Comparison of tumor-informed and tumor-naïve sequencing assays for ctDNA detection in breast cancer.

Analysis of circulating tumor DNA (ctDNA) to monitor cancer dynamics and detect minimal residual disease has been an area of increasing interest. Multiple methods have been proposed but few studies have compared the performance of different approaches. Here, we compare detection of ctDNA in serial plasma samples from patients with breast cancer using different tumor-informed and tumor-naïve assays designed to detect structural variants (SVs), single nucleotide variants (SNVs), and/or somatic copy-number aberrations, by multiplex PCR, hybrid capture, and different depths of whole-genome sequencing. Our results demonstrate that the ctDNA dynamics and allele fractions (AFs) were highly concordant when analyzing the same patient samples using different assays. Tumor-informed assays showed the highest sensitivity for detection of ctDNA at low concentrations. Hybrid capture sequencing targeting between 1,347 and 7,491 tumor-identified mutations at high depth was the most sensitive assay, detecting ctDNA down to an AF of 0.00024% (2.4 parts per million, ppm). Multiplex PCR targeting 21-47 tumor-identified SVs per patient detected ctDNA down to 0.00047% AF (4.7 ppm) and has potential as a clinical assay.

Role of germline variants in the metastasis of breast carcinomas.

Most cancer-related deaths in breast cancer patients are associated with metastasis, a multistep, intricate process that requires the cooperation of tumour cells, tumour microenvironment and metastasis target tissues. It is accepted that metastasis does not depend on the tumour characteristics but the host's genetic makeup. However, there has been limited success in determining the germline genetic variants that influence metastasis development, mainly because of the limitations of traditional genome-wide association studies to detect the relevant genetic polymorphisms underlying complex phenotypes. In this work, we leveraged the extreme discordant phenotypes approach and the epistasis networks to analyse the genotypes of 97 breast cancer patients. We found that the host's genetic makeup facilitates metastases by the dysregulation of gene expression that can promote the dispersion of metastatic seeds and help establish the metastatic niche-providing a congenial soil for the metastatic seeds.

ctDNA monitoring using patient-specific sequencing and integration of variant reads.

Circulating tumor-derived DNA (ctDNA) can be used to monitor cancer dynamics noninvasively. Detection of ctDNA can be challenging in patients with low-volume or residual disease, where plasma contains very few tumor-derived DNA fragments. We show that sensitivity for ctDNA detection in plasma can be improved by analyzing hundreds to thousands of mutations that are first identified by tumor genotyping. We describe the INtegration of VAriant Reads (INVAR) pipeline, which combines custom error-suppression methods and signal-enrichment approaches based on biological features of ctDNA. With this approach, the detection limit in each sample can be estimated independently based on the number of informative reads sequenced across multiple patient-specific loci. We applied INVAR to custom hybrid-capture sequencing data from 176 plasma samples from 105 patients with melanoma, lung, renal, glioma, and breast cancer across both early and advanced disease. By integrating signal across a median of >105 informative reads, ctDNA was routinely quantified to 1 mutant molecule per 100,000, and in some cases with high tumor mutation burden and/or plasma input material, to parts per million. This resulted in median area under the curve (AUC) values of 0.98 in advanced cancers and 0.80 in early-stage and challenging settings for ctDNA detection. We generalized this method to whole-exome and whole-genome sequencing, showing that INVAR may be applied without requiring personalized sequencing panels so long as a tumor mutation list is available. As tumor sequencing becomes increasingly performed, such methods for personalized cancer monitoring may enhance the sensitivity of cancer liquid biopsies.

Triple negative breast cancer subtypes and pathologic complete response rate to neoadjuvant chemotherapy.

Triple negative breast cancer (TNBC) is a heterogeneous disease with distinct molecular subtypes that differentially respond to chemotherapy and targeted agents. The purpose of this study is to explore the clinical relevance of Lehmann TNBC subtypes by identifying any differences in response to neoadjuvant chemotherapy among them. We determined Lehmann subtypes by gene expression profiling in paraffined pre-treatment tumor biopsies from 125 TNBC patients treated with neoadjuvant anthracyclines and/or taxanes +/- carboplatin. We explored the clinicopathological characteristics of Lehmann subtypes and their association with the pathologic complete response (pCR) to different treatments. The global pCR rate was 37%, and it was unevenly distributed within Lehmann's subtypes. Basal-like 1 (BL1) tumors exhibited the highest pCR to carboplatin containing regimens (80% vs 23%, p=0.027) and were the most proliferative (Ki-67>50% of 88.2% vs. 63.7%, p=0.02). Luminal-androgen receptor (LAR) patients achieved the lowest pCR to all treatments (14.3% vs 42.7%, p=0.045 when excluding mesenchymal stem-like (MSL) samples) and were the group with the lowest proliferation (Ki-67≤50% of 71% vs 27%, p=0.002). In our cohort, only tumors with LAR phenotype presented non-basal-like intrinsic subtypes (HER2-enriched and luminal A). TNBC patients present tumors with a high genetic diversity ranging from highly proliferative tumors, likely responsive to platinum-based therapies, to a subset of chemoresistant tumors with low proliferation and luminal characteristics.

Shallow whole genome sequencing for robust copy number profiling of formalin-fixed paraffin-embedded breast cancers.

