ALK alteration is a frequent event in aggressive breast cancers

One thousand and nine patients with breast cancer diagnosed between1990 and 2011 were selected from the files of the King Faisal Specialist Hospital and Research Centre (KFSHRC). The patients included in this study had their diagnosis, treatment and follow-up care in the Department of Surgical Oncology at KFSHRC. The histologic subtype of each breast tumor sample was determined according to World Health Organization (WHO) criteria. Waiver of consent was obtained for the study from the Institutional Review Board (IRB) and Research Ethics Committee (REC) of KFSHRC under Project RAC number 2040004 on breast cancer archival clinical samples. All samples were analyzed in a tissue microarray (TMA) format.

For each tumor, representative areas were selected mapped and a TMA was constructed using these mapped slides as a reference. The tissue microarrayer (Semiautomated Arrayer, CM1 Mirlacher, Neuenburg, Germany) was used and 0.6 mm diameter punches were obtained from the donor blocks. A map of the recipient block was prepared with coordinates and a number for each sample to correctly identify the tumor. The punched-out tissue cores from the donor block were inserted in the recipient block. The array blocks were incubated at 45 °C for 10 minutes to improve adhesion between cores and paraffin of the recipient block. They were cut at room temperature with a standard microtome (Thermo Shandon, Runcorn, UK) and slides were prepared using a tape-sectioning system (Instrumedics, Inc., St. Louis, MO, USA).

IHC and FISH staining were done on TMA slides cut from two replicas of TMA blocks for scoring to reduce the number of non-interpretable spots. Both the ALK IHC and ALK FISH non-interpretable spots were within the range of 10 % and the rest of the TMA spots were scored in concordance with earlier biomarker studies on TMAs [32].

FISH assay for ALK amplification and rearrangement was performed on a TMA format. The probe (BAC clone) corresponding to the ALK gene was selected by browsing Ensembl Genome Browser [33] and were purchased from the Children’s Hospital Oakland Research Institute (Oakland, CA USA). The BAC clone was cultured, DNA isolated and was labeled with digoxygenin (DIG) utilizing a DNA labeling kit from Roche (Roche, Hamburg, Germany). A commercially available centromeric probe for chromosome 2; CEP2 (Abbott Molecular, Abbott Park, IL, USA) was utilized as an internal control. FISH on a breast cancer TMA was performed as previously described [32]. Tissue samples were classified with an ALK/CEP2 ratio of 1.0 as normal; between 1.0 and 2.5 as having ALK gains; 2.5 and above as amplified [17]. A minimum of 25 cells were scored in a single microarray spot for the presence of both CEP2 and ALK signals. ALK gene amplifications and gains identified by the BAC clone RP11-328L16 were also further confirmed by the break-apart probe from Vysis (Abbott Molecular, Abbott Park, IL, USA) according to the manufacturer’s instructions using a BX51 Olympus fluorescence microscope (Olympus, Richardson, TX, USA). Briefly, in a normal cell with no gain or amplification, two fused red and green signals were observed. Increase in the number of red and green fused signals were considered to be amplifications and gains. An ALK break-apart probe set was also used to check for ALK gene rearrangement on breast cancer TMA slides. ALK rearrangement was termed as positive if >15 % of tumor cells showed split red and green signals and/or single red signals in addition to a single fused signal; otherwise, the specimen was classified as ALK FISH negative [26].

FISH analysis using the ALK probe was also performed on eight breast cancer cell lines (Cal-120, HCC-1937, EFM-19, HDQ-PI, CAL-51, MT-3, MDA-MB231 and MCF-10).

A Gentra DNA isolation kit (Gentra, Minneapolis, MN, USA) was utilized to extract DNA from paraffin-embedded breast cancer tissues using the manufacturer’s recommendations as described previously [34].

