The evolution of metastatic upper tract urothelial carcinoma through genomic-transcriptomic and single-cell protein markers analysis.

The molecular characteristics of metastatic upper tract urothelial carcinoma (UTUC) are not well understood, and there is a lack of knowledge regarding the genomic and transcriptomic differences between primary and metastatic UTUC. To address these gaps, we integrate whole-exome sequencing, RNA sequencing, and Imaging Mass Cytometry using lanthanide metal-conjugated antibodies of 44 tumor samples from 28 patients with high-grade primary and metastatic UTUC. We perform a spatially-resolved single-cell analysis of cancer, immune, and stromal cells to understand the evolution of primary to metastatic UTUC. We discover that actionable genomic alterations are frequently discordant between primary and metastatic UTUC tumors in the same patient. In contrast, molecular subtype membership and immune depletion signature are stable across primary and matched metastatic UTUC. Molecular and immune subtypes are consistent between bulk RNA-sequencing and mass cytometry of protein markers from 340,798 single cells. Molecular subtypes at the single-cell level are highly conserved between primary and metastatic UTUC tumors within the same patient.

Immunogenomic Landscape of Neuroendocrine Prostate Cancer.

PURPOSE: Patients with neuroendocrine prostate cancer (NEPC) are often managed with immunotherapy regimens extrapolated from small-cell lung cancer (SCLC). We sought to evaluate the tumor immune landscape of NEPC compared with other prostate cancer types and SCLC. EXPERIMENTAL DESIGN: In this retrospective study, a cohort of 170 patients with 230 RNA-sequencing and 104 matched whole-exome sequencing data were analyzed. Differences in immune and stromal constituents, frequency of genomic alterations, and associations with outcomes were evaluated. RESULTS: In our cohort, 36% of the prostate tumors were identified as CD8+ T-cell inflamed, whereas the remaining 64% were T-cell depleted. T-cell-inflamed tumors were enriched in anti-inflammatory M2 macrophages and exhausted T cells and associated with shorter overall survival relative to T-cell-depleted tumors (HR, 2.62; P < 0.05). Among all prostate cancer types in the cohort, NEPC was identified to be the most immune depleted, wherein only 9 out of the 36 total NEPC tumors were classified as T-cell inflamed. These inflamed NEPC cases were enriched in IFN gamma signaling and PD-1 signaling compared with other NEPC tumors. Comparison of NEPC with SCLC revealed that NEPC had poor immune content and less mutations compared with SCLC, but expression of checkpoint genes PD-L1 and CTLA-4 was comparable between NEPC and SCLC. CONCLUSIONS: NEPC is characterized by a relatively immune-depleted tumor immune microenvironment compared with other primary and metastatic prostate adenocarcinoma except in a minority of cases. These findings may inform development of immunotherapy strategies for patients with advanced prostate cancer.

Molecular Evolution of Classic Hodgkin Lymphoma Revealed Through Whole-Genome Sequencing of Hodgkin and Reed Sternberg Cells.

The rarity of malignant Hodgkin and Reed Sternberg (HRS) cells in classic Hodgkin lymphoma (cHL) limits the ability to study the genomics of cHL. To circumvent this, our group has previously optimized fluorescence-activated cell sorting to purify HRS cells. Using this approach, we now report the whole-genome sequencing landscape of HRS cells and reconstruct the chronology and likely etiology of pathogenic events leading to cHL. We identified alterations in driver genes not previously described in cHL, APOBEC mutational activity, and the presence of complex structural variants including chromothripsis. We found that high ploidy in cHL is often acquired through multiple, independent chromosomal gains events including whole-genome duplication. Evolutionary timing analyses revealed that structural variants enriched for RAG motifs, driver mutations in B2M, BCL7A, GNA13, and PTPN1, and the onset of AID-driven mutagenesis usually preceded large chromosomal gains. This study provides a temporal reconstruction of cHL pathogenesis. SIGNIFICANCE: Previous studies in cHL were limited to coding sequences and therefore not able to comprehensively decipher the tumor complexity. Here, leveraging cHL whole-genome characterization, we identify driver events and reconstruct the tumor evolution, finding that structural variants, driver mutations, and AID mutagenesis precede chromosomal gains. This article is highlighted in the In This Issue feature, p. 171.

Molecular Evaluation of Low-grade Low-stage Endometrial Cancer With and Without Recurrence.

