Shallow whole-genome sequencing of plasma cell-free DNA accurately differentiates small from non-small cell lung carcinoma

Background Accurate lung cancer classification is crucial to guide therapeutic decisions. However, histological subtyping by pathologists requires tumor tissue-a necessity that is often intrinsically associated with procedural difficulties. The analysis of circulating tumor DNA present in minimal-invasive blood samples, referred to as liquid biopsies, could therefore emerge as an attractive alternative. Methods Concerning adenocarcinoma, squamous cell carcinoma, and small cell carcinoma, our proof of concept study investigates the potential of liquid biopsy-derived copy number alterations, derived from single-end shallow whole-genome sequencing (coverage 0.1-0.5x), across 51 advanced stage lung cancer patients. Results Genomic abnormality testing reveals anomalies in 86.3% of the liquid biopsies (16/20 for adenocarcinoma, 13/16 for squamous cell, and 15/15 for small cell carcinoma). We demonstrate that copy number profiles from formalin-fixed paraffin-embedded tumor biopsies are well represented by their liquid equivalent. This is especially valid within the small cell carcinoma group, where paired profiles have an average Pearson correlation of 0.86 (95% CI 0.79-0.93). A predictive model trained with public data, derived from 843 tissue biopsies, shows that liquid biopsies exhibit multiple deviations that reflect histological classification. Most notably, distinguishing small from non-small cell lung cancer is characterized by an area under the curve of 0.98 during receiver operating characteristic analysis. Additionally, we investigated how deeper paired-end sequencing, which will eventually become feasible for routine diagnosis, empowers tumor read enrichment by insert size filtering: for all of the 29 resequenced liquid biopsies, the tumor fraction could be increased in silico, thereby "rescuing" three out of five cases with previously undetectable alterations. Conclusions Copy number profiling of cell-free DNA enables histological classification. Since shallow whole-genome sequencing is inexpensive and often fully operational at routine molecular laboratories, this finding has current diagnostic potential, especially for patients with lesions that are difficult to reach

Definitions of disease burden across the spectrum of metastatic castration-sensitive prostate cancer: comparison by disease outcomes and genomics

BACKGROUND: Several definitions have attempted to stratify metastatic castrate-sensitive prostate cancer (mCSPC) into low and high-volume states. However, at this time, comparison of these definitions is limited. Here we aim to compare definitions of metastatic volume in mCSPC with respect to clinical outcomes and mutational profiles. METHODS: We performed a retrospective review of patients with biochemically recurrent or mCSPC whose tumors underwent somatic targeted sequencing. 294 patients were included with median follow-up of 58.3 months. Patients were classified into low and high-volume disease per CHAARTED, STAMPEDE, and two numeric (≤3 and ≤5) definitions. Endpoints including radiographic progression-free survival (rPFS), time to development of castration resistance (tdCRPC), and overall survival (OS) were evaluated with Kaplan-Meier survival curves and log-rank test. The incidence of driver mutations between definitions were compared. RESULTS: Median OS and tdCRPC were shorter for high-volume than low-volume disease for all four definitions. In the majority of patients (84.7%) metastatic volume classification did not change across all four definitions. High volume disease was significantly associated with worse OS for all four definitions (CHAARTED: HR 2.89; p < 0.01, STAMPEDE: HR 3.82; p < 0.01, numeric ≤3: HR 4.67; p < 0.01, numeric ≤5: HR 3.76; p < 0.01) however, were similar for high (p = 0.95) and low volume (p = 0.79) disease across all four definitions. Those with discordant classification tended to have more aggressive clinical behavior and mutational profiles. Patients with low-volume disease and TP53 mutation experienced a more aggressive course with rPFS more closely mirroring high-volume disease. CONCLUSIONS: The spectrum of mCSPC was confirmed across four different metastatic definitions for clinical endpoints and genetics. All definitions were generally similar in classification of patients, outcomes, and genetic makeup. Given these findings, the simplicity of numerical definitions might be preferred, especially when integrating metastasis directed therapy. Incorporation of tumor genetics may allow further refinement of current metastatic definitions

