Creation and Validation of Patient-Derived Cancer Model Using Peritoneal and Pleural Effusion in Patients with Advanced Ovarian Cancer: An Early Experience.

Background: The application of personalized cancer treatment based on genetic information and surgical samples has begun in the field of cancer medicine. However, a biopsy may be painful for patients with advanced diseases that do not qualify for surgical resection. Patient-derived xenografts (PDXs) are cancer models in which patient samples are transplanted into immunodeficient mice. PDXs are expected to be useful for personalized medicine. The aim of this study was to establish a PDX from body fluid (PDX-BF), such as peritoneal and pleural effusion samples, to provide personalized medicine without surgery. Methods: PDXs-BF were created from patients with ovarian cancer who had positive cytology findings based on peritoneal and pleural effusion samples. PDXs were also prepared from each primary tumor. The pathological findings based on immunohistochemistry were compared between the primary tumor, PDX, and PDX-BF. Further, genomic profiles and gene expression were evaluated using DNA and RNA sequencing to compare primary tumors, PDXs, and PDX-BF. Results: Among the 15 patients, PDX-BF was established for 8 patients (5 high-grade serous carcinoma, 1 carcinosarcoma, 1 low-grade serous carcinoma, and 1 clear cell carcinoma); the success rate was 53%. Histologically, PDXs-BF have features similar to those of primary tumors and PDXs. In particular, PDXs-BF had similar gene mutations and expression patterns to primary tumors and PDXs. Conclusions: PDX-BF reproduced primary tumors in terms of pathological features and genomic profiles, including gene mutation and expression. Thus, PDX-BF may be a potential alternative to surgical resection for patients with advanced disease.

Consistency between Primary Uterine Corpus Malignancies and Their Corresponding Patient-Derived Xenograft Models.

Patient-derived xenograft (PDX) models retain the characteristics of tumors and are useful tools for personalized therapy and translational research. In this study, we aimed to establish PDX models for uterine corpus malignancies (UC-PDX) and analyze their similarities. Tissue fragments obtained from 92 patients with uterine corpus malignancies were transplanted subcutaneously into immunodeficient mice. Histological and immunohistochemical analyses were performed to compare tumors of patients with PDX tumors. DNA and RNA sequencing were performed to validate the genetic profile. Furthermore, the RNA in extracellular vesicles (EVs) extracted from primary and PDX tumors was analyzed. Among the 92 cases, 52 UC-PDX models were established, with a success rate of 56.5%. The success rate depended on tumor histology and staging. The pathological and immunohistochemical features of primary and PDX tumors were similar. DNA sequencing revealed similarities in gene mutations between the primary and PDX tumors. RNA sequencing showed similarities in gene expressions between primary and PDX tumors. Furthermore, the RNA profiles of the EVs obtained from primary and PDX tumors were similar. As UC-PDX retained the pathological and immunohistochemical features and gene profiles of primary tumors, they may provide a platform for developing personalized medicine and translational research.

The Impact of FGFR3 Alterations on the Tumor Microenvironment and the Efficacy of Immune Checkpoint Inhibitors in Bladder Cancer.

