High‐throughput DNA analyses in cancers allow the detection of molecular abnormalities that can guide patients' treatment. The collection of a tumour DNA is mainly performed on a biopsy, an invasive ...procedure for the patients. Another less invasive method consists in using a fine needle. We showed that fine‐needle biopsies are as suitable as a classical biopsy for DNA analysis.
High‐throughput molecular profiling of solid tumours using core needle biopsies (CNB) allows the identification of actionable molecular alterations, with around 70% success rate. Although several studies have demonstrated the utility of small biopsy specimens for molecular testing, there remains debate as to the sensitivity of the less invasive fine‐needle aspiration (FNA) compared to CNB to detect molecular alterations. We aimed to prospectively evaluate the potential of FNA to detect such alterations in various tumour types as compared to CNB in cancer patients included in the SHIVA02 trial. An in‐house amplicon‐based targeted sequencing panel (Illumina TSCA 99.3 kb panel covering 87 genes) was used to identify pathogenic variants and gene copy number variations (CNV) in concomitant CNB and FNA samples obtained from 61 patients enrolled in the SHIVA02 trial (NCT03084757). The main tumour types analysed were breast (38%), colon (15%), pancreas (11%), followed by cervix and stomach (7% each). We report 123 molecular alterations (85 variants, 23 amplifications and 15 homozygous deletions) among which 98 (80%) were concordant between CNB and FNA. The remaining discordances were mainly related to deletions status, yet undetected alterations were not exclusively specific to FNA. Comparative analysis of molecular alterations in CNB and FNA showed high concordance in terms of variants as well as CNVs identified. We conclude FNA could therefore be used in routine diagnostics workflow and clinical trials for tumour molecular profiling with the advantages of being minimally invasive and preserve tissue material needed for diagnostic, prognostic or theranostic purposes.
The triple-negative breast cancer (TN BC) subtype is the most aggressive form of invasive BC. Despite intensive efforts to improve BC treatments, patients with TN BC continue to exhibit poor ...survival, with half developing resistance to chemotherapy. Here we identify autophagy as a key mechanism in the progression and chemoresistance of a subset of TN tumors. We demonstrate that LC3B, a protein involved in autophagosome formation, is a reliable marker of poor prognosis in TN BC, validating this prognostic value at both the mRNA and protein levels in several independent cohorts. We also show that LC3B has no prognostic value for other BC subtypes (Luminal or HER2 BC), thus revealing a specific impact of autophagy on TN tumors. Autophagy is essential for the proliferative and invasive properties in 3D of TN BC cells characterized by high LC3B levels. Interestingly, the activity of the transcriptional co-activator YAP1 (Yes-associated protein 1) is regulated by the autophagy process and we identify YAP1 as a new actor in the autophagy-dependent proliferative and invasive properties of high-LC3B TN BC. Finally, inhibiting autophagy by silencing ATG5 or ATG7 significantly impaired high-LC3B TN tumor growth in vivo. Moreover, using a patient-derived TN tumor transplanted into mice, we show that an autophagy inhibitor, chloroquine, potentiates the effects of chemotherapeutic agents. Overall, our data identify LC3B as a new prognostic marker for TN BC and the inhibition of autophagy as a promising therapeutic strategy for TN BC patients.
A significant proportion of patients with oestrogen receptor (ER) positive breast cancers (BC) develop resistance to endocrine treatments (ET) and relapse with metastatic disease. Here we perform ...whole exome sequencing and gene expression analysis of matched primary breast tumours and bone metastasis-derived patient-derived xenografts (PDX). Transcriptomic analyses reveal enrichment of the G2/M checkpoint and up-regulation of Polo-like kinase 1 (PLK1) in PDX. PLK1 inhibition results in tumour shrinkage in highly proliferating CCND1-driven PDX, including different RB-positive PDX with acquired palbociclib resistance. Mechanistic studies in endocrine resistant cell lines, suggest an ER-independent function of PLK1 in regulating cell proliferation. Finally, in two independent clinical cohorts of ER positive BC, we find a strong association between high expression of PLK1 and a shorter metastases-free survival and poor response to anastrozole. In conclusion, our findings support clinical development of PLK1 inhibitors in patients with advanced CCND1-driven BC, including patients progressing on palbociclib treatment.
