Detection of circulating tumour DNA after neoadjuvant chemoradiotherapy in patients with locally advanced oesophageal cancer.

Active surveillance instead of standard surgery after neoadjuvant chemoradiotherapy (nCRT) has been proposed for patients with oesophageal cancer. Circulating tumour DNA (ctDNA) may be used to facilitate selection of patients for surgery. We show that detection of ctDNA after nCRT seems highly suggestive of major residual disease. Tumour biopsies and blood samples were taken before, and 6 and 12 weeks after, nCRT. Biopsies were analysed with regular targeted next-generation sequencing (NGS). Circulating cell-free DNA (cfDNA) was analysed using targeted NGS with unique molecular identifiers and digital polymerase chain reaction. cfDNA mutations matching pre-treatment biopsy mutations confirmed the presence of ctDNA. In total, 31 patients were included, of whom 24 had a biopsy mutation that was potentially detectable in cfDNA (77%). Pre-treatment ctDNA was detected in nine of 24 patients (38%), four of whom had incurable disease progression before surgery. Pre-treatment ctDNA detection had a sensitivity of 47% (95% CI 24-71) (8/17), specificity of 85% (95% CI 42-99) (6/7), positive predictive value (PPV) of 89% (95% CI 51-99) (8/9), and negative predictive value (NPV) of 40% (95% CI 17-67) (6/15) for detecting major residual disease (>10% residue in the resection specimen or progression before surgery). After nCRT, ctDNA was detected in three patients, two of whom had disease progression. Post-nCRT ctDNA detection had a sensitivity of 21% (95% CI 6-51) (3/14), specificity of 100% (95% CI 56-100) (7/7), PPV of 100% (95% CI 31-100) (3/3), and NPV of 39% (95% CI 18-64) (7/18) for detecting major residual disease. The addition of ctDNA to the current set of diagnostics did not lead to more patients being clinically identified with residual disease. These results indicate that pre-treatment and post-nCRT ctDNA detection may be useful in identifying patients at high risk of disease progression. The addition of ctDNA analysis to the current set of diagnostic modalities may not improve detection of residual disease after nCRT. © 2022 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Characterization of the mechanism by which the RB/E2F pathway controls expression of the cancer genomic DNA deaminase APOBEC3B.

APOBEC3B (A3B)-catalyzed DNA cytosine deamination contributes to the overall mutational landscape in breast cancer. Molecular mechanisms responsible for A3B upregulation in cancer are poorly understood. Here we show that a single E2F cis-element mediates repression in normal cells and that expression is activated by its mutational disruption in a reporter construct or the endogenous A3B gene. The same E2F site is required for A3B induction by polyomavirus T antigen indicating a shared molecular mechanism. Proteomic and biochemical experiments demonstrate the binding of wildtype but not mutant E2F promoters by repressive PRC1.6/E2F6 and DREAM/E2F4 complexes. Knockdown and overexpression studies confirm the involvement of these repressive complexes in regulating A3B expression. Altogether, these studies demonstrate that A3B expression is suppressed in normal cells by repressive E2F complexes and that viral or mutational disruption of this regulatory network triggers overexpression in breast cancer and provides fuel for tumor evolution.

Expression of hypoxia-induced proteins in ductal carcinoma in situ and invasive cancer of the male breast.

AIMS: The aim of this study was to determine the role of hypoxia in male breast carcinogenesis by evaluating the expression of the hypoxia-related proteins, hypoxia-inducible factor-1α (HIF-1α), carbonic anhydrase IX (CAIX) and glucose transporter-1 (Glut-1), in ductal carcinoma in situ (DCIS) of the male breast in relation to invasive cancer (IC). METHODS: Tumour tissue blocks of 18 cases of pure DCIS, 58 DCIS cases adjacent to IC (DCIS-AIC) and the 58 IC cases were stained by immunohistochemistry for HIF-1α, CAIX and Glut-1, and expression frequencies and patterns (diffuse and/or perinecrotic) were noted. RESULTS: HIF-1α overexpression was observed in 61.1% (11/18) of pure DCIS, in 37.9% (22/58) of DCIS-AIC and in 36.2% (21/58) of IC cases (not significant (n.s.)). CAIX overexpression was observed in 16.7% (3/18) of pure DCIS, in 37.9% (22/58) of DCIS-AIC and in 24.1% (14/58) of IC cases (n.s.). Glut-1 overexpression was observed in 61.1% (11/18) of pure DCIS, in 75.9% (44/58) of DCIS-AIC and in 62.1% (36/58) of IC cases (n.s.). Expression of hypoxia-related proteins was seen around necrosis in a little over one-third of DCIS cases, and often coincided with expression in adjacent IC when present. All these observations indicate that the hypoxia response is already at its maximum in the preinvasive DCIS stage. CONCLUSIONS: In conclusion, male DCIS frequently shows activated hypoxia response, comparable to male IC. This indicates that the activated hypoxia response previously seen in male IC is not a late bystander but likely a genuine carcinogenetic event.