Unraveling the genomic underpinnings of advanced prostate cancer

Metastatic hormone-sensitive prostate cancer (mHSPC) is a highly heterogeneous malignancy with varied outcomes observed across patients. Genomic features have been associated with differential clinical characteristics and treatment outcomes, but it is not yet clear to what degree these features should guide clinical decision-making in mHSPC. Precision medicine in mHSPC care is today confounded by the multifocality and molecular heterogeneity often seen in PCa. In this thesis, we aim to resolve the genomic drivers of mHSPC disease and dissect the tumor heterogeneity with an ultimate goal of enhancing the integration of genomics in cancer practice. In a first phase, a thorough review of the literature to summarize the current understanding of genomic alterations in mHSPC in terms of both the prevalence of alterations involving specific pathways and the relationship of these alterations with clinical features was performed. This study showed that genomic alterations may have prognostic and predictive implications. Another key finding of this study was that genomic data from mHSPC patients varied greatly in terms of the clinical states of patients and the source of material, and that the current study designs, in which only one sample from the resected tumor is sequenced, are not optimized for the study of multifocality. In a second phase, we assessed the prognostic and predictive ability of a genomic signature to risk stratify outcomes for patients with oligometastatic PCa recurrences, by pooling the only two prospective randomized trials, STOMP and ORIOLE, of metastasis-directed therapy (MDT) versus observation in oligometastatic HSPC. In this study, we observed a high-risk mutational signature consisting of pathogenic alterations in ATM, BRCA1/2, Rb1 and TP53 that is highly prognostic and predictive in this patient population suggesting that future trials should integrate these biomarkers to better understand their role in patient selection. Next, we investigated whether lung involvement should be considered as a proxy of more indolent disease in patients with presenting with lung recurrences. To address this question, we determined the genomic alterations that characterize lung-recurrent mHSPC (i.e. patients with lung metastases after curative-intent treatment for PCa) through multi-region profiling of both primary and metastatic lung samples. We found that the presence of lung recurrences associates with clinical and genomic indolence, proposing the site of metastatic recurrence as stratification factor for future predictive models in mHPSC. A second aim was to develop a practical strategy to reliably genotype patients with metastases at first diagnosis of prostate cancer, which is known as de novo mHSPC. First, we explored the extent to which a single diagnostic biopsy captures the metastatic genotype in synchronous metastatic tissue/ctDNA. Clinically relevant alterations were often missed in these single-biopsy approaches due to the heterogeneity within the primary and metastases. Exploration of the relationship between primary and metastatic tumors revealed little genomic evolution in lung-recurrent mHSPC patients, whereas we found extensive polyclonal seeding in a significant proportion of the de novo mHSPC patients

Comparative analysis of somatic variant calling on matched FF and FFPE WGS samples

BACKGROUND: Research grade Fresh Frozen (FF) DNA material is not yet routinely collected in clinical practice. Many hospitals, however, collect and store Formalin Fixed Paraffin Embedded (FFPE) tumor samples. Consequently, the sample size of whole genome cancer cohort studies could be increased tremendously by including FFPE samples, although the presence of artefacts might obfuscate the variant calling. To assess whether FFPE material can be used for cohort studies, we performed an in-depth comparison of somatic SNVs called on matching FF and FFPE Whole Genome Sequence (WGS) samples extracted from the same tumor. METHODS: Four variant callers (i.e. Strelka2, Mutect2, VarScan2 and Shimmer) were used to call somatic variants on matching FF and FFPE WGS samples from a metastatic prostate tumor. Using the variants identified by these callers, we developed a heuristic to maximize the overlap between the FF and its FFPE counterpart in terms of sensitivity and precision. The proposed variant calling approach was then validated on nine matched primary samples. Finally, we assessed what fraction of the discrepancy could be attributed to intra-tumor heterogeneity (ITH), by comparing the overlap in clonal and subclonal somatic variants. RESULTS: We first compared variants between an FF and an FFPE sample from a metastatic prostate tumor, showing that on average 50% of the calls in the FF are recovered in the FFPE sample, with notable differences between callers. Combining the variants of the different callers using a simple heuristic, increases both the precision and the sensitivity of the variant calling. Validating the heuristic on nine additional matched FF-FFPE samples, resulted in an average F1-score of 0.58 and an outperformance of any of the individual callers. In addition, we could show that part of the discrepancy between the FF and the FFPE samples can be attributed to ITH. CONCLUSION: This study illustrates that when using the correct variant calling strategy, the majority of clonal SNVs can be recovered in an FFPE sample with high precision and sensitivity. These results suggest that somatic variants derived from WGS of FFPE material can be used in cohort studiesUGent Bijzonder onderzoeksfonds, Agentschap voor Innovatie door Wetenschap en Technologie (IWT) [NEMOA] and Fonds Wetenschappelijk Onderzoek.https://bmcmedgenomics.biomedcentral.compm2020Genetic