Women diagnosed with high-grade serous ovarian cancers (HGSOC) are still likely to exhibit a bad prognosis, particularly when suffering from HGSOC of the Mesenchymal molecular subtype (50% cases). ...These tumors show a desmoplastic reaction with accumulation of extracellular matrix proteins and high content of cancer-associated fibroblasts. Using patient-derived xenograft mouse models of Mesenchymal and Non-Mesenchymal HGSOC, we show here that HGSOC exhibit distinct stiffness depending on their molecular subtype. Indeed, tumor stiffness strongly correlates with tumor growth in Mesenchymal HGSOC, while Non-Mesenchymal tumors remain soft. Moreover, we observe that tumor stiffening is associated with high stromal content, collagen network remodeling, and MAPK/MEK pathway activation. Furthermore, tumor stiffness accompanies a glycolytic metabolic switch in the epithelial compartment, as expected based on Warburg's effect, but also in stromal cells. This effect is restricted to the central part of stiff Mesenchymal tumors. Indeed, stiff Mesenchymal tumors remain softer at the periphery than at the core, with stromal cells secreting high levels of collagens and showing an OXPHOS metabolism. Thus, our study suggests that tumor stiffness could be at the crossroad of three major processes, i.e. matrix remodeling, MEK activation and stromal metabolic switch that might explain at least in part Mesenchymal HGSOC aggressiveness.
Abstract
Background
Early luminal breast cancer (BC) represents 70% of newly diagnosed BC cases. Among them, small (under 2 cm) BC without lymph node metastasis (classified as T1N0) have been rarely ...studied, as their prognosis is generally favorable. Nevertheless, up to 5% of luminal T1N0 BC patients relapse with distant metastases that ultimately prove fatal. The aim of our work was to identify the mechanisms involved in metastatic recurrence in these patients.
Methods
Our study addresses the role that autonomous and non-autonomous tumor cell features play with regard to distant recurrence in early luminal BC patients. We created a cohort of T1N0 luminal BC patients (tumors between 0.5–2 cm without lymph node metastasis) with metastatic recurrence (“cases”) and corresponding “controls” (without relapse) matched 1:1 on main prognostic factors: age, grade, and proliferation. We deciphered different characteristics of cancer cells and their tumor micro-environment (TME) by deep analyses using immunohistochemistry. We performed in vitro functional assays and highlighted a new mechanism of cooperation between cancer cells and one particular subset of cancer-associated fibroblasts (CAF).
Results
We found that specific TME features are indicative of relapse in early luminal BC. Indeed, quantitative histological analyses reveal that “cases” are characterized by significant accumulation of a particular CAF subset (CAF-S1) and decrease in CD4
+
T lymphocytes, without any other association with immune cells. In multivariate analysis, TME features, in particular CAF-S1 enrichment, remain significantly associated with recurrence, thereby demonstrating their clinical relevance. Finally, by performing functional analyses, we demonstrated that CAF-S1 pro-metastatic activity is mediated by the CDH11/osteoblast cadherin, consistent with bones being a major site of metastases in luminal BC patients.
Conclusions
This study shows that distant recurrence in T1N0 BC is strongly associated with the presence of CAF-S1 fibroblasts. Moreover, we identify CDH11 as a key player in CAF-S1-mediated pro-metastatic activity. This is independent of tumor cells and represents a new prognostic factor. These results could assist clinicians in identifying luminal BC patients with high risk of relapse. Targeted therapies against CAF-S1 using anti-FAP antibody or CDH11-targeting compounds might help in preventing relapse for such patients with activated stroma.
Invasion of cancer cells into collagen-rich extracellular matrix requires membrane-tethered membrane type 1-matrix metalloproteinase (MT1-MMP) as the key protease for collagen breakdown. ...Understanding how MT1-MMP is delivered to the surface of tumor cells is essential for cancer cell biology. In this study, we identify ARF6 together with c-Jun NH2-terminal kinase-interacting protein 3 and 4 (JIP3 and JIP4) effectors as critical regulators of this process. Silencing ARF6 or JIP3/JIP4 in breast tumor cells results in MT1-MMP endosome mispositioning and reduces MT1-MMP exocytosis and tumor cell invasion. JIPs are recruited by Wiskott-Aldrich syndrome protein and scar homologue (WASH) on MT1-MMP endosomes on which they recruit dynein-dynactin and kinesin-1. The interaction of plasma membrane ARF6 with endosomal JIPs coordinates dynactin-dynein and kinesin-1 activity in a tug-of-war mechanism, leading to MT1-MMP endosome tubulation and exocytosis. In addition, we find that ARF6, MT1-MMP, and kinesin-1 are up-regulated in high-grade triple-negative breast cancers. These data identify a critical ARF6-JIP-MT1-MMP-dynein-dynactin-kinesin-1 axis promoting an invasive phenotype of breast cancer cells.
