Loss of heterozygosity on chromosome 11p15 during histological progression in microdissected ductal carcinoma of the breast

Abstract

Microdissection of histologically identifiable components from formalin-fixed, paraffin-embedded tissue sections allows molecular genetic analyses to be correlated directly with pathological findings. In this study, we have characterized loss of heterozygosity (LOH) at chromosome 11p15 at different stages of progression in microdissected tumor components from 115 ductal carcinomas of the breast. Microdissected foci of intraductal, infiltrating, and metastatic tumors were analyzed to determine the stage of progression at which LOH at 11p15 occurs. LOH was detected in 43 (37%) of 115 cases. Foci of intraductal carcinoma could be microdissected from 85 cases, of which 30 (35%) showed LOH at some stage of progression. LOH was detected in the intraductal component in 26 of these 30 cases. Interstitial deletions were characterized by using a panel of 10 highly polymorphic markers. The smallest region of overlap (SRO) for LOH at 11p15 was bounded by the markers D11S4046 and D11S1758. LOH at 11p15.5 showed no correlation with estrogen receptor status, the presence of positive lymph nodes, tumor size, histological grade, or long-term survival. We conclude that 11p15 LOH usually occurs early in breast cancer development but less frequently does not develop until the infiltrating or metastatic stages of tumor progression.

Figure 1.

Location of markers used in this study and candidate tumor suppressor genes. The upper map shows the position of several genes, including the candidate tumor suppressor genes H19 and KIP2, along the region of approximately 6 Mb analyzed in this study. The lower map shows the positions of the genetic markers used. The map positions of the Genethon markers are from the sex-averaged Genethon Human Linkage Map. The distances between Genethon markers are indicated below the map in centiMorgans. The marker D11S837 (ST5 gene) has been localized within the centromeric group of BWS breakpoints. The locations of the markers D11S988 and TH are from the CHLC/GDB map. The upper and lower maps are shown in approximate alignment. The KVLQT1 gene occupies approximately 300 kb and encompasses the more telomeric group of BWS breakpoints. The TH gene is known to be closely linked to D11S1318, but the order of these two loci is not known with certainty. The H19 gene has been identified on a 100-kb bacterial artificial chromosome clone that also contains the marker D11S4046, suggesting close proximity of these loci in the genome.

Figure 2.

LOH occurring at different stages in the progression of breast cancer. Alleles showing loss in tumor specimens are indicated by arrows. Allele ratios were determined by quantitating the bands on a Molecular Dynamics Storm system, calculating the ratio of (lost allele)/(retained allele) and dividing by the same ratio obtained with the normal tissue. In case 72, LOH was first seen in invasive tumor at 11p15 but in intraductal at 17q. In case 112, LOH was first detected in the metastasis. In case 77, LOH was not seen in primary tumor but was present in a pleural metastasis 6 years later. In case 90, LOH was present in primary tumor but not in a contralateral tumor (labeled recurrence) 4 years later. In case 50, LOH was detected at 11p15 and 17q in invasive tumor but only at 17q in metastasis.

Figure 3.

Sublocalization of the minimal region of LOH at 11p15 in breast cancer. Results obtained with the 43 cases showing LOH with at least one marker are presented. Solid circles, LOH; shaded circles, retention of heterozygosity; open circles, uninformative (homozygous or nonamplifiable); M, microsatellite instability. The solid bar to the right of the figure indicates the minimal shared region of LOH inferred from this data set.

Figure 4.

Patterns of LOH at 11p15 in breast cancer. A: Case 38, showing LOH at the distal group of markers, retention of heterozygosity at D11S1758, D11S4146, and D11S988, defining centromeric border of SRO. B: Case 93, with LOH at proximal and distal markers with retention of heterozygosity between these two regions. C: Case 124, with retention of heterozygosity at D11S4046, defining telomeric border of SRO. N, normal tissue; T, tumor. Arrowheads indicate the allele lost in assays showing LOH. When LOH was present, quantitation was as in Figure 2 ▶ . In cases where there was no LOH, the numerator and denominator of the reported allele ratio were chosen so as to yield a number less than the ideal value of 1.0.

Figure 5.

Cumulative proportion surviving (Kaplan-Meier). Survival curves were generated for cases with (——) and without (- - -) LOH at 11p15.