Male breast cancer precursor lesions: analysis of the EORTC 10085/TBCRC/BIG/NABCG International Male Breast Cancer Program.

In men, data regarding breast cancer carcinogenesis are limited. The aim of our study was to describe the presence of precursor lesions adjacent to invasive male breast cancer, in order to increase our understanding of carcinogenesis in these patients. Central pathology review was performed for 1328 male breast cancer patients, registered in the retrospective joint analysis of the International Male Breast Cancer Program, which included the presence and type of breast cancer precursor lesions. In a subset, invasive breast cancer was compared with the adjacent precursor lesion by immunohistochemistry (n=83) or targeted next generation sequencing (n=7). Additionally, we correlated the presence of ductal carcinoma in situ with outcome. A substantial proportion (46.2%) of patients with invasive breast cancer also had an adjacent precursor lesion, mainly ductal carcinoma in situ (97.9%). The presence of lobular carcinoma in situ and columnar cell-like lesions were very low (<1%). In the subset of invasive breast cancer cases with adjacent ductal carcinoma in situ (n=83), a complete concordance was observed between the estrogen receptor, progesterone receptor, and HER2 status of both components. Next generation sequencing on a subset of cases with invasive breast cancer and adjacent ductal carcinoma in situ (n=4) showed identical genomic aberrations, including PIK3CA, GATA3, TP53, and MAP2K4 mutations. Next generation sequencing on a subset of cases with invasive breast cancer and an adjacent columnar cell-like lesion showed genomic concordance in two out of three patients. A multivariate Cox model for survival showed a trend that the presence of ductal carcinoma in situ was associated with a better overall survival, in particular in the Luminal B HER2+ subgroup. In conclusion, ductal carcinoma in situ is the most commonly observed precursor lesion in male breast cancer and its presence seems to be associated with a better outcome, in particular in Luminal B HER2+ cases. The rate of lobular carcinoma in situ and columnar cell-like lesions adjacent to male breast cancer is very low, but our findings support the role of columnar cell-like lesions as a precursor of male breast cancer.

Cell-free DNA mutations as biomarkers in breast cancer patients receiving tamoxifen.

The aim was to identify mutations in serum cell-free DNA (cfDNA) associated with disease progression on tamoxifen treatment in metastatic breast cancer (MBC). Sera available at start of therapy, during therapy and at disease progression were selected from 10 estrogen receptor (ER)-positive breast cancer patients. DNA from primary tumor and normal tissue and cfDNA from minute amounts of sera were analyzed by targeted next generation sequencing (NGS) of 45 genes (1,242 exons). At disease progression, stop-gain single nucleotide variants (SNVs) for CREBBP (1 patient) and SMAD4 (1 patient) and non-synonymous SNVs for AKAP9 (1 patient), PIK3CA (2 patients) and TP53 (2 patients) were found. Mutations in CREBBP and SMAD4 have only been occasionally reported in breast cancer. All mutations, except for AKAP9, were also present in the primary tumor but not detected in all blood specimens preceding progression. More sensitive detection by deeper re-sequencing and digital PCR confirmed the occurrence of circulating tumor DNA (ctDNA) and these biomarkers in blood specimens.

SIAH2 protein expression in breast cancer is inversely related with ER status and outcome to tamoxifen therapy.

Our previous study demonstrated that high mRNA levels for Seven in Absentia Homolog 2 (SIAH2) correlated with high Estrogen Receptor (ER) mRNA levels and with longer progression-free survival (PFS) after first-line tamoxifen. Others showed high SIAH2 protein levels in ER-negative breast cancer associated with an unfavorable relapse-free survival. In the current study, we investigated SIAH2 protein expression to clarify the discrepancy between protein and mRNA findings and to determine its diagnostic value in breast cancer patients. Tissue microarrays (TMAs) containing core specimens of primary breast tumors were immunohistochemically stained for SIAH2 protein. The TMAs analyzed a cohort of 746 patients with primary breast cancer (PBC) and a cohort of 245 patients with ER-positive metastatic breast cancer (MBC) treated with first-line tamoxifen. SIAH2 staining was scored for intensity and proportion of positive tumor cells and evaluated for its relationship with metastasis-free survival (MFS) and PFS. Multivariate survival analyses included traditional prognostic or predictive factors, respectively. The PBC-cohort had 263 patients with high SIAH2 protein expression and decreased expression of ER protein and mRNA levels (P = 0.005 and P = 0.003, respectively). High SIAH2 levels correlated with significant unfavorable MFS in lymph node negative, ER-positive breast cancer patients. The MBC-cohort had 86 patients with increased SIAH2 protein expression. High SIAH2 expression was associated with an unfavorable PFS after first-line tamoxifen in multivariate analyses (HR = 1.45; 95% CI, 1.07-1.96; P = 0.015). In conclusion, SIAH2 protein expression is especially observed in ER-negative tumors. Its prognostic value in breast cancer does not add to current prognostic markers. The proportion of SIAH2-positive cells can be used as biomarker to predict tamoxifen treatment failure in MBC patients.

