Genomic Landscape of Chinese Clear Cell Renal Cell Carcinoma Patients With Venous Tumor Thrombus Identifies Chromosome 9 and 14 Deletions and Related Immunosuppressive Microenvironment

BackgroundClear cell renal cell carcinoma (ccRCC) with venous tumor thrombus (VTT) is associated with a poor clinical outcome. Although several studies have examined the genomic features of ccRCC, the genetic profile of VTT along with its matched primary tumor has not been fully elucidated.Materials and methodsSamples of VTT tissues and matched primary tumor tissues from ccRCC patients (n = 25), as well as primary tumor tissues from patients without VTT (n = 25) were collected and analyzed using whole-exome sequencing. Four additional ccRCC patients who were unfit for surgery were treated with an anti-programmed death receptor-1 (PD-1) monoclonal antibody (Toripalimab, 240 mg, Q3W, IV).ResultsBy comparing the primary kidney tumors from ccRCC patients with or without VTT, a relatively higher prevalence of BAP1 and KDM5C alterations were found in ccRCC patients with VTT, and these alterations were associated with worse overall survival in the kidney renal clear cell carcinoma (KIRC) database. Based on subclone analysis, VTT was predicted to primarily originate directly from the primary renal mass. A significantly higher prevalence of CELSR2 and TET2 alterations were identified in the VTTs compared with the matched primary tumors. An increased prevalence of DNA damage repair genes, especially those involved in homologous recombination repair and non-homologous end joining, was found in ccRCC patients with VTT. Notably, VTT was characterized by the increase incidence of copy number loss in the whole exome (p < 0.05), particularly in the chromosome 9 and 14 regions. Deletion of chromosome 9 and 14 was associated with worse survival, unfavorable clinical features, and the presence of an immunosuppressive microenvironment, which was characterized by higher infiltration of regulatory T cells, follicular helper T cells, and resting mast cells, but lower counts of resting CD4 memory T cells and CD8 positive T cells. A significantly lower count of CD4+ and CD8+ tumor-infiltrated lymphocytes was identified in the VTT samples comparing with matched primary tumor. Of note, three out of the four ccRCC patients with VTT in our cohort who were treated with the anti-PD-1 therapy exhibited remarkable remission in the renal mass but no notable shrinkage in the VTT mass.ConclusionOur study revealed the genetic profile of Chinese ccRCC patients with VTT, and identified multiple features associated with known poor outcomes, including gene alterations and copy number loss. The deletions in chromosomes 9 and 14, and the associated immunosuppressive microenvironment may indicate limited sensitivity to anti-PD-1/PD-L1 monotherapy in VTT.

MBRS-20. CSF-DERIVED CIRCULATING TUMOR DNA AS A BIOMARKER FOR DISEASE PROGRESSION AND TUMOR EVOLUTION IN MEDULLOBLASTOMA

BACKGROUND Cell-free DNA (cfDNA) profiling has been shown to carry utility as a clinically relevant biomarker in a variety of cancers, but studies in pediatric brain tumors, including medulloblastoma, are scarce. We hereby evaluated the actionability of profiling cfDNA from cerebrospinal fluid (CSF) based on a multi-institutional cohort of children with medulloblastoma. METHODS 103 children aged ≥ 3 years with medulloblastoma harboring chromosomal aneuploidy enrolled on two prospective therapeutic trials were included. cfDNA was extracted from CSF obtained longitudinally, and profiled by low-coverage whole-genome sequencing (lcWGS) for annotating copy-number variants (CNVs). cfDNA-derived CNVs were compared against patient-matched primary tumor-derived CNVs and correlated with outcome. cfDNA profiles at diagnosis and relapse were compared to evaluate tumor evolution. RESULTS Tumor-derived somatic CNVs were detected in 72% of baseline cfDNA samples, with higher detection rate in samples from patients with metastatic disease than those without (90% versus 50%, chi-square p=0.001). Longitudinal profiling of cfDNA revealed correlation between CNV detectability and clinical course, with detection of tumor-derived CNVs in cfDNA samples predating radiographic progression for ≥ 3 months in 62% of relapsing patients. Presence of cfDNA-derived CNVs in CSF collected during chemotherapy and at the end of therapy was significantly associated with inferior PFS (log-rank p<0.0001 for both time-points). Comparison of CNV profiles from cfDNA at baseline and relapse revealed molecular divergence in a subset of patients. CONCLUSION These results carry major implications and supports the incorporation of cfDNA profiling in upcoming medulloblastoma protocols for more sensitive and accurate disease monitoring and personalization of treatment.