Inferring the genomic landscape of lymph-node positive high-risk prostate cancer

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Whole slide imaging-based prediction of TP53 mutations identifies an aggressive disease phenotype in prostate cancer

In prostate cancer, there is an urgent need for objective prognostic biomarkers that identify the metastatic potential of a tumor at an early stage. While recent analyses indicated TP53 mutations as candidate biomarkers, molecular profiling in a clinical setting is complicated by tumor heterogeneity. Deep learning models that predict the spatial presence of TP53 mutations in whole slide images (WSI) offer the potential to mitigate this issue. To assess the potential of WSIs as proxies for spatially resolved profiling and as biomarkers for aggressive disease, we developed TiDo, a deep learning model that achieves state-of-the-art performance in predicting TP53 mutations from WSIs of primary prostate tumors. In an independent multifocal cohort, the model showed successful generalization at both the patient and lesion level. Analysis of model predictions revealed that false positive (FP) predictions could at least partially be explained by TP53 deletions, suggesting that some FP carry an alteration that leads to the same histological phenotype as TP53 mutations. Comparative expression and histologic cell type analyses identified a TP53-like cellular phenotype triggered by expression of pathways affecting stromal composition. Together, these findings indicate that WSI-based models might not be able to perfectly predict the spatial presence of individual TP53 mutations but they have the potential to elucidate the prognosis of a tumor by depicting a downstream phenotype associated with aggressive disease biomarkers

Oligometastatic prostate cancer : the game is afoot

Oligometastatic prostate cancer represents an intermediate state between a localized tumor and widespread metastatic disease. Its specific clinical features suggest the existence of a distinct biology which still needs to be elucidated. New imaging techniques like prostate specific membrane antigen (PSMA) PET scans have shown to perform well in the staging and restaging of this category of patients, at different phases of disease evolution. Despite limited prospective evidence, metastasis-directed therapies (MDT) are emerging as valid treatment options able to postpone systemic therapies and probably improve survival outcome. The aim of this review is to shed light on the clinical scenario of prostate cancer patients with limited metastatic disease burden and highlight the role of MDT strategies in this setting

Shallow whole-genome sequencing : a useful, easy to apply molecular technique for CNA detection on FFPE tumor tissue : a glioma-driven study

Copy number alterations (CNAs) have increasingly become part of the diagnostic algorithm of glial tumors. Alterations such as homozygous deletion of CDKN2A/B, 7 +/ 10 - chromosome copy number changes or EGFR amplification are predictive of a poor prognosis. The codeletion of chromosome arms 1p and 19q, typically associated with oligodendroglioma, implies a more favorable prognosis. Detection of this codeletion by the current diagnostic standard, being fluorescence in situ hybridization (FISH), is sometimes however subject to technical and interpretation problems. In this study, we evaluated CNA detection by shallow whole-genome sequencing (sWGS) as an inexpensive, complementary molecular technique. A cohort of 36 glioma tissue samples, enriched with "difficult" and "ambiguous" cases, was analyzed by sWGS. sWGS results were compared with FISH assays of chromosomes 1p and 19q. In addition, CNAs relevant to glioblastoma diagnosis were explored. In 4/36 samples, EGFR (7p11.2) amplifications and homozygous loss of CDKN2A/B were identified by sWGS. Six out of 8 IDH-wild-type glioblastomas demonstrated a prognostic chromosome 7/chromosome 10 signature. In 11/36 samples, local interstitial and terminal 1p/19q alterations were detected by sWGS, implying that FISH's targeted nature might promote false arm-level extrapolations. In this cohort, differences in overall survival between patients with and without codeletion were better pronounced by the sequencing-based distinction (likelihood ratio of 7.48) in comparison to FISH groupings (likelihood ratio of 0.97 at diagnosis and 1.79 +/- 0.62 at reobservation), suggesting sWGS is more accurate than FISH. We recognized adverse effects of tissue block age on FISH signals. In addition, we show how sWGS reveals relevant aberrations beyond the 1p/19q state, such as EGFR amplification, combined gain of chromosome 7 and loss of chromosome 10, and homozygous loss of CDKN2A/B. The findings presented by this study might stimulate implementation of sWGS as a complementary, easy to apply technique for copy number detection