Low-grade, low-stage endometrioid carcinomas (LGLS EC) demonstrate 5-yr survival rates up to 95%. However, a small subset of these tumors recur, and little is known about prognostic markers or established mutation profiles associated with recurrence. The goal of the current study was to identify the molecular profiles of the primary carcinomas and the genomic differences between primary tumors and subsequent recurrences. Four cases of LGLS EC with recurrence and 8 cases without recurrence were evaluated via whole-exome sequencing. Three of the 4 recurrent tumors were evaluated via Oncomine Comprehensive Assay. The resulting molecular profiles of the primary and recurrent tumors were compared. Two of the 3 recurrent cases showed additional mutations in the recurrence. One recurrent tumor included an additional TP53 mutation and the other recurrent tumor showed POLE and DDR2 kinase gene mutation. The POLE mutation occurred outside the exonuclease domain. PIK3CA mutations were detected in 4 of 4 primary LGLS EC with recurrence and in 3 of 8 disease-free cases. LGLS EC with recurrence showed higher MSIsensor scores compared with LGLS without recurrence. The level of copy number gains in LGLS EC with recurrence was larger than LGLS EC without recurrence. This pilot study showed 1 of 3 recurrent cases gained a mutation associated with genetic instability (TP53) and 1 of them also acquired a mutation in the DDR2 kinase, a potential therapeutic target. We also noted a higher level of copy number gains, MSIsensor scores and PIK3CA mutations in the primary tumors that later recurred.

Validation of a Circulating Tumor DNA-Based Next-Generation Sequencing Assay in a Cohort of Patients with Solid tumors: A Proposed Solution for Decentralized Plasma Testing.

BACKGROUND: Characterization of circulating tumor DNA (ctDNA) has been integrated into clinical practice. Although labs have standardized validation procedures to develop single locus tests, the efficacy of on-site plasma-based next-generation sequencing (NGS) assays still needs to be proved. MATERIALS AND METHODS: In this retrospective study, we profiled DNA from matched tissue and plasma samples from 75 patients with cancer. We applied an NGS test that detects clinically relevant alterations in 33 genes and microsatellite instability (MSI) to analyze plasma cell-free DNA (cfDNA). RESULTS: The concordance between alterations detected in both tissue and plasma samples was higher in patients with metastatic disease. The NGS test detected 77% of sequence alterations, amplifications, and fusions that were found in metastatic samples compared with 45% of those alterations found in the primary tumor samples (p = .00005). There was 87% agreement on MSI status between the NGS test and tumor tissue results. In three patients, MSI-high ctDNA correlated with response to immunotherapy. In addition, the NGS test revealed an FGFR2 amplification that was not detected in tumor tissue from a patient with metastatic gastric cancer, emphasizing the importance of profiling plasma samples in patients with advanced cancer. CONCLUSION: Our validation experience of a plasma-based NGS assay advances current knowledge about translating cfDNA testing into clinical practice and supports the application of plasma assays in the management of oncology patients with metastatic disease. With an in-house method that minimizes the need for invasive procedures, on-site cfDNA testing supplements tissue biopsy to guide precision therapy and is entitled to become a routine practice. IMPLICATIONS FOR PRACTICE: This study proposes a solution for decentralized liquid biopsy testing based on validation of a next-generation sequencing (NGS) test that detects four classes of genomic alterations in blood: sequence mutations (single nucleotide substitutions or insertions and deletions), fusions, amplifications, and microsatellite instability (MSI). Although there are reference labs that perform single-site comprehensive liquid biopsy testing, the targeted assay this study validated can be established locally in any lab with capacity to offer clinical molecular pathology assays. To the authors' knowledge, this is the first report that validates evaluating an on-site plasma-based NGS test that detects the MSI status along with common sequence alterations encountered in solid tumors.

Molecular medicine tumor board: whole-genome sequencing to inform on personalized medicine for a man with advanced prostate cancer.

PURPOSE: Molecular profiling of cancer is increasingly common as part of routine care in oncology, and germline and somatic profiling may provide insights and actionable targets for men with metastatic prostate cancer. However, all reported cases are of deidentified individuals without full medical and genomic data available in the public domain. PATIENT AND METHODS: We present a case of whole-genome tumor and germline sequencing in a patient with advanced prostate cancer, who has agreed to make his genomic and clinical data publicly available. RESULTS: We describe an 84-year-old Caucasian male with a Gleason 10 oligometastastic hormone-sensitive prostate cancer. Whole-genome sequencing provided insights into his tumor's underlying mutational processes and the development of an SPOP mutation. It also revealed an androgen-receptor dependency of his cancer which was reflected in his durable response to radiation and hormonal therapy. Potentially actionable genomic lesions in the tumor were identified through a personalized medicine approach for potential future therapy, but at the moment, he remains in remission, illustrating the hormonal sensitivity of his SPOP-driven prostate cancer. We also placed this patient in the context of a large prostate-cancer cohort from the PCAWG (Pan-cancer Analysis of Whole Genomes) group. In this comparison, the patient's cancer appears typical in terms of the number and type of somatic mutations, but it has a somewhat larger contribution from the mutational process associated with aging. CONCLUSION: We combined the expertise of medical oncology and genomics approaches to develop a molecular tumor board to integrate the care and study of this patient, who continues to have an outstanding response to his combined modality treatment. This identifiable case potentially helps overcome barriers to clinical and genomic data sharing.

