Of 90 families with breast and colorectal cancer, 53 families (59%) were classified as Lynch syndrome on the basis of a germline mutation (n=52) or multiple MSH2/MSH6 deficient tumors within the family (n=1). shows the distribution of families amongst the four causative MMR genes (MLH1, MSH2, MSH6 and PMS2). In thirty-seven remaining families, Lynch syndrome could be excluded as no evidence of MMR deficiency from IHC or MSI testing was found, nor were any MMR mutations identified where tested. In 30 families Lynch syndrome was excluded on the basis of IHC and MSI testing of several family members with no evidence of MMR deficiency. The remaining seven families showed multiple MLH1-deficient colorectal and/or endometrial cancers associated with MLH1 methylation and/or somatic BRAF mutation (CRC only) with no evidence of germline mutations in MLH1 either by direct sequencing or MLPA. Only one of these seven families met modified Amsterdam criteria (ACII). None of the 61 breast cancers in this study, which could be analyzed, demonstrated the V600E activating mutation in BRAF.
Characteristics of families related to mutation in a particular MMR gene
Abnormal immunostaining for MMR protein was observed for 18/107 breast cancers (17%) ( and Supplementary Figure 1
and Supplementary Figure 2
). In twelve cases, tumors showed loss of MSH2 and MSH6 proteins, in five cases MLH1 and PMS2 were absent, and in one case, MSH6 only was absent. Microsatellite instability testing was performed for 89 breast cancers and was concordant with IHC results in 85 cases (96%). All but one of the MMR deficient breast cancers arose in families meeting ACII. Of the 18 participants whose breast cancers showed loss of one or more MMR proteins on IHC, 16 tested positive for germline mutations in the DNA MMR genes MLH1
consistent with both their tumor immunodeficiency as well as their respective family mutation, whilst a further individual who was deceased was found to be an obligate carrier of her family mutation (). The remaining case demonstrating immunohistochemical loss of MSH2 and MSH6 arose in a family in which no MSH2
mutation has been identified to date but which has three affected kindred members where their tumors demonstrate commensurate loss of MMR proteins.
(A) – (D) illustrate a poorly differentiated ductal carcinoma showing retention of MLH1 (A) and PMS2 (D) expression but loss of MSH2 (B) and MSH6 (C) staining in tumor cells.
Mutation status of individuals with MMR deficient breast cancers
Overall, 18/35 known MMR mutation carriers with breast cancer (51%) produced a breast cancer that was MMR deficient. Of the 89 breast cancers with normal immunohistochemistry, 13 arose in individuals from families where MLH1 mutations were identified, 20 in individuals with a family MSH2 mutation, 5 with MSH6 mutations and 3 with PMS2. Of these 41, 17 individuals tested carried the family mutation, suggesting that only a proportion of breast cancers in mutation carriers are associated with MMR deficiency.
Ten of the 18 individuals showing loss of one or more MMR proteins in their breast cancers had other primary tumors as summarized in . In three of the ten cases, the breast cancer was the first diagnosed malignancy, preceding the second cancer by between 2 and 27 years. In the other patients, the breast cancers were diagnosed between 1 and 42 years after the first cancer. In all cases but one (a meningioma), the non-breast cancers tested showed the same pattern of MMR protein deficiency as the breast tumors, a finding which supports the premise of this report, namely that the breast cancers in mutation carriers that are MMR deficient are likely to have developed in association with the germline mutation carried. Importantly, in eight cases of breast cancer in a proven mutation carrier, breast cancer was the only cancer documented.
Multiple MMR deficient tumors in MMR deficient breast cancer patients
There was no statistical difference in age of presentation between the MMR deficient breast cancers (mean = 57.5 ± 8.1 yr, range 43.4 – 75.0 yr) and MMR intact BCs (mean = 55.8 ± 11.9 yr, range 36.1 – 86.7 yr) (p=0.56), nor between the MMR deficient BC group and MMR intact known mutation carriers (57.1 ± 12.0 yr, range 36.1 – 80.5 yr) (p=0.90). Similarly, no difference in mean age of presentation was observed between the five BC cases which were MMR deficient in MLH1 germline mutation carriers and the twelve cases occurring in MSH2 carriers (58.0 yr vs. 57.5 yr) (p=0.90). The average age of the 11 individuals with the primary or only cancer being a breast cancer with MMR deficiency was 53.7 ± 6.0 yr.
In 104 breast cancers that underwent pathology review, histological differences for invasive BCs only were compared, with twelve cases of ductal carcinoma in
situ excluded from analysis. Specifically, MMR deficient invasive breast cancers (n=16) were more likely to be estrogen- and progesterone receptor negative (p=0.031 and p=0.022 respectively), have peritumoral lymphocytes (p=0.015), to have confluent necrosis (p=0.002), to have growth in solid sheets (p<0.001), and to have a higher mitotic rate (p=0.002) when compared to MMR-proficient BCs (n=79). In addition, MMR deficient breast cancers less frequently had contiguous in situ
disease (p=0.038). No statistically significant association was seen between MMR status and tumor type, size, lymphovascular invasion, node status, prominent eosinophilic nucleoli, or tumoral calcification ( and Supplementary Table 1
). No statistical differences were observed for clinicopathological features between the MMR-proficient invasive breast tumors arising in known carriers of germline MMR gene mutations and tumors from the non-Lynch syndrome group (data not shown), and therefore all MMR-intact tumors were considered together as the reference group for comparison with MMR-deficient invasive cancers. There were, however, significant differences in growth in solid sheets (p=0.002), and the presence of pushing margins (p=0.042), and confluent necrosis (p=0.017) and residual carcinoma in situ
(p=0.004) between MMR deficient and intact invasive cancers amongst proven carriers of MMR gene germline mutations ().
Histological features in MMR deficient vs. MMR proficient invasive breast cancers
Histological features in MMR deficient vs. MMR proficient invasive breast cancers from MMR germline mutation carriers
Four tumors displayed typical BRCA1
histological phenotype (characterized by high grade, high mitotic index, pushing margin, growth in solid sheets, and the presence of lymphocytic infiltrate and tumor necrosis (28
)), and two of these tumors (50%) showed loss of MSH2 and MSH6.
The two cases of ductal carcinoma in situ which exhibited loss of MMR expression were both of solid type, but there was no significant difference overall between DCIS type (cribriform, solid, papillary or clinging) and MMR expression when including in situ disease accompanied by an invasive component (p = 0.45) (data not shown). Lobular carcinoma in situ was present in eight cases accompanying invasive disease. There was no statistically significant difference between MMR deficient and proficient breast cancers and over-expression of p53 (p = 0.39).
There was a trend for individuals with a MMR deficient breast cancer to have also developed an early onset colorectal cancer, or was a first degree relative of someone so affected (n= 15) when compared to individuals with a MMR proficient BC having more distantly related cases of early-onset CRC (n=3) (p=0.11, 21% vs. 9%, respectively). Of the thirteen cases in which the same individual had both early-onset colorectal cancer and breast cancer, five (39%) showed mismatch repair deficiency. There was no statistical difference between degree of kinship between the breast and early onset CRC patients within individual families and whether the pedigree satisfied the modified Amsterdam criteria (p = 0.17).