In human cancer, early systemic spread of tumor cells is recognized as a leading cause of death. Adjuvant therapies are administered to patients after complete resectioning of their primary tumors to eradicate the few residual and latent metastatic cells. These therapeutic regimens, however, are currently designed without direct information about the presence or nature of the latent cells. To address this problem, we developed a PCR-based technique to analyze the transcriptome of individual tumor cells isolated from the bone marrow of cancer patients. From the same cells, genomic aberrations were identified by comparative genomic hybridization. The utility of this approach for understanding the biology of occult disseminated cells and for the identification of new therapeutic targets is demonstrated here by the detection of frequent extracellular matrix metalloproteinase inducer (EMMPRIN; CD147) expression which was verified by immunostaining.
Adult-onset urticaria pigmentosa/mastocytosis in the skin almost always persists throughout life. The prevalence of systemic mastocytosis in such patients is not precisely known. Bone marrow biopsies from 59 patients with mastocytosis in the skin and all available skin biopsies (n ¼ 27) were subjected to a meticulous cytological, histological, immunohistochemical, and molecular analysis for the presence of WHO-defined diagnostic criteria for systemic mastocytosis: compact mast cell infiltrates (major criterion); atypical mast cell morphology, KIT D816V, abnormal expression of CD25 by mast cells, and serum tryptase levels 420 ng/ml (minor criteria). Systemic mastocytosis is diagnosed when the major diagnostic criterion plus one minor criterion or at least three minor criteria are fulfilled. Systemic mastocytosis was confirmed in 57 patients (97%) by the diagnosis of compact mast cell infiltrates plus at least one minor diagnostic criterion (n ¼ 42, 71%) or at least three minor diagnostic criteria (n ¼ 15, 25%). In two patients, only two minor diagnostic criteria were detectable, insufficient for the diagnosis of systemic mastocytosis. By the use of highly sensitive molecular methods, including the analysis of microdissected mast cells, KIT D816V was found in all 58 bone marrow biopsies investigated for it but only in 74% (20/27) of the skin biopsies. It is important to state that even in cases with insufficient diagnostic criteria for systemic mastocytosis, KIT D816V-positive mast cells were detected in the bone marrow. This study demonstrates, for the first time, that almost all patients with adult-onset mastocytosis in the skin, in fact, have systemic mastocytosis with cutaneous involvement.
Only few selected cancer cells drive tumor progression and are responsible for therapy resistance. Their specific genomic characteristics, however, are largely unknown because high-resolution genome analysis is currently limited to DNA pooled from many cells. Here, we describe a protocol for array comparative genomic hybridization (array CGH), which enables the detection of DNA copy number changes in single cells. Combining a PCR-based whole genome amplification method with arrays of highly purified BAC clones we could accurately determine known chromosomal changes such as trisomy 21 in single leukocytes as well as complex genomic imbalances of single cell line cells. In single T47D cells aberrant regions as small as 1–2 Mb were identified in most cases when compared to non-amplified DNA from 106 cells. Most importantly, in single micrometastatic cancer cells isolated from bone marrow of breast cancer patients, we retrieved and confirmed amplifications as small as 4.4 and 5 Mb. Thus, high-resolution genome analysis of single metastatic precursor cells is now possible and may be used for the identification of novel therapy target genes.