Integrative molecular analysis of metastatic hepatocellular carcinoma

Background Hepatocellular carcinoma (HCC) is the major type of primary liver cancer. Intrahepatic metastasis, such as portal vein tumor thrombosis (PVTT), strongly indicates poor prognosis of HCC. But now, there are limited understandings of the molecular features and mechanisms of those metastatic HCCs. Methods To characterize the molecular alterations of the metastatic HCCs, we implemented an integrative analysis of the copy number variations (CNVs), DNA methylations and transcriptomes of matched adjacent normal, primary tumor and PVTT samples from 19 HCC patients. Results CNV analysis identified a frequently amplified focal region chr11q13.3 and a novel deletion peak chr19q13.41 containing three miRNAs. The integrative analysis with RNA-seq data suggests that CNVs and differential promoter methylations regulate distinct oncogenic processes. Then, we used individualized differential analysis to identify the differentially expressed genes between matched primary tumor and PVTT of each patient. Results show that 5 out of 19 studied patients acquire evidential progressive alterations of gene expressions (more than 1000 differentially expressed genes were identified in each patient). While, another subset of eight patients have nearly identical gene expressions between the corresponding matched primary tumor and PVTT. Twenty genes were found to be recurrently and progressively differentially expressed in multiple patients. These genes are mainly associated with focal adhesion, xenobiotics metabolism by cytochrome P450 and amino acid metabolism. For several differentially expressed genes in metabolic pathways, their expressions are significantly associated with overall survivals and vascular invasions of HCC patients. The following transwell assay experiments validate that they can regulate invasive phenotypes of HCC cells. Conclusions The metastatic HCCs with PVTTs have significant molecular alterations comparing with adjacent normal tissues. The recurrent alteration patterns are similar to several previously published general HCC cohorts, but usually with higher severity. By an individualized differential analysis strategy, the progressively differentially expressed genes between the primary tumor and PVTT were identified for each patient. A few patients aquire evidential progressive alterations of gene expressions. And, experiments show that several recurrently differentially expressed genes can strongly regulate HCC cell invasions.

Ultra-deep amplicon sequencing to identify actionable mutations in matched plasma/tumor specimens from 44 patients with colorectal cancer of UICC stage III and IV.

3634 Background: Circulating cell-free DNA (cf-DNA) isolated from plasma samples of cancer patients (pts) is a promising source for noninvasive examination of tumor-specific mutation patterns. We examined the efficiency of ultra-deep amplicon sequencing (UDAS) of cf-DNA isolated from plasma and tumor DNA isolated from matched primary tumor tissue of pts with colorectal cancer (CRC). Methods: Blood was drawn prior to surgery from 44 pts: 20 male, 24 female; 44-79 years of age (median: 67.5); 11 stage III, 33 stage IV; 36 colon, 8 rectum tumors; no neo-adjuvant therapy. Cf-DNA was isolated from 2 ml of EDTA plasma. UDAS was applied to 72 DNA samples (44 plasma, 28 matched snap-frozen primary tumors) using the MiSeq platform (Illumina). A panel of 49 highly multiplexed amplicons was designed (Life Technologies) representing 9 cancer genes frequently mutated in CRC. For 16 primary tumors the mutation profile was determined using the TruSeq Amplicon Cancer Panel (Illumina). Results: Median cf-DNA yield was 310 ng / 2 ml plasma (1ng – 6.600 ng). There was no significant relation between cf-DNA yield and any clinical characteristic. Median amplicon coverage was 20.162 reads per bp (2.913 - 115.782). 33/49 amplicons (67%) had a coverage of > 10.000 reads. Altogether 61 high quality COSMIC-cited mutations were confirmed in plasma of 29/44 (66%) pts: 24/33 (73%) pts in stage IV, 5/11 (45%) pts in stage III. Confirmed mutations are: APC-17; BRAF-2, FBXW7-1, KRAS-15, NRAS-1, PIK3CA-4, SMAD4-2, and TP53-19 mutations. No high quality mutations were found in CTNNB1 and HRAS. Moreover two pts exhibited four high quality plasma mutations which were not detected in the matched primary tumor: APC (R216X), KRAS (Q61K), and SMAD4 (D355E); PIK3CA (E545K). Conclusions: Ultra deep amplicon sequencing is suitable to detect mutations in plasma samples of CRC pts with a high concordance to matched primary tumors. The concordance rate can be further increased by extending the spectrum of analyzed mutations or by the enrichment of cf-DNA tumor copies. This method could be applied to detect and monitor metastasis thus opening a new paradigm for the selection of pts for targeted therapies.