Hepatocellular Carcinoma Immune Microenvironment Analysis: A Comprehensive Assessment with Computational and Classical Pathology.

PURPOSE: The spatial variability and clinical relevance of the tumour immune microenvironment (TIME) are still poorly understood for hepatocellular carcinoma (HCC). Here we aim to develop a deep learning (DL)-based image analysis model for the spatial analysis of immune cell biomarkers, and microscopically evaluate the distribution of immune infiltration. EXPERIMENTAL DESIGN: Ninety-two HCC surgical liver resections and 51 matched needle biopsies were histologically classified according to their immunophenotypes: inflamed, immune-excluded, and immune-desert. To characterise the TIME on immunohistochemistry (IHC)-stained slides, we designed a multi-stage DL algorithm, IHC-TIME, to automatically detect immune cells and their localisation in TIME in tumour-stromal, centre-border segments. RESULTS: Two models were trained to detect and localise the immune cells on IHC-stained slides. The framework models, i.e. immune cell detection models and tumour-stroma segmentation, reached 98% and 91% accuracy, respectively. Patients with inflamed tumours showed better recurrence-free survival than those with immune-excluded or immune desert tumours. Needle biopsies were found to be 75% accurate in representing the immunophenotypes of the main tumour. Finally, we developed an algorithm that defines immunophenotypes automatically based on the IHC-TIME analysis, achieving an accuracy of 80%. CONCLUSIONS: Our DL-based tool can accurately analyse and quantify immune cells on IHC-stained slides of HCC. The microscopical classification of the TIME can stratify HCCs according to the patient prognosis. Needle biopsies can provide valuable insights for TIME-related prognostic prediction, albeit with specific constraints. The computational pathology tool provides a new way to study the HCC TIME.

Ribosomal protein RPL39L is an efficiency factor in the cotranslational folding of a subset of proteins with alpha helical domains.

Increasingly many studies reveal how ribosome composition can be tuned to optimally translate the transcriptome of individual cell types. In this study, we investigated the expression pattern, structure within the ribosome and effect on protein synthesis of the ribosomal protein paralog 39L (RPL39L). With a novel mass spectrometric approach we revealed the expression of RPL39L protein beyond mouse germ cells, in human pluripotent cells, cancer cell lines and tissue samples. We generated RPL39L knock-out mouse embryonic stem cell (mESC) lines and demonstrated that RPL39L impacts the dynamics of translation, to support the pluripotency and differentiation, spontaneous and along the germ cell lineage. Most differences in protein abundance between WT and RPL39L KO lines were explained by widespread autophagy. By CryoEM analysis of purified RPL39 and RPL39L-containing ribosomes we found that, unlike RPL39, RPL39L has two distinct conformations in the exposed segment of the nascent peptide exit tunnel, creating a distinct hydrophobic patch that has been predicted to support the efficient co-translational folding of alpha helices. Our study shows that ribosomal protein paralogs provide switchable modular components that can tune translation to the protein production needs of individual cell types.

In vivo interaction screening reveals liver-derived constraints to metastasis.

It is estimated that only 0.02% of disseminated tumour cells are able to seed overt metastases1. While this suggests the presence of environmental constraints to metastatic seeding, the landscape of host factors controlling this process remains largely unclear. Here, combining transposon technology2 and fluorescence niche labelling3, we developed an in vivo CRISPR activation screen to systematically investigate the interactions between hepatocytes and metastatic cells. We identify plexin B2 as a critical host-derived regulator of liver colonization in colorectal and pancreatic cancer and melanoma syngeneic mouse models. We dissect a mechanism through which plexin B2 interacts with class IV semaphorins on tumour cells, leading to KLF4 upregulation and thereby promoting the acquisition of epithelial traits. Our results highlight the essential role of signals from the liver parenchyma for the seeding of disseminated tumour cells before the establishment of a growth-promoting niche. Our findings further suggest that epithelialization is required for the adaptation of CRC metastases to their new tissue environment. Blocking the plexin-B2-semaphorin axis abolishes metastatic colonization of the liver and therefore represents a therapeutic strategy for the prevention of hepatic metastases. Finally, our screening approach, which evaluates host-derived extrinsic signals rather than tumour-intrinsic factors for their ability to promote metastatic seeding, is broadly applicable and lays a framework for the screening of environmental constraints to metastasis in other organs and cancer types.