Pathology archives with linked clinical data are an invaluable resource for translational research, with the limitation that most cancer samples are formalin-fixed paraffin-embedded (FFPE) tissues. Therefore, FFPE tissues are an important resource for genomic profiling studies but are under-utilised due to the low amount and quality of extracted nucleic acids. We profiled the copy number landscape of 356 breast cancer patients using DNA extracted FFPE tissues by shallow whole genome sequencing. We generated a total of 491 sequencing libraries from 2 kits and obtained data from 98.4% of libraries with 86.4% being of good quality. We generated libraries from as low as 3.8 ng of input DNA and found that the success was independent of input DNA amount and quality, processing site and age of the fixed tissues. Since copy number alterations (CNA) play a major role in breast cancer, it is imperative that we are able to use FFPE archives and we have shown in this study that sWGS is a robust method to do such profiling.

Male breast cancer: correlation between immunohistochemical subtyping and PAM50 intrinsic subtypes, and the subsequent clinical outcomes.

Male breast cancer is a rare disease that is still poorly understood. It is mainly classified by immunohistochemistry as a luminal disease. In this study, we assess for the first time the correlation between molecular subtypes based on a validated six-marker immunohistochemical panel and PAM50 signature in male breast cancer, and the subsequent clinical outcome of these different subtypes. We collected 67 surgical specimens of invasive male breast cancer from four different Spanish pathology laboratories. Immunohistochemical staining for the six-marker panel was performed on tissue microarrays. PAM50 subtypes were determined in a research-use-only nCounter Analysis System. We explored the association of immunohistochemical and PAM50 subtypes. Overall survival and disease-free survival were analyzed in the different subtypes of each classification. The distribution of tumor molecular subtypes according PAM50 was: 60% luminal B, 30% luminal A and 10% human epidermal growth factor receptor 2 (Her2) enriched. Only one Her2-enriched tumor was also positive by immunohistochemistry and was treated with trastuzumab. None of the tumors were basal-like. Using immunohistochemical surrogates, 51% of the tumors were luminal B, 44% luminal A, 4% triple-negative and 1% Her2-positive. The clinicopathological characteristics did not differ significantly between immunohistochemical and PAM50 subtypes. We found a significant worse overall survival in Her2-enriched compared with luminal tumors. Male breast cancer seems to be mainly a genomic luminal disease with a predominance of the luminal B subtype. In addition, we found a proportion of patients with Her2-negative by immunohistochemistry but Her2-enriched profile by PAM50 tumors with a worse outcome compared with luminal subtypes that may benefit from anti-Her2 therapies.

Proliferation Determined by Ki-67 Defines Different Pathologic Response to Neoadjuvant Trastuzumab-Based Chemotherapy in HER2-Positive Breast Cancer.

BACKGROUND: This study aimed to assess the role of proliferation measured by Ki-67 as a predictive factor for pathologic complete response (pCR) to trastuzumab-based chemotherapy in patients with human epidermal growth factor receptor 2 (HER2)-positive (HER2(+)) breast cancer (BC). METHODS: A total of 81 patients with HER2(+) BC were treated with a sequential schedule consisting of 4 cycles of cyclophosphamide (600 mg/m(2)) and doxorubicin (60 mg/m(2)) every 3 weeks, followed by 4 cycles of weekly paclitaxel (80 mg/m(2)) or docetaxel (100 mg/m(2)) every 3 weeks combined with trastuzumab (loading dose of 8 mg/kg and then 6 mg/kg every 3 weeks) as neoadjuvant treatment. Histologic subgroups classified by hormone receptor (HR) expression and Ki-67 index were 17% HR(+)/Ki-67 ≥ 50%, 41% HR(+)/Ki-67 < 50%, 25% HR-negative (HR(-)) Ki-67 ≥ 50%, and 17% HR(-)/Ki-67 < 50%. RESULTS: pCR, defined as the absence of invasive cells in the breast and axillary lymph node, was achieved in 33 patients (41%). The median Ki-67 expression was significantly higher in tumors with pCR (53%) compared with tumors without pCR (30%) (P < .001). Receiver operating characteristic (ROC) curve methodology suggested that 50% was the optimal Ki-67 cutoff point to best identify patients who achieved a pCR. The pCR rate was significantly different between histologic subgroups: HR(-)/Ki-67 ≥ 50% (70%), HR(+)/Ki-67 ≥ 50% (71%), HR(-)/Ki-67 < 50% (22%), and HR(+)/Ki-67 < 50% (18%) (P < .001). A multivariate analysis revealed that a Ki-67 marker ≥ 50% was the only independent predictive factor of pCR (P = .003; odds ratio [OR], 0.133; 95% confidence interval [CI], 0.036-0.5). The median follow-up was 32 months (range, 14-48 months). Patients who achieved a pCR had significantly lower recurrence (P = .001) and higher overall survival (OS) (P = .013) compared with those who did not. There were no statistically significant differences in disease-free survival (DFS) and OS in relation to HRs, the Ki-67 marker as a continuous or categorical variable, and histologic subgroups. CONCLUSION: Proliferation determined by Ki-67 expression ≥ 50% was an independent predictive factor for pCR in patients with HER2(+) BC treated with trastuzumab-based chemotherapy.

The seed and soil hypothesis revisited: current state of knowledge of inherited genes on prognosis in breast cancer.

The crucial event in the course of malignancies such as breast cancer is its metastatic spread from the primary tumor of origin to distant organs. The natural history of a tumor is determined by the expression of its genes, and in this sense, knowledge has advanced dramatically in recent decades. However, much less is known about the role that the patient plays in the behavior of a tumor. In this article, we review the evidence regarding the genetic background of the host in metastatic tumor dissemination, providing information from epidemiological studies as well as from animal models and human studies. Undoubtedly, the elucidation of possible interpersonal variability in susceptibility to developing metastases would significantly contribute to improve management of cancer patients.