HER2-positive breast cancer has long proven to be a clinically distinct class of breast cancers for which several targeted therapies are now available. However, resistance to the treatment associated ...with specific gene expressions or mutations has been observed, revealing the underlying diversity of these cancers. Therefore, understanding the full extent of the HER2-positive disease heterogeneity still remains challenging. Here we carry out an in-depth genomic characterization of 64 HER2-positive breast tumour genomes that exhibit four subgroups, based on the expression data, with distinctive genomic features in terms of somatic mutations, copy-number changes or structural variations. The results suggest that, despite being clinically defined by a specific gene amplification, HER2-positive tumours melt into the whole luminal-basal breast cancer spectrum rather than standing apart. The results also lead to a refined ERBB2 amplicon of 106 kb and show that several cases of amplifications are compatible with a breakage-fusion-bridge mechanism.
Metaplastic breast carcinoma (MBC) is a rare and aggressive histologic type of breast cancer, predominantly of triple-negative phenotype, and characterized by the presence of malignant cells showing ...squamous and/or mesenchymal differentiation. We sought to define the repertoire of somatic genetic alterations and the mutational signatures of MBCs.
Whole-exome sequencing was performed in 35 MBCs, with 16, 10, and 9 classified as harboring chondroid, spindle, and squamous metaplasia as the predominant metaplastic component. The genomic landscape of MBCs was compared with that of triple-negative invasive ductal carcinomas of no special type (IDC-NST) from The Cancer Genome Atlas. Wnt and PI3K/AKT/mTOR pathway activity was assessed using a qPCR assay.
MBCs harbored complex genomes with frequent
(69%) mutations. In contrast to triple-negative IDC-NSTs, MBCs more frequently harbored mutations in
(29%),
(11%),
(11%),
(11%), and
(11%).
mutations were not found in MBCs with chondroid metaplasia. Compared with triple-negative IDC-NSTs, MBCs significantly more frequently harbored mutations in PI3K/AKT/mTOR pathway-related (57% vs. 22%) and canonical Wnt pathway-related (51% vs. 28%) genes. MBCs with somatic mutations in PI3K/AKT/mTOR or Wnt pathway-related genes displayed increased activity of the respective pathway.
MBCs are genetically complex and heterogeneous, and are driven by a repertoire of somatic mutations distinct from that of triple-negative IDC-NSTs. Our study highlights the genetic basis and the importance of PI3K/AKT/mTOR and Wnt pathway dysregulation in MBCs and provides a rationale for the metaplastic phenotype and the reported responses to PI3K/AKT/mTOR inhibitors in these tumors.
.
Human papillomavirus (HPV) is found in 90% of squamous cell carcinomas of the anal canal (SCCA). We investigated the clinical validity of HPV circulating tumor DNA (ctDNA) detection in patients ...enrolled in the Epitopes-HPV02 trial that demonstrated the efficacy of docetaxel, cisplatin, and 5-FU as first-line chemotherapy in advanced SCCA.
According to the protocol, serum samples were collected before chemotherapy and on completion of chemotherapy. HPV16 ctDNA was quantified by droplet digital PCR (ddPCR) and correlated with prospectively registered patient characteristics and outcomes. A landmark was set at the time of chemotherapy completion for postchemotherapy progression-free survival (PFS) analyses.
Among 57 patients with HPV16-related advanced SCCA, HPV ctDNA was detected in 91.1% (95% confidence interval, 81.1-96.2) of baseline samples. Baseline HPV ctDNA levels were not associated with any patient characteristics; baseline ctDNA level below the cutoff obtained by AUC (area under the curve) was associated with a longer PFS (HR = 2.1;
= 0.04). Among the 36 patients who completed 5 months of chemotherapy, residual HPV ctDNA was detected after chemotherapy in 38.9% of patients. Residual HPV ctDNA detected at chemotherapy completion was associated with shorter postchemotherapy PFS (median PFS 3.4 months vs. not reached; HR = 5.5;
< 0.001) and a reduction of 1-year overall survival rate (OR = 7.0;
= 0.02).
This prospective study in advanced SCCA demonstrated a significant prognostic impact of HPV ctDNA level before first-line chemotherapy and HPV ctDNA negativity after chemotherapy completion. With a limited cost and short turnaround, this assay is a promising tool to optimize the therapeutic management of SCCA.
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