptor-binding SET-domain protein 1 (NSD1) gene. Recently, Hollink et al identified NUP98-NSD1 gene fusion by high-resolution genome-wide copy number analysis and reverse transcription (RT)-PCR in pediatric and adult AML patients, and reported that NUP98-NSD1 gene fusion was a predictor of poor prognosis. Thus, we explored the frequency, gene expression pattern, and clinical impact of NUP98-NSD1 in a large cohort of Japanese pediatric AML patients. Methods From January 2000 to December 2002, 318 patients were newly diagnosed with de novo AML. Of these, samples from 124 patients whose gene expression profiling data were available were analyzed, including 10 patients with FAB-M3 and 6 patients with Down syndrome (DS) who were treated on different treatment protocols. For the detection of NUP98-NSD1 gene fusion, direct sequencing was performed using an ABI PRISM 310 Genetic Analyzer (Applied Biosystems). Gene expression profiling data, which were performed before, were available for these 124 patients. Hierarchical clustering analysis was performed using Cluster and Tree View software. Result NUP98-NSD1 fusion transcript was detected in 6 (4.8%) of 124 pediatric AML patients and a characteristic gene expression pattern related to these patients was also detected. Further hierarchical clustering analysis using the 87 probe sets which were differentially expressed in 6 NUP98-NSD1-positive patients did not detect a simple cluster of 6 NUP98-NSD1-positive patients but a relatively large cluster including 18 NUP98-NSD1-negative patients. When compared with 100 NUP98-NSD1 signature-negative patients, the 24 NUP98-NSD1 signature-positive patients frequently had normal karyotype (62.5%), and del(9q) (8.3%), but did not have any favorable chromosomal translocations such as t(8;21), inv(16) and t(15;17). The frequencies of M4 and M5 subtypes (14/24, P < 0.001) were also higher than those in NUP98-NSD1 signature-negative patients. In addition, they frequently had FLT3-ITD (12/24, P < 0.001), MLL-PTD (7/24, P = 0.124), NPM1 (3/24, P = 0.007) and WT1 (5/24, P = 0.037) mutations. In total, NUP98-NSD1 related gene expression signature (NUP98-NSD1 signature) represented 19% (24/124) of total patients and 58% (15/26) of cytogenetically normal patients in our cohort. Their 4-year overall survival (OS) and event-free survival (EFS) were poor when compared to 100 NUP98-NSD1 signature-negative patients (37.5% vs. 86.0% and 37.5% vs. 72.0%). Conclusion Our results indicated that Patients with NUP98-NSD1 related gene expression signature had extremely poor clinical outcome, irrespective of the presence of NUP98-NSD1 gene fusion. Most of the patients displaying the NUP98-NSD1 signature have been classified into an intermediate risk group, but their unfavorable outcome suggests that a high risk group is a more suitable stratification. These results suggest the presence of a new poor prognostic subgroup which revealed the value of the NUP98-NSD1 signature as prognostic markers in pediatric AML. Although further investigation is necessary, we believe that our work contributes to improve the risk stratification in pediatric AML. Disclosures: No relevant conflicts of interest to declare.