Extracellular Vesicle DNA Extraction and Sequencing in Ancient Serum Samples From Patients With Breast Cancer.

BACKGROUND/AIM: Extracellular vesicle DNA (EV-DNA) has emerged as a novel biomarker for tumor mutation detection using liquid biopsies, exhibiting biological advantages compared to cell-free DNA (cfDNA). This study assessed the feasibility of EV-DNA and cfDNA extraction and sequencing in old serum samples of patients with breast cancer (BC). PATIENTS AND METHODS: A total of 28 serum samples of 27 patients with corresponding clinical information were collected between 1983 and 1991. EV-DNA was extracted using Exo-GAG kit (Nasabiotech) and cfDNA using QIAsymphony DSP Virus/Pathogen Midi Kit (Qiagen), respectively. Subsequently, 10 matched samples (EV-DNA n=5, cfDNA n=5) of five patients were subjected to sequencing using the Oncomine™ Breast cfDNA Research Assay v2 (Thermo Fisher Scientific). RESULTS: Samples were collected on median 1.9 years after primary diagnosis [interquartile range (IQR)=0.2-7.2]. Median follow-up was 9.5 years (IQR=5.2-14.2). Median age of serum samples was 36.1 years (IQR=34.5-37.3). EV-DNA and cfDNA were extracted from 100% (28/28) of the included samples. Both, DNA quantity and concentration were comparable between EV-DNA and cfDNA. Sequencing was successfully performed in 100% (10/10) of the included samples. Two matched analyses yielded equivalent results in EV-DNA and cfDNA (no mutations, n=1; PIK3CA mutation, n=1), whilst in two analyses, PIK3CA mutation was only found in cfDNA, and in one analysis, a TP53 mutation was only found in EV-DNA. CONCLUSION: EV-DNA extraction and sequencing in old serum samples of patients with BC is feasible and has the potential to address clinically relevant questions in longitudinal studies.

Tracing Tumor Heterogeneity of Pleomorphic Carcinoma of the Lung.

INTRODUCTION: Pulmonary pleomorphic carcinoma (PPC) is an aggressive and highly heterogeneous NSCLC whose underlying biology is still poorly understood. METHODS: A total of 42 tumor areas from 20 patients with PPC were microdissected, including 39 primary tumors and three metastases, and the histologically distinct components were subjected to whole exome sequencing separately. We further performed in silico analysis of microdissected bulk RNA sequencing and methylation data of 28 samples from 14 patients with PPC. We validated our findings using immunohistochemistry. RESULTS: The epithelial and the sarcomatoid components of PPCs shared a large number of genomic alterations. Most mutations in cancer driver genes were clonal and truncal between the two components of PPCs suggesting a common ancestor. The high number of alterations in the RTK-RAS pathway suggests that it plays an important role in the evolution of PPC. The metastases morphologically and genetically resembled the epithelial or the sarcomatoid components of the tumor. The transcriptomic and epigenetic profiles of the sarcomatoid components of PPCs with matched squamous-like or adenocarcinoma-like components differed from each other, and they shared more similarities to their matched epithelial components. NCAM1/CD56 was preferentially expressed in the sarcomatoid component of squamous-like PPCs, whereas CDH1/E-Cadherin expression was down-regulated in the sarcomatoid component of most PPCs. CONCLUSION: Lung adenocarcinoma-like PPCs are mainly driven by RTK-RAS signaling, whereas epithelial-mesenchymal transition programs as highlighted by increased NCAM1 and decreased CDH1 expression govern the epithelial-sarcomatoid transition between the clonally related tumor components. Several alterations in PPCs pinpoint therapeutic opportunities.

