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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.

// 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.

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.

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.