Allelotype analysis of flow-sorted breast cancer cells demonstrates genetically related diploid and aneuploid subpopulations in primary tumors and lymph node metastases

Abstract

Flow cytometric DNA content measurements have demonstrated extensive DNA ploidy heterogeneity in primary breast carcinomas. However, little is known at the molecular level about the clonal relationship between these tumor cell subpopulations, or about the molecular genetic changes associated with aneuploidization. We have used flow cytometric cell sorting to dissect some of this complexity by isolating clonal subpopulations in breast carcinomas for comparative molecular genetic analysis. Clonal subpopulations were isolated from 12 primary breast carcinomas and 5 lymph node metastases from 4 cases based on DNA content and cytokeratin 8/18 labeling. DNA from these clones was screened for allelic imbalances with 92 polymorphic microsatellite markers mapped to 39 different chromosome arms. Diploid and aneuploid populations were concurrently present in 11 out of 12 primary tumors. The DNA ploidy status of primary tumors was identical to that of the related lymph node metastases. Allelic imbalance was present in 10 out of 11 diploid clones (mean, 3.4 ± 4.2). All allelic imbalances observed in the diploid clones recurred in the cognate aneuploid clones, but were, in the latter, accompanied by additional allelic imbalances at other loci and/or chromosome arms (mean, 10.9 ± 5.8). In only two of the four metastatic cases did the allelotypes of metastatic clones show small differences relative to their cognate primary tumors. The primary diploid tumor clone recurred in all lymph node metastases. This study indicates that the majority of allelic imbalances in breast carcinomas are established during generation of DNA ploidy diversity. Recurrence of the allelic imbalances in diploid clones in the aneuploid clones suggests linear tumor progression, whereas the simultaneous presence of early diploid and advanced aneuploid clones in both primary and metastatic tumor sites suggests that acquisition of metastatic propensity can be an early event in the genetic progression of breast cancer. Genes Chromosomes Cancer 28:173–183, 2000. © 2000 Wiley‐Liss, Inc.

Characterisation of p53 status at the gene, chromosomal and protein levels in oesophageal adenocarcinoma

“…Tumour samples not only contain contaminating normal cells, but are also genetically heterogeneous; therefore, a deletion would have to be present in a substantial number of cells to be detected by LOH analysis. In addition, most tumours are aneuploid and considerable LOH frequency differences have been detected between flowsorted aneuploid and diploid cells (Bonsing et al, 2000). LOHbased studies therefore tend to be inconsistent, particularly as definitions for LOH thresholds are variable (Tomlinson et al, 2002).…”

Loss of heterozygosity analysis: Practically and conceptually flawed?

“…Most carcinomas, however, are polyploid and aneuploid (Tirkkonen et al, 1998;Forozan et al, 2000;Abdel-Rahman et al, 2001). There can also be considerable intratumor heterogeneity in karyotype, resulting in a marked difference in LOH frequency between flow-sorted diploid and aneuploid cells (Bonsing et al, 2000). If the two hits have occurred before polyploidy, then the cancer will still show loss of one allele, subject to the influences of contaminating normal tissue in the sampled tumor.…”

Multiple genetic changes are associated with mammary tumorigenesis in Brca1 conditional knockout mice

“…In sporadic forms of breast cancer, a number of ampli®ed or deleted chromosome regions have been identi®ed using techniques, which test for allelic imbalance (Bieche et al, 1999;Bonsing et al, 2000;Orsetti et al, 1999;Osborne and Hamshere, 2000;Rodriguez et al, 2000;Sta et al, 2000). In high-grade breast cancer, chromosome regions that are ampli®ed leading to protein overexpression, often include genes coding ErbB2, c-Myc and Cyclin D1 (Brison, 1993).…”