Mutations are abundantly present in tissues of healthy individuals, including the breast epithelium. Yet it remains unknown whether mutant cells directly induce lesion formation or first spread, leading to a field of mutant cells that is predisposed towards lesion formation. To study the clonal and spatial relationships between morphologically normal breast epithelium adjacent to pre-cancerous lesions, we developed a three-dimensional (3D) imaging pipeline combined with spatially resolved genomics on archival, formalin-fixed breast tissue with the non-obligate breast cancer precursor ductal carcinoma in situ (DCIS). Using this 3D image-guided characterization method, we built high-resolution spatial maps of DNA copy number aberration (CNA) profiles within the DCIS lesion and the surrounding normal mammary ducts. We show that the local heterogeneity within a DCIS lesion is limited. However, by mapping the CNA profiles back onto the 3D reconstructed ductal subtree, we find that in eight out of 16 cases the healthy epithelium adjacent to the DCIS lesions has overlapping structural variations with the CNA profile of the DCIS. Together, our study indicates that pre-malignant breast transformations frequently develop within mutant clonal fields of morphologically normal-looking ducts. © 2024 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

UNASSIGNED: According to GLOBOCAN 2020, lymphoma ranked as the 9th most common cancer and the 12th leading cause of cancer-related deaths worldwide. Traditional diagnostic methods rely on the invasive excisional lymph node biopsy, which is an invasive approach with some limitations. Most lymphoma patients are diagnosed at an advanced stage since they are asymptomatic at the beginning, which has significantly impacted treatment efficacy and prognosis of the disease.
UNASSIGNED: This study assessed the performance and utility of a newly developed blood-based assay (SeekInCare) for lymphoma early detection. SeekInCare utilized protein tumor markers and a comprehensive set of cancer-associated genomic features, including copy number aberration (CNA), fragment size (FS), end motif, and lymphoma-related virus, which were profiled by shallow WGS of cfDNA.
UNASSIGNED: Protein marker CA125 could be used for lymphoma detection independent of gender, and the sensitivity was 27.8% at specificity of 98.0%. After integrating these multi-dimensional features, 77.8% sensitivity was achieved at specificity of 98.0%, while its NPV and PPV were both more than 92% for lymphoma detection. The sensitivity of early-stage (I-II) lymphoma was up to 51.3% (47.4% and 55.0% for stage I and II respectively). After 2 cycles of treatment, the molecular response of SeekInCare was correlated with the clinical outcome.
UNASSIGNED: In summary, a blood-based assay can be an alternative to detect lymphoma with adequate performance. This approach becomes particularly valuable in cases where obtaining tissue biopsy is difficult to obtain or inconclusive.

Genomic instability is an important hallmark of cancer and more recently has been identified in others like neurodegenrative diseases. Chromosomal instability, as a measure of genomic instability, has been used to characterize clinical and biological phenotypes associated with these diseases by measuring structural and numerical chromosomal alterations. There have been multiple chromosomal instability scores developed across many studies in the literature; however, these scores have not been compared because of the lack of a single tool available to calculate and facilitate these various metrics. Here, we provide an R package CINmetrics, that calculates six different chromosomal instability scores and allows direct comparison between them. We also demonstrate how these scores differ by applying CINmetrics to breast cancer data from The Cancer Genome Atlas (TCGA). The package is available on CRAN at https://cran.rproject.org/package=CINmetrics and on GitHub at https://github.com/lasseignelab/CINmetrics.