The quantitative PCR (qPCR) technique was utilized to validate the copy number variation of the ALK gene observed by FISH. A few representatives DNA samples from formalin-fixed paraffin-embedded samples (FFPE) of breast cancer with normal and increased ALK copy number by FISH were selected for validation by quantitative real-time (qRT) PCR. DNA content was normalized to that of long interspersed element 1 (LINE1), a repetitive element for which copy number per haploid genome is similar in both the normal DNA sample and DNA from cancer cells. Primers were designed by Primer Express software v3.0 (Applied Biosystems, Foster City, CA) to hybridize to sequences of genomic DNA for ALK. The primers to the genomic sequences for ALK and LINE1 are as follows:

The PCR conditions and Light Cycler PCR protocol were used as previously described [35]. The Pfaffl method for relative quantification was used to calculate the fold change for breast cancer samples that showed normal copy number and amplification of ALK gene [36].

For IHC staining, TMA slides were processed and stained manually as described earlier [36]. A list of antibodies and their dilutions are listed in Table S1 in Additional file 1. For ALK IHC, primary ALK antibody (cloneD5F3; CST; 1:100 dilutions; Dako Target Retrieval pH9) was applied and incubated overnight. Visualization of the antigen antibody reaction was done using an enhanced polymer-based detection system, Envision Plus Dual Link System-HRP for 1 hour. Diaminobenzidene (Dako, Glostrup, Denmark) was employed for 5 minutes as the chromogen. A known CD30-positive ALCL case was utilized as a positive control and the negative control used was a mouse immunoglobulin G1 serum substitution for the primary antibody (ALK) [37]. The cutoff for estrogen receptor (ER) and progesterone receptor (PR) was taken as 1 % nuclear staining. Human epidermal growth factor receptor 2 (HER2) overexpression was assessed according to American Society of Clinical Oncology/College of American Pathologists (ASCO/CAP) guidelines [38]. The cutoff for high Ki67 was taken as more than 10 % nuclear staining [39]. For p-AKT immunoscoring only intensity scores were considered. Tumors with intensity 2+/3+ were considered positive and intensity 0/1+ were taken as negative [40].

For ALK IHC H-score (range 0–300) was obtained by adding the sum of scores obtained for each intensity and proportion of area stained [35]. X-tile plots were constructed for assessment of biomarker and optimization of cutoff points based on outcome as has been described earlier [41]. Breast cancers were categorized into two groups based on X-tile plots: one with complete absence (H score = 0) and the other with ALK expression (H score >0).

Two pathologists (SB and SP) independently performed IHC scoring for biomarkers and for discrepant scores consensus was established by reviewing the slides together.

Proteins from MDA-MB231 and MCF-12A cells were isolated as described previously [42]. Twenty micrograms of isolated proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon, EMD Millipore, Billerica, MA, USA). Immunoblotting was performed using ALK (Santa Cruz Biotechnology Inc., Dallas, TX, USA) and beta-actin (Cell Signaling Technology, Boston, MA, USA) antibodies and visualized by the enhanced chemiluminescence (Amersham, Pittsburg, PA, USA) method.

MDA-MB231 and MCF12A cells were suspended in buffer A containing 10mM HEPES, 10mM KCl, 0.1mM EDTA, 0.1mM EGTA, 1mM DTT and the recommended amount of protease inhibitors and incubated on ice for 15 minutes. After incubation, cells were lysed with 10 % NP-40 and vortexed for 10 seconds. Cells were spun for 30 seconds at 14,000 rpm and supernatants containing nuclear-free cytosolic extracts were isolated. The remaining pellet was lysed in protein lysis buffer containing 0.4mM NaCl and nuclear extracts were isolated. Twenty micrograms of isolated proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) membrane (Immobilon, EMD Millipore, Billerica, MA, USA). Immunoblotting were performed using ALK and alpha-tubulin (Cell Signaling Technology, Boston, MA, USA) antibodies and visualized by the enhanced chemiluminescence (Amersham, Pittsburg, PA, USA) method.