A Hepatocellular Cancer Patient-derived Organoid Xenograft Model to Investigate Impact of Liver Regeneration on Tumor Growth.

Recurrence poses a notable challenge after hepatocellular carcinoma (HCC) treatment, impacting more than 70% of patients who undergo surgical resection. Recurrence stems from undetected micro-metastasis or de novo cancer, potentially triggered by postsurgical liver regeneration. Prior research employed HCC cell lines in orthotopic models to study the impact of liver regeneration, but their limited validity prompted the need for a more representative model. Here, we introduce a novel approach utilizing patient-derived HCC organoids to investigate the influence of liver regeneration on HCC. Patient tumor tissues are processed to create tumor organoids, embedded in a three-dimensional basement membrane matrix, and cultured in a liver-specific medium. One million organoids are injected into the right superior lobe (RSL) of immunodeficient mice, confirming macroscopic tumor growth through sonography. The intervention group undergoes resection of the left lateral lobe (LLL) (30% of total liver volume) or additionally, the middle lobe (ML) (65% of total liver volume) to induce liver regeneration within the tumor site. The control group experiences re-laparotomy without liver tissue resection. After 2 weeks, both groups undergo tumor and normal tissue explantation. In conclusion, this patient-derived HCC organoid model offers a robust platform to investigate the impact of liver regeneration post-cancer resection. Its multi-cellular composition, genetic diversity, and prolonged culture capabilities make it an invaluable tool for studying HCC recurrence mechanisms and potential interventions.

Analysis of off-tumour toxicities of T-cell-engaging bispecific antibodies via donor-matched intestinal organoids and tumouroids.

Predicting the toxicity of cancer immunotherapies preclinically is challenging because models of tumours and healthy organs do not typically fully recapitulate the expression of relevant human antigens. Here we show that patient-derived intestinal organoids and tumouroids supplemented with immune cells can be used to study the on-target off-tumour toxicities of T-cell-engaging bispecific antibodies (TCBs), and to capture clinical toxicities not predicted by conventional tissue-based models as well as inter-patient variabilities in TCB responses. We analysed the mechanisms of T-cell-mediated damage of neoplastic and donor-matched healthy epithelia at a single-cell resolution using multiplexed immunofluorescence. We found that TCBs that target the epithelial cell-adhesion molecule led to apoptosis in healthy organoids in accordance with clinical observations, and that apoptosis is associated with T-cell activation, cytokine release and intra-epithelial T-cell infiltration. Conversely, tumour organoids were more resistant to damage, probably owing to a reduced efficiency of T-cell infiltration within the epithelium. Patient-derived intestinal organoids can aid the study of immune-epithelial interactions as well as the preclinical and clinical development of cancer immunotherapies.

Patient-derived organoids identify tailored therapeutic options and determinants of plasticity in sarcomatoid urothelial bladder cancer.

Sarcomatoid Urothelial Bladder Cancer (SARC) is a rare and aggressive histological subtype of bladder cancer for which therapeutic options are limited and experimental models are lacking. Here, we report the establishment of a long-term 3D organoid-like model derived from a SARC patient (SarBC-01). SarBC-01 emulates aggressive morphological, phenotypical, and transcriptional features of SARC and harbors somatic mutations in genes frequently altered in sarcomatoid tumors such as TP53 (p53) and RB1 (pRB). High-throughput drug screening, using a library comprising 1567 compounds in SarBC-01 and conventional urothelial carcinoma (UroCa) organoids, identified drug candidates active against SARC cells exclusively, or UroCa cells exclusively, or both. Among those, standard-of-care chemotherapeutic drugs inhibited both SARC and UroCa cells, while a subset of targeted drugs was specifically effective in SARC cells, including agents targeting the Glucocorticoid Receptor (GR) pathway. In two independent patient cohorts and in organoid models, GR and its encoding gene NR3C1 were found to be significantly more expressed in SARC as compared to UroCa, suggesting that high GR expression is a hallmark of SARC tumors. Further, glucocorticoid treatment impaired the mesenchymal morphology, abrogated the invasive ability of SARC cells, and led to transcriptomic changes associated with reversion of epithelial-to-mesenchymal transition, at single-cell level. Altogether, our study highlights the power of organoids for precision oncology and for providing key insights into factors driving rare tumor entities.