Cell-free DNA (cfDNA) provides a convenient diagnosis avenue for noninvasive cancer detection. The current methods are focused on identifying circulating tumor DNA (ctDNA)s genomic aberrations, e.g. mutations, copy number aberrations (CNAs) or methylation changes. In this study, we report a new computational method that unifies two orthogonal pieces of information, namely methylation and CNAs, derived from whole-genome bisulfite sequencing (WGBS) data to quantify low tumor content in cfDNA. It implements a Bayes model to enrich ctDNA from WGBS data based on hypomethylation haplotypes, and subsequently, models CNAs for cancer detection. We generated WGBS data in a total of 262 samples, including high-depth (>20×, deduped high mapping quality reads) data in 76 samples with matched triplets (tumor, adjacent normal and cfDNA) and low-depth (~2.5×, deduped high mapping quality reads) data in 186 samples. We identified a total of 54 Mb regions of hypomethylation haplotypes for model building, a vast majority of which are not covered in the HumanMethylation450 arrays. We showed that our model is able to substantially enrich ctDNA reads (tens of folds), with clearly elevated CNAs that faithfully match the CNAs in the paired tumor samples. In the 19 hepatocellular carcinoma cfDNA samples, the estimated enrichment is as high as 16 fold, and in the simulation data, it can achieve over 30-fold enrichment for a ctDNA level of 0.5% with a sequencing depth of 600×. We also found that these hypomethylation regions are also shared among many cancer types, thus demonstrating the potential of our framework for pancancer early detection.

Lung cancer is a common disease with a poor prognosis. Genomic alterations involving the KRAS gene are common in lung carcinomas, although much is unknown about how different mutations, deletions, and expressions influence the disease course. The first approval of a KRAS-directed inhibitor was recently approved by the FDA. Mutations in the KRAS gene have been associated with poor prognosis for lung adenocarcinomas, but implications of the loss of heterozygosity (LOH) of KRAS have not been investigated. In this study, we have assessed the LOH of KRAS in early-stage lung adenocarcinoma by analyzing DNA copy number profiles and have investigated the effect on patient outcome in association with mRNA expression and somatic hotspot mutations. KRAS mutation was present in 36% of cases and was associated with elevated mRNA expression. LOH in KRAS was associated with a favorable prognosis, more prominently in KRAS mutated than in wild-type patients. The presence of both LOH and mutation in KRAS conferred a better prognosis than KRAS mutation alone. For wild-type tumors, no difference in prognosis was observed between patients with and without LOH in KRAS. Our study indicates that LOH in KRAS is an independent prognostic factor that may refine the existing prognostic groups of lung adenocarcinomas.

Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related death worldwide, and heterogeneity of HCC is the major barrier in improving patient outcome. To stratify HCC patients with different degrees of malignancy and provide precise treatment strategies, we reconstructed the tumor evolution trajectory with the help of scRNA-seq data and established a 30-gene prognostic model to identify the malignant state in HCC. Patients were divided into high-risk and low-risk groups. C-index and receiver operating characteristic (ROC) curve confirmed the excellent predictive value of this model. Downstream analysis revealed the underlying molecular and functional characteristics of this model, including significantly higher genomic instability and stronger proliferation/progression potential in the high-risk group. In summary, we established a novel prognostic model to overcome the barriers caused by HCC heterogeneity and provide the possibility of better clinical management for HCC patients to improve their survival outcomes.

Cancer can develop from an accumulation of alterations, some of which cause a nonmalignant cell to transform to a malignant state exhibiting increased rate of cell growth and evasion of growth suppressive mechanisms, eventually leading to tissue invasion and metastatic disease. Triple-negative breast cancers (TNBC) are heterogeneous and are clinically characterized by the lack of expression of hormone receptors and human epidermal growth factor receptor 2 (HER2), which limits its treatment options. Since tumor evolution is driven by diverse cancer cell populations and their microenvironment, it is imperative to map TNBC at single-cell resolution. Here, we describe an experimental procedure for isolating a single-cell suspension from a TNBC patient-derived xenograft, subjecting it to single-cell RNA sequencing using droplet-based technology from 10× Genomics and analyzing the transcriptomic data at single-cell resolution to obtain inferred copy number aberration profiles, using scCNA. Data obtained using this single-cell RNA sequencing experimental and analytical methodology should enhance our understanding of intratumor heterogeneity which is key for identifying genetic vulnerabilities and developing effective therapies.

