Introduction 1 2 FANCA FANCB FANCC FANCD1 BRCA2 FANCD2 FANCE FANCF FANCG FANCJ FANCL FANCM 3 18 2 10 BRCA1 BRCA2 10 19 20 21 22 23 10 24 Materials and methods Tissue microarray 25 1 26 27 Table 1 Patient characteristics and histology of 120 sporadic and hereditary breast cancers  Sporadic BRCA1 mutation Total No. of patients 96 24 120 Age    Mean 65 42 62 Lymph node status    Negative 58 9 67    Positive 38 11 49 Tumor size    Mean 2.34 2.78 2.40 Histological type    Ductal 85 17 102    Lobular 7 1 8    Medullary 1 2 3    Tubular 1 – 1    Cribriform 1 – 1    Apocrine 1 – 1    Metaplastic – 4 4 28 Immunohistochemistry neu 29 23 2 2 neu 30 Statistics R p t neu Results 1 Fig. 1 Examples of FANCD2 staining in sporadic (A/B, A = negative control) and BRCA1-related breast cancers (C/D, C = negative control)  p p p p p 2 neu. Table 2 Mean percentage of FANCD2-positive cells in high- and low-level expression groups of hormone receptors, growth factor receptors, Cyclin D1, and p53 in sporadic and hereditary breast cancers  Number (%) Mean (SE) of FANCD2% p t Total 117 (100)   ER    Low 56 (48) 14 (2.5) 0.019    High 61 (52) 8 (1.6)  PR    Low 81 (69) 17 (1.9) 0.016    High 36 (31) 12 (1.9)  neu    Negative 93 (79) 16 (1.6) 0.797    Positive 24 (21) 17 (3.5)  EGF receptor    Negative 78 (66) 13 (1.5) 0.002    Positive 39 (33) 19 (3.1)  Cyclin D1    Low 85 (73) 12 (1.9) 0.002    High 32 (27) 7 (2.6)  p53    Low 88 (75) 8 (1.3) <0.001    High 29 (25) 19 (4.3)  ER PR 3 R p R p R p R p R p R p R p 2 p p p p Table 3 Correlation between mean percentage of FANCD2-expressing cells and other continuous clinicopathological variables in sporadic and hereditary breast cancers Feature R p a 0.506 <0.001 a 0.502 <0.001 a 0.482 <0.001 Age −0.197 0.033 ER −0.221 0.017 PR −0.204 0.028 Tumor size 0.018 0.852 p27 −0.074 0.430 p21 −0.171 0.065 p53 0.379 <0.001 Cyclin D1 −0.126 0.176 ER PR a Fig. 2 Top left Top right Bottom left  In the regression analysis between the percentages of FANCD2-expressing cells and Ki-67 and Cyclin A, the completely negative FANCD2 cases were excluded. These FANCD2-negative cases had Ki-67 values between 1 and 65 (mean 16%), Cyclin A values between 0 and 50 (mean 10%), and MAI values between 0 and 37 (mean 11), indicating that these cases had (sometimes even high) FANCD2-independent proliferation. p 3 p Fig. 3 p N  Discussion 21 22 24 23 It appeared that 19% of sporadic breast cancers completely lacked FANCD2 expression. Yet, these FANCD2-negative cases had high mean Ki-67, Cyclin A, and MAI values, indicating that the low FANCD2 levels in these cases cannot be explained by low proliferation. The fact that these FANCD2-negative cases stained for other proteins makes it quite unlikely that the FANCD2 negativity is due to fixation problems. In the FANCD2-negative cases, FANCD2 inactivation may, in view of its important function, have been a hit in carcinogenesis. BRCA1 germline mutation-related breast cancers showed lack of FANCD2 expression in only 9.5% of cases, which fits with the concept that a major hit in an important pathway (in these cases the BRCA1 germline mutation) is usually not associated with further hits in this pathway. It is yet unclear what the mechanism behind the lack of FANCD2 expression in these cases is. It needs to be further studied whether there are inactivating somatic mutations in these cases or whether promoter methylation plays a role. 23 31 33 34 The negative relation between FANCD2 and age can likely be explained by the fact that BRCA1-related patients that have higher FANCD2 expression are younger. Within the light of the above observations, the question remains why FANCD2 patients do not seem to be predisposed to breast cancer in clinical practice. FA itself is a rare genetic disease where the complementation group D2 constitutes only 1–2% of all FA cases and these patients generally have a more severe clinical course. They may therefore simply not live to get breast cancer in an apparent increased frequency. Our results do not indicate that somatic (epi)genetic changes in FANCD2 are a frequent secondary carcinogenetic event in BRCA1 germline-mutated patients, although this needs to be confirmed in a larger study group. In conclusion, FANCD2 expression is absent in 10–20% of sporadic and BRCA1-related breast cancers, indicating that somatic inactivating (epi)genetic events in FANCD2 may be important in both sporadic and hereditary breast carcinogenesis. FANCD2 is of independent prognostic value in sporadic breast cancer.