High density of CXCL12-positive immune cell infiltration predicts chemosensitivity and recurrence-free survival in ovarian carcinoma.

BACKGROUND: Ovarian carcinoma is the most lethal gynecologic malignancy because of its late diagnosis, extremely high recurrence rate, and limited curative treatment options. In clinical practice, high-grade serous carcinoma (HGSC) predominates due to its frequency, high aggressiveness, and rapid development of drug resistance. Recent evidence suggests that CXCL12 is an important immunological factor in ovarian cancer progression. Therefore, we investigated the predictive and prognostic significance of the expression of this chemokine in tumor and immune cells in patients with HGSC. METHODS: We studied a cohort of 47 primary high-grade serous ovarian carcinomas and their associated recurrences. A tissue microarray was constructed to evaluate the CXCL12 immunostained tumor tissue. CXCL12 expression was evaluated and statistically analyzed to correlate clinicopathologic data, overall survival, and recurrence-free survival. RESULTS: A high proportion of CXCL12 + positive immune cells in primary ovarian serous carcinoma correlated significantly with chemosensitivity (p = 0.005), overall survival (p = 0.021), and longer recurrence-free survival (p = 0.038). In recurrent disease, high expression of CXCL12 was also correlated with better overall survival (p = 0.040). Univariate and multivariate analysis revealed that high CXCL12 + tumor-infiltrating immune cells (TICs) (HR 0.99, p = 0.042, HR 0.99, p = 0.023, respectively) and combined CXCL12 + /CD66b + infiltration (HR 0.15, p = 0.001, HR 0.13, p = 0.001, respectively) are independent favorable predictive markers for recurrence-free survival. CONCLUSION: A high density of CXCL12 + TICs predicts a good response to chemotherapy, leading to a better overall survival and a longer recurrence-free interval. Moreover, with concomitant high CXCL12/CD66b TIC density, it is an independent favorable predictor of recurrence-free survival in patients with ovarian carcinoma.

Fragment-sequencing unveils local tissue microenvironments at single-cell resolution.

Cells collectively determine biological functions by communicating with each other-both through direct physical contact and secreted factors. Consequently, the local microenvironment of a cell influences its behavior, gene expression, and cellular crosstalk. Disruption of this microenvironment causes reciprocal changes in those features, which can lead to the development and progression of diseases. Hence, assessing the cellular transcriptome while simultaneously capturing the spatial relationships of cells within a tissue provides highly valuable insights into how cells communicate in health and disease. Yet, methods to probe the transcriptome often fail to preserve native spatial relationships, lack single-cell resolution, or are highly limited in throughput, i.e. lack the capacity to assess multiple environments simultaneously. Here, we introduce fragment-sequencing (fragment-seq), a method that enables the characterization of single-cell transcriptomes within multiple spatially distinct tissue microenvironments. We apply fragment-seq to a murine model of the metastatic liver to study liver zonation and the metastatic niche. This analysis reveals zonated genes and ligand-receptor interactions enriched in specific hepatic microenvironments. Finally, we apply fragment-seq to other tissues and species, demonstrating the adaptability of our method.

Arginine reprograms metabolism in liver cancer via RBM39.