Limitations of Detecting Genetic Variants from the RNA Sequencing Data in Tissue and Fine-Needle Aspiration Samples.

Background: Genetic profiling of resected tumor or biopsy samples is increasingly used for cancer diagnosis and therapy selection for thyroid and other cancer types. Although mutations occur in cell DNA and are typically detected using DNA sequencing, recent attempts focused on detecting pathogenic variants from RNA. The aim of this study was to determine the completeness of capturing mutations using RNA sequencing (RNA-Seq) in thyroid tissue and fine-needle aspiration (FNA) samples. Methods: To compare the detection rate of mutations between DNA sequencing and RNA-Seq, 35 tissue samples were analyzed in parallel by whole-exome DNA sequencing (WES) and whole-transcriptome RNA-Seq at two study sites. Then, DNA and RNA from 44 thyroid FNA samples and 47 tissue samples were studied using both targeted DNA sequencing and RNA-Seq. Results: Of 162 genetic variants identified by WES of DNA in 35 tissue samples, 77 (48%) were captured by RNA-Seq, with a detection rate of 49% at site 1 and 46% at site 2 and no difference between thyroid and nonthyroid samples. Targeted DNA sequencing of 91 thyroid tissue and FNA samples detected 118 pathogenic variants, of which 57 (48%) were identified by RNA-Seq. For DNA variants present at >10% allelic frequency (AF), the detection rate of RNA-Seq was 62%, and for those at low (5-10%) AF, the detection rate of RNA-Seq was 7% (p < 0.0001). For common oncogenes (BRAF and RAS), 94% of mutations present at >10% AF and 11% of mutations present at 5-10% AF were captured by RNA-Seq. As expected, none of TERT promoter mutations were identified by RNA-Seq. The rate of mutation detection by RNA-Seq was lower in FNA samples than in tissue samples (32% vs. 49%, p = 0.02). Conclusions: In this study, RNA-Seq analysis detected only 46-49% of pathogenic variants identifiable by sequencing of tumor DNA. Detection of mutations by RNA-Seq was more successful for mutations present at a high allelic frequency. Mutations were more often missed by RNA-Seq when present at low frequency or when tested on FNA samples. All TERT mutations were missed by RNA-Seq. These data suggest that RNA-Seq does not detect a significant proportion of clinically relevant mutations and should be used with caution in clinical practice for detecting DNA mutations.

Integration of whole-exome and anchored PCR-based next generation sequencing significantly increases detection of actionable alterations in precision oncology.

BACKGROUND: Frequency of clinically relevant mutations in solid tumors by targeted and whole-exome sequencing is ∼30%. Transcriptome analysis complements detection of actionable gene fusions in advanced cancer patients. Goal of this study was to determine the added value of anchored multiplex PCR (AMP)-based next-generation sequencing (NGS) assay to identify further potential drug targets, when coupled with whole-exome sequencing (WES). METHODS: Selected series of fifty-six samples from 55 patients enrolled in our precision medicine study were interrogated by WES and AMP-based NGS. RNA-seq was performed in 19 cases. Clinically relevant and actionable alterations detected by three methods were integrated and analyzed. RESULTS: AMP-based NGS detected 48 fusions in 31 samples (55.4%); 31.25% (15/48) were classified as targetable based on published literature. WES revealed 29 samples (51.8%) harbored targetable alterations. TMB-high and MSI-high status were observed in 12.7% and 1.8% of cases. RNA-seq from 19 samples identified 8 targetable fusions (42.1%), also captured by AMP-based NGS. When number of actionable fusions detected by AMP-based NGS were added to WES targetable alterations, 66.1% of samples had potential drug targets. When both WES and RNA-seq were analyzed, 57.8% of samples had targetable alterations. CONCLUSIONS: This study highlights importance of an integrative genomic approach for precision oncology, including use of different NGS platforms with complementary features. Integrating RNA data (whole transcriptome or AMP-based NGS) significantly enhances detection of potential targets in cancer patients. In absence of fresh frozen tissue, AMP-based NGS is a robust method to detect actionable fusions using low-input RNA from archival tissue.