ABSTRACT
Single cell analysis techniques have great potential in the cancer genomics field. The detection and characterization of circulating tumour cells are important for identifying metastatic disease at an early stage and monitoring it. This protocol is based on transcript profiling using Reverse Transcriptase Multiplex Ligation-dependent Probe Amplification (RT-MLPA), which is a specific method for simultaneous detection of multiple mRNA transcripts. Because of the small amount of (circulating) tumour cells, a pre-amplification reaction is performed after reverse transcription to generate a sufficient number of target molecules for the MLPA reaction. We designed a highly sensitive method for detecting and quantifying a panel of seven genes whose expression patterns are associated with breast cancer, and optimized the method for single cell analysis. For detection we used a fluorescence-dependent semi-quantitative method involving hybridization of unique barcodes to an array. We evaluated the method using three human breast cancer cell lines and identified specific gene expression profiles for each line. Furthermore, we applied the method to single cells and confirmed the heterogeneity of a cell population. Successful gene detection from cancer cells in human blood from metastatic breast cancer patients supports the use of RT-MLPA as a diagnostic tool for cancer genomics.
Subject(s)
Multiplex Polymerase Chain Reaction/methods , Neoplasms/diagnosis , Neoplasms/genetics , Single-Cell Analysis/methods , Case-Control Studies , Cell Line, Tumor , Gene Expression Profiling , Humans , Multiplex Polymerase Chain Reaction/standards , Neoplastic Cells, Circulating , Reproducibility of Results , Sensitivity and Specificity , Sequence Analysis, RNAABSTRACT
The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.
Subject(s)
Ataxia Telangiectasia Mutated Proteins/genetics , Gene Expression Regulation, Leukemic , Multiplex Polymerase Chain Reaction/methods , Mutation , Reverse Transcriptase Polymerase Chain Reaction/methods , Tumor Suppressor Protein p53/genetics , Antineoplastic Agents/pharmacology , Ataxia Telangiectasia Mutated Proteins/metabolism , Biological Assay , DNA Damage , Doxorubicin/pharmacology , Drug Resistance, Neoplasm/genetics , Epigenesis, Genetic , Gamma Rays , Humans , Leukemia, Lymphocytic, Chronic, B-Cell/drug therapy , Leukemia, Lymphocytic, Chronic, B-Cell/genetics , Leukemia, Lymphocytic, Chronic, B-Cell/metabolism , Leukemia, Lymphocytic, Chronic, B-Cell/pathology , RNA, Neoplasm/genetics , Sensitivity and Specificity , Tumor Suppressor Protein p53/metabolism , Vidarabine/analogs & derivatives , Vidarabine/pharmacologyABSTRACT
BACKGROUND: We have previously reported an array comparative genomic hybridization profile that identifies triple-negative breast cancers (TNBC), with BRCA1 dysfunction and a high sensitivity to intensified dose bifunctional alkylating agents. To determine the effect of conventional-dose chemotherapy in patients with this so-called BRCA1-like profile, clinical characteristics and survival were studied in a large group of TNBC patients. PATIENTS AND METHODS: DNA was isolated and BRCA1-like status was assessed in 101 patients with early-stage TNBC receiving adjuvant cyclophosphamide-based chemotherapy. Clinical characteristics and survival were compared between BRCA1-like and non-BRCA1-like groups. Results Sixty-six tumors (65%) had a BRCA1-like profile. Patients with BRCA1-like tumors tended to be younger and had more often node-negative disease (P = 0.06 and P = 0.03, respectively). Five-year recurrence-free survival was 80% for the BRCA1-like group and 75% for the non-BRCA1-like group (P = 0.35). T stage was the only variable significantly associated with survival. CONCLUSIONS: BRCA1-like tumors share clinical features, like young age at diagnosis and similar nodal status, with breast cancers in BRCA1 mutation carriers. Their prognosis is similar to that of non-BRCA1-like tumors when conventional-dose chemotherapy is administered. TNBCs that are classified as BRCA1-like may contain a defect in homologous recombination and could, in theory, benefit from the addition of poly ADP ribose polymerase inhibitors.
Subject(s)
Antineoplastic Combined Chemotherapy Protocols/therapeutic use , BRCA1 Protein/genetics , Breast Neoplasms/metabolism , Neoplasm Recurrence, Local/prevention & control , Adult , Aged , Breast Neoplasms/drug therapy , Breast Neoplasms/mortality , Chemotherapy, Adjuvant , Cyclophosphamide/therapeutic use , Disease-Free Survival , Docetaxel , Epirubicin/therapeutic use , Female , Fluorouracil/therapeutic use , Follow-Up Studies , Humans , Kaplan-Meier Estimate , Methotrexate/therapeutic use , Middle Aged , Proportional Hazards Models , Receptor, ErbB-2/metabolism , Receptors, Estrogen/metabolism , Receptors, Progesterone/metabolism , Taxoids/administration & dosage , Treatment Outcome , Young AdultABSTRACT
BACKGROUND: Tumors with homologous recombination deficiency (HRD), such as BRCA1-associated breast cancers, are not able to reliably repair DNA double-strand breaks (DSBs) and are therefore highly sensitive to both DSB-inducing chemotherapy and poly (ADP-ribose) polymerase inhibitors. We have studied markers that may indicate the presence of HRD in HER2-negative breast cancers and related them to neoadjuvant chemotherapy response. PATIENTS AND METHODS: Array comparative genomic hybridization (aCGH), BRCA1 promoter methylation, BRCA1 messenger RNA (mRNA) expression and EMSY amplification were assessed in 163 HER2-negative pretreatment biopsies from patients scheduled for neoadjuvant chemotherapy. RESULTS: Features of BRCA1 dysfunction were frequent in triple-negative (TN) tumors: a BRCA1-like aCGH pattern, promoter methylation and reduced mRNA expression were observed in, respectively, 57%, 25% and 36% of the TN tumors. In ER+ tumors, a BRCA2-like aCGH pattern and the amplification of the BRCA2 inhibiting gene EMSY were frequently observed (43% and 13%, respectively) and this BRCA2-like profile was associated with a better response to neoadjuvant chemotherapy. CONCLUSIONS: Abnormalities associated with BRCA1 inactivation are present in about half of the TN breast cancers but were not predictive of chemotherapy response. In ER+/HER2- tumors, a BRCA2-like aCGH pattern was predictive of chemotherapy response. These findings should be confirmed in independent series.