Metabolic reprogramming is a hallmark of cancer. However, mechanisms underlying metabolic reprogramming and how altered metabolism in turn enhances tumorigenicity are poorly understood. Here, we report that arginine levels are elevated in murine and patient hepatocellular carcinoma (HCC), despite reduced expression of arginine synthesis genes. Tumor cells accumulate high levels of arginine due to increased uptake and reduced arginine-to-polyamine conversion. Importantly, the high levels of arginine promote tumor formation via further metabolic reprogramming, including changes in glucose, amino acid, nucleotide, and fatty acid metabolism. Mechanistically, arginine binds RNA-binding motif protein 39 (RBM39) to control expression of metabolic genes. RBM39-mediated upregulation of asparagine synthesis leads to enhanced arginine uptake, creating a positive feedback loop to sustain high arginine levels and oncogenic metabolism. Thus, arginine is a second messenger-like molecule that reprograms metabolism to promote tumor growth.

Colitis Is Associated with Loss of the Histidine Phosphatase LHPP and Upregulation of Histidine Phosphorylation in Intestinal Epithelial Cells.

Protein histidine phosphorylation (pHis) is a posttranslational modification involved in cell cycle regulation, ion channel activity and phagocytosis. Using novel monoclonal antibodies to detect pHis, we previously reported that the loss of the histidine phosphatase LHPP (phospholysine phosphohistidine inorganic pyrophosphate phosphatase) results in elevated pHis levels in hepatocellular carcinoma. Here, we show that intestinal inflammation correlates with the loss of LHPP in dextran sulfate sodium (DSS)-treated mice and in inflammatory bowel disease (IBD) patients. Increased histidine phosphorylation was observed in intestinal epithelial cells (IECs), as determined by pHis immunofluorescence staining of colon samples from a colitis mouse model. However, the ablation of Lhpp did not cause increased pHis or promote intestinal inflammation under physiological conditions or after DSS treatment. Our observations suggest that increased histidine phosphorylation plays a role in colitis, but the loss of LHPP is not sufficient to increase pHis or to cause inflammation in the intestine.

Early Age of Onset Is an Independent Predictor for a Worse Response to Neoadjuvant Therapies in Sporadic Rectal Cancer Patients.

The incidence of rectal cancer (RC) is increasing in the population aged ≤ 49 (early-onset RC-EORC). EORC patients are more likely to present with locally advanced disease at diagnosis than late-onset RC (LORC; aged ≥ 50) patients. As a consequence, more EORC patients undergo neoadjuvant therapies. The response to treatment in EORC patients is still unknown. This study aims to explore the effect of age of onset on the pathological response to neoadjuvant therapies in sporadic locally advanced RC (LARC) patients. Based on an institutional prospectively maintained database, LARC patients undergoing neoadjuvant therapies and radical surgery between January 2010 and December 2022 were allocated to the EORC and LORC groups. The primary endpoint was the rate of incomplete response (Dworak 0-2). A total of 326 LORC and 79 EORC patients were included. Pre-neoadjuvant tumor features were comparable. A significantly higher rate of incomplete response was observed in EORC patients (49% vs. 35%; p = 0.028). From multivariable analysis, early age of onset, smoking and extramural invasion presented as independent risk factors for a worse response. This study demonstrates that an early age of onset is related to a worse response and calls for different multimodal strategies in this group of patients.

Cylindroma of the breast with CYLD gene mutation: a case report and review of the literature.

BACKGROUND: Cylindroma of the breast is a rare benign neoplasm. Since its first description in 2001, 20 cases have been reported in the literature. METHODS AND RESULTS: We report another case of this rare tumor in a 60-year-old woman with demonstration of the underlying molecular alteration. Histologically, the tumor showed the typical "jigsaw" pattern of a dual population of cells with a triple-negative phenotype. The pathognomonic mutation of the CYLD gene mutation was detected by whole exome sequencing. Cylindromas show morphological overlap with the solid-basaloid variant of adenoid cystic carcinoma, which renders this differential diagnosis difficult. However, distinction of these two lesions is of outmost importance, since cylindromas, in contrast to solid-basaloid variant of adenoid cystic carcinoma, behave in an entirely benign fashion. CONCLUSIONS: Careful evaluation of morphological features such as mitotic figures and cellular atypia is crucial in the diagnostic work-up of triple-negative breast lesions. It is important to keep cylindroma in mind as a pitfall and possible differential diagnosis for the solid-basaloid variant of adenoid cystic carcinoma. Molecular detection of CYLD gene mutation is helpful in cases with ambiguous histology. With this case report, we aim to contribute to a better understanding of mammary cylindroma and facilitate the diagnosis of this rare entity.