BACKGROUND: Currently, only limited knowledge is available regarding the phenotypic association between fibroblast growth factor receptor 3 (FGFR3) alterations and the tumor microenvironment (TME) in bladder cancer (BLCA). METHODS: A multi-omics analysis on 389 BLCA and 35 adjacent normal tissues from a cohort of OMPU-NCC Consortium Japan was retrospectively performed by integrating the whole-exome and RNA-sequence dataset and clinicopathological record. A median follow-up duration of all BLCA cohort was 31 months. RESULTS: FGFR3 alterations (aFGFR3), including recurrent mutations and fusions, accounted for 44% of non-muscle invasive bladder cancer (NMIBC) and 15% of muscle-invasive bladder cancer (MIBC). Within MIBC, the consensus subtypes LumP was significantly more prevalent in aFGFR3, whereas the Ba/Sq subtype exhibited similarity between intact FGFR3 (iFGFR3) and aFGFR3 cases. We revealed that basal markers were significantly increased in MIBC/aFGFR3 compared to MIBC/iFGFR3. Transcriptome analysis highlighted TIM3 as the most upregulated immune-related gene in iFGFR3, with differential immune cell compositions observed between iFGFR3 and aFGFR3. Using EcoTyper, TME heterogeneity was discerned even within aFGFR cases, suggesting potential variations in the response to checkpoint inhibitors (CPIs). Among 72 patients treated with CPIs, the objective response rate (ORR) was comparable between iFGFR3 and aFGFR3 (20% vs 31%; p = 0.467). Strikingly, a significantly higher ORR was noted in LumP/aFGFR3 compared to LumP/iFGFR3 (50% vs 5%; p = 0.022). This trend was validated using data from the IMvigor210 trial. Additionally, several immune-related genes, including IDO1, CCL24, IL1RL1, LGALS4, and NCAM (CD56) were upregulated in LumP/iFGFR3 compared to LumP/aFGFR3 cases. CONCLUSIONS: Differential pathways influenced by aFGFR3 were observed between NMIBC and MIBC, highlighting the upregulation of both luminal and basal markers in MIBC/aFGFR3. Heterogeneous TME was identified within MIBC/aFGFR3, leading to differential outcomes for CPIs. Specifically, a favorable ORR in LumP/aFGFR3 and a poor ORR in LumP/iFGFR3 were observed. We propose TIM3 as a potential target for iFGFR3 (ORR: 20%) and several immune checkpoint genes, including IDO1 and CCL24, for LumP/iFGFR3 (ORR: 5%), indicating promising avenues for precision immunotherapy for BLCA.

Tertiary lymphoid structure and neutrophil-lymphocyte ratio coordinately predict outcome of pembrolizumab.

Emerging evidence suggests that the presence of tertiary lymphoid structures (TLS) and neutrophil-lymphocyte ratio (NLR) in peripheral blood is associated with the treatment response to checkpoint inhibitors (CPIs), whereas there is limited knowledge regarding whether these factors reciprocally impact the treatment outcomes of CPIs in metastatic urothelial carcinoma (mUC). Herein, we investigated treatment outcomes of platinum-refractory mUC patients (50 cases with whole-exome and transcriptome sequencing) treated with pembrolizumab. The pathological review identified 24% of cases of TLS in the specimens. There was no significant difference in the NLR between the TLS- and TLS+ groups (p = 0.153). In the lower NLR group, both overall survival and progression-free survival were significantly longer in patients with TLS than in those without TLS, whereas the favorable outcomes associated with TLS were not observed in patients in the higher NLR group. We explored transcriptomic differences in UC with TLS. The TLS was comparably observed between luminal (20%) and basal (25%) tumor subtypes (p = 0.736). Exploring putative immune-checkpoint genes revealed that ICOSLG (B7-H2) was significantly increased in tumors with lower NLR. KRT expression levels exhibited higher basal cell markers (KRT5 and KRT17) in the higher NLR group and lower differentiated cell markers (KRT8 and KRT18) in patients with TLS. In conclusion, the improved outcomes of pembrolizumab treatment in mUC are restricted to patients with lower NLR. Our findings begin to elucidate a distinct molecular pattern for the presence of TLS according to the NLR in peripheral blood.

NeoRdRp: A Comprehensive Dataset for Identifying RNA-dependent RNA Polymerases of Various RNA Viruses from Metatranscriptomic Data.

