Abstract P3-10-10: STATUS OF ANAPLASTIC LYMPHOMA KINASE (ALK) GENE IN INFLAMMATORY BREAST CARCINOMA

Abstract

Abstract The ALK gene located on chromosome 2p23 belongs to the insulin receptor superfamily and encodes a receptor tyrosine kinase that is normally expressed in brain, testis and small intestine. There is limited data regarding ALK gene rearrangements in breast cancers. We performed a comprehensive investigation of ALK in IBC using array comparative genomic hybridization (CGH), transcriptional profiling, fluorescence in situ hybridization (FISH) and immunohistochemistry (IHC) for ALK protein expression. Materials and Methods: We used formalin fixed and paraffin embedded core biopsies of IBC for IHC and FISH and microdissected frozen tissues for array CGH and mRNA analysis. ALK protein was evaluated by IHC using primary antibody against ALK (DAKO cytomation). The Vysis ALK Break Apart FISH probe kit (Abbott Laboratories) was used to detect chromosome 2p23 gene rearrangements. Genomic DNA was applied to whole genome DNA microarray (Link Genomics) which covered the entire human genome using 12310 individually amplified bacterial artificial chromosome clones (BAC) clones. Thresholds of gains and losses were set at 1.2 and 0.8 respectively. Analysis of mRNA expression was performed by labeling cRNA from the specimens to an oligo-nucleotide microarray (whole genome 4×44K Agilent technologies), scanned with an Agilent microarray scanner and analyzed with features extraction software (Agilent Technologies). Results: We performed IHC and FISH for ALK in 29 IBC patients (ER/PR +, HER2− in 12, HER2+ in 13, triple negative in 4). Array CGH and mRNA analysis was conducted in 20 of these 29 cases. All the 29 cases were negative for ALK protein expression by IHC. FISH analysis revealed intact bi-color flanking signals in all the tumor cell nuclei from the 29 specimens indicating absence of EML4-ALK gene rearrangements in any of the cases. We found mild gain of intact ALK signals in a small percentage of interphase cells in 90% of the cases. FISH for CEP2 performed in 8 specimens showed normal CEP2 levels in 6/8 indicating mild amplification of the ALK gene in these cases. A total of 20 DNAs analyzed by array CGH focusing on the 2 BACs that covered the ALK gene region showed no gain in 17 cases indicating no change in copy number of ALK, gain in 1 BAC in 2 cases and gain of 2 BACS in 1 case. Transcriptional profiling revealed that mRNA expression of ALK was not significantly higher than the 5 normal breast controls (P >0.05, t test) including the 3 patients with possible mild increase in copy numbers of ALK gene by array CGH. Conclusion: Gene rearrangements involving the ALK gene were not noted in any of the IBC patients. Mild gain of intact ALK signals as evidenced by FISH suggests low level amplification in majority of IBC patients. The lack of gain in copy numbers of ALK gene in 85% of the IBC patients together with the absence of significant elevation of mRNA levels and absence of ALK protein expression by IHC suggests absence of transcription and translation of the mild amplification of the ALK gene in patients with IBC. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P3-10-10.