PI3K inhibition circumvents resistance to SHP2 blockade in metastatic triple-negative breast cancer.

The protein tyrosine phosphatase SHP2 activates oncogenic pathways downstream of most receptor tyrosine kinases (RTK) and has been implicated in various cancer types, including the highly aggressive subtype of triple-negative breast cancer (TNBC). Although allosteric inhibitors of SHP2 have been developed and are currently being evaluated in clinical trials, neither the mechanisms of the resistance to these agents, nor the means to circumvent such resistance have been clearly defined. The PI3K signaling pathway is also hyperactivated in breast cancer and contributes to resistance to anticancer therapies. When PI3K is inhibited, resistance also develops for example via activation of RTKs. We therefore assessed the effect of targeting PI3K and SHP2 alone or in combination in preclinical models of metastatic TNBC. In addition to the beneficial inhibitory effects of SHP2 alone, dual PI3K/SHP2 treatment decreased primary tumor growth synergistically, blocked the formation of lung metastases, and increased survival in preclinical models. Mechanistically, transcriptome and phospho-proteome analyses revealed that resistance to SHP2 inhibition is mediated by PDGFRß-evoked activation of PI3K signaling. Altogether, our data provide a rationale for co-targeting of SHP2 and PI3K in metastatic TNBC.

Minor intron splicing is critical for survival of lethal prostate cancer.

The evolutionarily conserved minor spliceosome (MiS) is required for protein expression of ∼714 minor intron-containing genes (MIGs) crucial for cell-cycle regulation, DNA repair, and MAP-kinase signaling. We explored the role of MIGs and MiS in cancer, taking prostate cancer (PCa) as an exemplar. Both androgen receptor signaling and elevated levels of U6atac, a MiS small nuclear RNA, regulate MiS activity, which is highest in advanced metastatic PCa. siU6atac-mediated MiS inhibition in PCa in vitro model systems resulted in aberrant minor intron splicing leading to cell-cycle G1 arrest. Small interfering RNA knocking down U6atac was ∼50% more efficient in lowering tumor burden in models of advanced therapy-resistant PCa compared with standard antiandrogen therapy. In lethal PCa, siU6atac disrupted the splicing of a crucial lineage dependency factor, the RE1-silencing factor (REST). Taken together, we have nominated MiS as a vulnerability for lethal PCa and potentially other cancers.

PI5P4Kα supports prostate cancer metabolism and exposes a survival vulnerability during androgen receptor inhibition.

Phosphatidylinositol (PI)regulating enzymes are frequently altered in cancer and have become a focus for drug development. Here, we explore the phosphatidylinositol-5-phosphate 4-kinases (PI5P4K), a family of lipid kinases that regulate pools of intracellular PI, and demonstrate that the PI5P4Kα isoform influences androgen receptor (AR) signaling, which supports prostate cancer (PCa) cell survival. The regulation of PI becomes increasingly important in the setting of metabolic stress adaptation of PCa during androgen deprivation (AD), as we show that AD influences PI abundance and enhances intracellular pools of PI-4,5-P2. We suggest that this PI5P4Kα-AR relationship is mitigated through mTORC1 dysregulation and show that PI5P4Kα colocalizes to the lysosome, the intracellular site of mTORC1 complex activation. Notably, this relationship becomes prominent in mouse prostate tissue following surgical castration. Finally, multiple PCa cell models demonstrate marked survival vulnerability following stable PI5P4Kα inhibition. These results nominate PI5P4Kα as a target to disrupt PCa metabolic adaptation to castrate resistance.