A 3'-untranslated region KRAS variant and triple-negative breast cancer: a case-control and genetic analysis.

BACKGROUND: We previously identified a functional variant in a let-7 microRNA (miRNA) complementary site in the 3'-untranslated region of the KRAS oncogene (rs61764370) which is associated with cancer. We aimed to investigate the association of this KRAS variant with breast cancer and tumour biology. METHODS: We assessed frequency distributions of the KRAS variant in 415 patients with histologically confirmed breast cancer and 457 controls from Connecticut, USA (study group 1) and association of this variant with breast-cancer subtypes in 690 Irish women with known oestrogen receptor (ER), progesterone receptor (PR), and HER2 statuses, and 360 controls (study group 2). We pooled data for study groups 1 and 2 with a cohort of 140 women with triple-negative breast cancer and 113 controls to assess the association of the KRAS variant with triple-negative breast cancer risk, and genome-wide mRNA and specific miRNA expression in patients with triple-negative breast cancer. FINDINGS: Although frequency distributions of the KRAS variant in study group 1 did not differ between all genotyped individuals, eight (33%) of 24 premenopausal women with ER/PR-negative cancer had the KRAS variant, compared with 27 (13%) of 201 premenopausal controls (p=0.015). In study group 2, the KRAS variant was significantly enriched in women with triple-negative breast cancer (19 [21%] of 90 cases) compared with 64 (13%) of 478 for luminal A, 13 (15%) of 87 for luminal B, and two (6%) of 35 for HER2-positive subgroups (p=0.044). Multivariate analysis in the pooled study groups showed that the KRAS variant was associated with triple-negative breast cancer in premenopausal women (odds ratio 2.307, 95% CI 1.261-4.219, p=0.0067). Gene-expression analysis of triple-negative breast-cancer tumours suggested that KRAS-variant positive tumours have significantly altered gene expression, and are enriched for the luminal progenitor and BRCA1 deficiency signatures. miRNA analysis suggested reduced levels of let-7 miRNA species in KRAS-variant tumours. INTERPRETATION: The KRAS variant might be a genetic marker for development of triple-negative breast cancer in premenopausal women, and altered gene and miRNA expression signatures should enable molecular and biological stratification of patients with this subgroup of breast cancer. FUNDING: US National Institutes of Health.

Clinical significance of the nuclear receptor co-regulator DC-SCRIPT in breast cancer: an independent retrospective validation study.

INTRODUCTION: In this study we aimed to validate the prognostic value of DC-SCRIPT mRNA expression in a large independent breast cancer cohort. In addition, since DC-SCRIPT is a transcriptional co-regulator of nuclear receptors, we explored its prognostic value in relation to estrogen-receptor-α (ESR1) and -ß (ESR2) and evaluated its predictive value for response to tamoxifen treatment. METHODS: DC-SCRIPT mRNA levels were measured by real-time PCR in 1,505 primary invasive breast cancers and associated with outcome (disease-free survival (DFS), metastasis-free survival (MFS) and overall survival (OS)) using univariate and multivariable Cox regression analysis. Logistic and Cox regressions were used to associate DC-SCRIPT levels with clinical benefit and progression-free survival (PFS) for 296 patients treated with first-line systemic tamoxifen for advanced disease. RESULTS: In univariate and multivariable analysis higher DC-SCRIPT levels were associated with a favorable outcome for both the entire cohort and patients with lymph node-negative (LNN) disease that did not receive adjuvant therapy (DFS, MFS and OS; all, P < 0.001). This association was most pronounced in small (pT1) tumors, in ESR1-positive tumors and in tumors with low ESR2 expression. For first-line endocrine therapy for advanced disease no predictive association was seen with clinical benefit or PFS. CONCLUSIONS: This study provides a higher level of evidence that DC-SCRIPT is indeed an independent, pure prognostic, factor for primary breast cancer and shows that DC-SCRIPT mRNA expression is most informative for either ESR1-positive and/or ESR2-low pT1 tumors.

DNA methylation as a biomarker in breast cancer.

In cancer, epigenetic changes such as covalent addition of methyl groups to the genomic DNA itself are more prominent than genetic changes. Cytosine-phosphate-guanosine (CpG) methylation negatively affects gene transcription of an adjacent gene. It is thought that DNA methylation significantly contributes to all hallmarks of cancer. Next to being a potential therapy target, DNA methylation is an emerging field of biomarkers. Technically, DNA provides a stable and robust analyte that is particularly suitable for clinical applications. Moreover, there are numerous potential human DNA sources that could facilitate integration of methylation tests in clinical practice. In breast cancer, DNA methylation has shown promise as a potential biomarker for early detection, therapy monitoring, assessment of prognosis or prediction of therapy response. In particular, paired-like homeodomain transcription factor 2 (PITX2) DNA methylation has been validated using a robust assay for paraffin-embedded tissue for clinically relevant outcome prediction in early breast cancer patients treated by adjuvant tamoxifen therapy.