The diagnostic accuracy of the multigene panel test (MPT) and OncoScan™ in the determination of HER2 amplification in breast tumors remains controversial. In the present study, HER2 copy number was analyzed using both MPT and OncoScan™ in 45 breast tumors and was compared with that in fluorescent in situ hybridization (FISH) analysis. Tumors with low cellularity were examined using tumor cell enrichment and fluorescence‑activated cell sorting. Both MPT and OncoScan™ exhibited significant correlations with FISH with respect to the determination of HER2 amplification in breast tumors. However, the correlation coefficient was significantly higher for the comparison of MPT and FISH (r=0.770) compared with that between OncoScan™ and FISH (r=0.564). The accuracy of MPT (93.3%) was slightly higher compared with that in OncoScan™ (84.4%) in determining the HER2 status, which was mostly explained by the higher sensitivity of MPT in tumors with low cellularity (83.3 vs. 33.3%), but not in those with high cellularity (81.8 vs. 72.7%). The specificity was 100% for both tests. The MPT exhibited higher sensitivity in the determination of the amplification of other genes, including MYC, fibroblast growth factor receptor 1 and GATA binding protein 3 in tumors with low cellularity compared with that in tumors with high cellularity. OncoScan™ exhibited low sensitivity without tumor cell enrichment. The results suggested that MPT could be a promising method to determine HER2 status in breast tumors and that it could exhibit improved accuracy compared with that in OncoScan™ in tumors with low cellularity.

Circulating tumor cells (CTCs) are a rare population of cells found in the bloodstream and represent key players in the metastatic cascade. Their analysis has proved to provide further core information concerning the tumor. Herein, we aim at investigating CTCs isolated from a 32-year-old patient diagnosed with triple negative spindle-shaped metaplastic breast cancer (MpBC), a rare tumor poorly responsive to therapies and with a dismal prognosis. The molecular analysis performed on the primary tumor failed to underline effective actionable targets to address the therapeutic strategy. Besides the presence of round-shaped CTCs, cells with a spindle shape were present as well, and through molecular analysis, we confirmed their malignant nature. This aspect was coherent with the primary tumor histology, proving that CTCs are released regardless of their morphology. Copy number aberration (CNA) profiling and variant analysis using next-generation sequencing (NGS) showed that these cells did not harbor the alterations exhibited by the primary tumor (PIK3CA G1049A mutation, MYC copy number gain). However, despite the great heterogeneity observed, the amplification of regions involved in metastasis emerged (8q24.22-8q24.23). Our findings support the investigation of CTCs to identify alterations that could have a role in the metastatic process. To the best of our knowledge, this is the first examination of CTCs in an MpBC patient.

Copy number gain (CNG) and/or protein overexpression of ErbB2 have been observed in human breast cancer patients and are associated with poor prognosis. Similarly, ErbB2 overexpression has also been observed in canine mammary carcinoma; however, data on ErbB2 copy number is limited. The purposes of this study were to evaluate ErbB2 copy number in dogs with mammary carcinoma and to investigate associations of ErbB2 CNG with ErbB2 expression, histological and clinical characteristics, and survival. DNA samples were isolated from 59 formalin-fixed paraffin-embedded canine mammary gland tissues (34 carcinoma, 14 adenoma, and 11 normal). Using a digital PCR assay, the ErbB2 copy number in these samples was determined as compared to a reference gene on canine chromosome 8. ErbB2 CNG was detected in 14/34 (41%) carcinomas and 2/14 (14%) adenomas. ErbB2 overexpression was observed in 3/34 (9%) carcinomas but not in adenomas. Neither ErbB2 CNG nor ErbB2 overexpression were detected in the normal controls. There was no significant association of the ErbB2 CNG with histological and clinical characteristics such as age, neutered status, histological grade, tumor size, lymph node involvement, distant metastasis, and clinical stage in the dogs with mammary carcinoma. The presence of ErbB2 CNG, but not ErbB2 overexpression, was significantly related to the shorter overall survival. These findings suggest that ErbB2 CNG is a prognostic factor in dogs with mammary carcinoma.