RNA viruses are distributed throughout various environments, and most have recently been identified by metatranscriptome sequencing. However, due to the high nucleotide diversity of RNA viruses, it is still challenging to identify novel RNA viruses from metatranscriptome data. To overcome this issue, we created a dataset of RNA-dependent RNA polymerase (RdRp) domains that are essential for all RNA viruses belonging to Orthornavirae. Genes with RdRp domains from various RNA viruses were clustered based on amino acid sequence similarities. A multiple sequence alignment was generated for each cluster, and a hidden Markov model (HMM) profile was created when the number of sequences was greater than three. We further refined 426 HMM profiles by detecting RefSeq RNA virus sequences and subsequently combined the hit sequences with the RdRp domains. As a result, 1,182 HMM profiles were generated from 12,502 RdRp domain sequences, and the dataset was named NeoRdRp. The majority of NeoRdRp HMM profiles successfully detected RdRp domains, specifically in the UniProt dataset. Furthermore, we compared the NeoRdRp dataset with two previously reported methods for RNA virus detection using metatranscriptome sequencing data. Our methods successfully identified the majority of RNA viruses in the datasets; however, some RNA viruses were not detected, similar to the other two methods. NeoRdRp may be repeatedly improved by the addition of new RdRp sequences and is applicable as a system for detecting various RNA viruses from diverse metatranscriptome data.

Validation of a Patient-Derived Xenograft Model for Cervical Cancer Based on Genomic and Phenotypic Characterization.

Patient-derived xenograft (PDX) models are useful tools for preclinical drug evaluation, biomarker identification, and personalized medicine strategies, and can be developed by the heterotopic or orthotopic grafting of surgically resected tumors into immunodeficient mice. We report the PDX models of cervical cancer and demonstrate the similarities among original and different generations of PDX tumors. Fresh tumor tissues collected from 22 patients with primary cervical cancer were engrafted subcutaneously into NOD.CB17-PrkdcSCID/J mice. Histological and immunohistochemical analyses were performed to compare primary and different generations of PDX tumors. DNA and RNA sequencing were performed to verify the similarity between the genetic profiles of primary and PDX tumors. Total RNA in extracellular vesicles (EVs) released from primary and PDX tumors was also quantified to evaluate gene expression. The total tumor engraftment rate was 50%. Histologically, no major differences were observed between the original and PDX tumors. Most of the gene mutations and expression patterns related to carcinogenesis and infiltration were similar between the primary tumor and xenograft. Most genes associated with carcinogenesis and infiltration showed similar expression levels in the primary tumor and xenograft EVs. Therefore, compared with primary tumors, PDX models could be potentially more useful for translational research.

Efficacy of pembrolizumab and comprehensive CD274/PD-L1 profiles in patients previously treated with chemoradiation therapy as radical treatment in bladder cancer.

BACKGROUND: Chemoradiation therapy (CRT) has been increasingly reported as a possible alternative to total cystectomy (TC) for localized bladder cancer (BC). Pembrolizumab is the standard of care for platinum-refractory metastatic urothelial carcinoma, although it is unknown whether the efficacy of pembrolizumab in patients previously treated with curative CRT varies from the results of benchmark trials. METHODS: We retrospectively assessed whether the survival benefit of pembrolizumab differs between patients previously treated with TC or CRT as radical treatment. A total of 212 patient records were collected for a logistic regression propensity score model. An independent dataset with next-generation sequencing (n=289) and PD-L1 Combined Positive Score (CPS: n=266) was analyzed to assess whether CRT-recurrent tumor harbors distinct CD274/PD-L1 profiles. RESULTS: Propensity score matching was performed using putative clinical factors, from which 30 patients in each arm were identified as pair-matched groups. There was no significant difference in overall survival from the initiation of pembrolizumab (p=0.80) and objective response rate (p=0.59) between CRT and TC treatment groups. In the independent 289 BC cohort, 22 samples (7.6%) were collected as CRT-recurrent tumors. There was no significant difference in CD274 mRNA expression level between CRT-naïve and CRT-recurrent tumors. The compositions of CD274 isoforms were comparable among all isoforms detected from RNAseq between CRT-naïve (n=267) and CRT-recurrent (n=22) tumors. No actionable exonic mutation in CD274 was detected in CRT-recurrent tumors. PD-L1 CPS was positively correlated with CD274 mRNA expression level, and PD-L1 CPS was comparable between CRT-naïve and CRT-recurrent tumors. CONCLUSIONS: The efficacy of pembrolizumab for patients previously treated with CRT was similar to those treated with TC. The enhanced tumor regression by combining programmed cell death protein 1/PD-L1 inhibitor and CRT might be expected only in the concurrent administration.