DNA Methylation Landscapes of Prostate Cancer Brain Metastasis Are Shaped by Early Driver Genetic Alterations.

Metastases from primary prostate cancers to rare locations, such as the brain, are becoming more common due to longer life expectancy resulting from improved treatments. Epigenetic dysregulation is a feature of primary prostate cancer, and distinct DNA methylation profiles have been shown to be associated with the mutually exclusive SPOP-mutant or TMPRSS2-ERG fusion genetic backgrounds. Using a cohort of prostate cancer brain metastases (PCBM) from 42 patients, with matched primary tumors for 17 patients, we carried out a DNA methylation analysis to examine the epigenetic distinction between primary prostate cancer and PCBM, the association between epigenetic alterations and mutational background, and particular epigenetic alterations that may be associated with PCBM. Multiregion sampling of PCBM revealed epigenetic stability within metastases. Aberrant methylation in PCBM was associated with mutational background and PRC2 complex activity, an effect that is particularly pronounced in SPOP-mutant PCBM. While PCBM displayed a CpG island hypermethylator phenotype, hypomethylation at the promoters of genes involved in neuroactive ligand-receptor interaction and cell adhesion molecules such as GABRB3, CLDN8, and CLDN4 was also observed, suggesting that cells from primary tumors may require specific reprogramming to form brain metastasis. This study revealed the DNA methylation landscapes of PCBM and the potential mechanisms and effects of PCBM-associated aberrant DNA methylation. SIGNIFICANCE: DNA methylation analysis reveals the molecular characteristics of PCBM and may serve as a starting point for efforts to identify and target susceptibilities of these rare metastases.

PipeIT2: A tumor-only somatic variant calling workflow for molecular diagnostic Ion Torrent sequencing data.

Precision oncology relies on the accurate identification of somatic mutations in cancer patients. While the sequencing of the tumoral tissue is frequently part of routine clinical care, the healthy counterparts are rarely sequenced. We previously published PipeIT, a somatic variant calling workflow specific for Ion Torrent sequencing data enclosed in a Singularity container. PipeIT combines user-friendly execution, reproducibility and reliable mutation identification, but relies on matched germline sequencing data to exclude germline variants. Expanding on the original PipeIT, here we describe PipeIT2 to address the clinical need to define somatic mutations in the absence of germline control. We show that PipeIT2 achieves a > 95% recall for variants with variant allele fraction >10%, reliably detects driver and actionable mutations and filters out most of the germline mutations and sequencing artifacts. With its performance, reproducibility, and ease of execution, PipeIT2 is a valuable addition to molecular diagnostics laboratories.

Cell-Free DNA Genomic Profiling and Its Clinical Implementation in Advanced Prostate Cancer.

Most men with prostate cancer (PCa), despite potentially curable localized disease at initial diagnosis, progress to metastatic disease. Despite numerous treatment options, choosing the optimal treatment for individual patients remains challenging. Biomarkers guiding treatment sequences in an advanced setting are lacking. To estimate the diagnostic potential of liquid biopsies in guiding personalized treatment of PCa, we evaluated the utility of a custom-targeted next-generation sequencing (NGS) panel based on the AmpliSeq HD Technology. Ultra-deep sequencing on plasma circulating free DNA (cfDNA) samples of 40 metastatic castration-resistant PCa (mCRPC) and 28 metastatic hormone-naive PCa (mCSPC) was performed. CfDNA somatic mutations were detected in 48/68 (71%) patients. Of those 68 patients, 42 had matched tumor and cfDNA samples. In 21/42 (50%) patients, mutations from the primary tumor tissue were detected in the plasma cfDNA. In 7/42 (17%) patients, mutations found in the primary tumor were not detected in the cfDNA. Mutations from primary tumors were detected in all tested mCRPC patients (17/17), but only in 4/11 with mCSPC. AR amplifications were detected in 12/39 (31%) mCRPC patients. These results indicate that our targeted NGS approach has high sensitivity and specificity for detecting clinically relevant mutations in PCa.