BackgroundThe transition from ductal carcinoma in situ (DCIS) to invasive breast carcinoma (IBC) is an important step during breast carcinogenesis. Understanding its molecular changes may help to identify high-risk DCIS that progress to IBC. Here, we describe a transcriptomic profiling analysis of matched formalin-fixed and paraffin-embedded (FFPE) DCIS and IBC components of individual breast tumours, containing both tumour compartments. The study was performed to validate progression-associated transcripts detected in an earlier gene profiling project using fresh frozen breast cancer tissue. In addition, FFPE tissues from patients with pure DCIS (pDCIS) were analysed to identify candidate transcripts characterizing DCIS with a high or low risk of progressing to IBC.MethodsFifteen laser microdissected pairs of DCIS and IBC were profiled by Illumina DASL technology and used for expression validation by qPCR. Differential expression was independently validated using further 25 laser microdissected DCIS/IBC sample pairs. Additionally, laser microdissected epithelial cells from 31 pDCIS were investigated for expression of candidate transcripts using qPCR.ResultsMultiple statistical calculation methods revealed 1784 mRNAs which are differentially expressed between DCIS and IBC (P < 0.05), of which 124 have also been identified in the gene profiling project using fresh frozen breast cancer tissue. Nine mRNAs that had been selected from the gene list obtained using fresh frozen tissues by applying pathway and network analysis (MMP11, GREM1, PLEKHC1, SULF1, THBS2, CSPG2, COL10A1, COL11A1, KRT14) were investigated in tissues from the same 15 microdissected specimens and the 25 independent tissue samples by qPCR. All selected transcripts were also detected in tumour cells from pDCIS. Expression of MMP11 and COL10A1 increased significantly from pDCIS to DCIS of DCIS/IBC mixed tumours.ConclusionWe confirm differential expression of progression-associated transcripts in FFPE breast cancer samples which might mediate the transition from DCIS to IBC. MMP11 and COL10A1 may characterize pure DCIS with a high risk developing IDC.Electronic supplementary materialThe online version of this article (10.1186/s12920-018-0403-5) contains supplementary material, which is available to authorized users.
The need to analyze rare cells is based on the nature of tissue differentiation and regeneration, the initiation and propagation of disease processes in multicellular organisms, and the functional diversity of individual cells. Gene transcription is the most important regulatory mechanism by which a phenotype and functional state of a cell is determined. Therefore, procedures for the qualitative and quantitative assessment of mRNA abundance are important. This unit presents a protocol for semi-quantitative analysis of gene expression of a single cell and quantitative representation of expressed genes from >10 to 30 cells. A basic protocol for array hybridization on nylon filters is provided because such filters are available in every laboratory. Tissue samples contain many different cell types in variable amounts, so their analysis may require microdissection; a protocol for obtaining cryosections is given. Finally, a simple procedure to prepare the data for statistical analysis is also provided.
A fundamental and clinically important step during breast tumourigenesis is the transition from ductal carcinoma in situ (DCIS) to invasive ductal carcinoma (IDC). Improved knowledge of this transition from pre-invasive to invasive breast cancer will pave the way for novel preventative and therapeutic strategies. We have previously reported on differential expression of the miRNA hsa-miR-199a-5p in 15 matched pairs of DCIS and IDC areas isolated by laser capture microdissection (LCM) from formalin fixed and paraffin embedded (FFPE) breast cancer tissues using Illumina miRNA BeadChip microarray platform. Differential expression of hsa-miR-199a-5p was validated by quantitative RT-PCR in additional independent DCIS/IDC sample pairs from 25 breast cancer patients. Knock down of hsa-miR-199a-5p in invasive MDA-MB-231 and TMX2-28 breast cancer cells using a specific inhibitor significantly reduced invasiveness by approx. 73% and 71%, respectively (P <0.01 and P<0.05). Now we report on experiments to validate differential expression of hsa-miR-199a-5p using a new platform – NanoString® (nCounter® miRNA Expression Assay, NanoString Technologies®). The NanoString® System is an automated, digital detection and counting system which uses a novel barcoding technology to directly profile up to 800 miRNAs simultaneously from a single sample. Total RNA from 6 DCIS/IDC FFPE tumours was used for miRNA expression analysis. This analysis resulted in 10 differentially expressed miRNAs including hsa-miR-199a-5p which is upregulated in IDC (P <0.05). Besides hsa-mir-199a-5p, hsa-miR-222 is significantly differentially expressed between DCIS and IDC which could be found in both expression data sets (Illumina® and NanoString®). In this project we identified candidate progression-associated miRNAs which are differentially expressed between DCIS and IDC. Hsa-miR-199a-5p was validated in an independent sample cohort and its expression was further verified using the new miRNA expression analysis platform NanoString®. Hsa-miR-199a5-p is influencing in vitro cell invasiveness and may therefore be a potential drugable regulator of tumour progression and invasion in breast cancer. Citation Format: Fehm T, Schultz S, Bartsch H, Petat-Dutter K, Kahlert S, Sotlar K, Niederacher D, Neubauer H. Verification of the breast cancer progression-associated miRNA hsa-miR-199a-5p using NanoString® platform. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P4-09-09.
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