MYC amplification in subtypes of breast cancers in African American women

This retrospective study was based on anonymized, formalin-fixed, paraffin-embedded FFPE archival BCa tissues (e.g. triple negative BCa, Luminal A, Luminal B, and HER2 positive) were obtained from Howard University Hospital Department of Pathology in Washington, DC. They were collected over a ten year period of time ranging from 2000 to 2010. IRB approval for the conduct of this study was received from Howard University’s Institutional Review Board and designated as exempt 15CMED-53. A total of 70 samples were analyzed. Samples were selected based on the availability and adequacy of FFPE tissue blocks for further study. Five μm thick sections were cut and areas of well-preserved carcinoma in a Hematoxylin and Eosin (H&E) orienting slide from each block were marked for study through hybridization and microscopy. Demographic and clinic-pathological data were obtained through the Howard University Cancer Center Tumor Registry.

Fluorescence in situ hybridization (FISH) techniques were used to assess MYC amplification in tumor specimens as previously described in our previous study (18). Briefly, the orange fluorophore directly-labeled MYC probe (CymoGen DX, NY USA) and a green labeled centromere 8 probe (Empire Genomics, NY, USA) were used to make a FISH probe mix. The slides were then baked overnight at 60 °C to adhere the tissues to the slides. Slides were subsequently deparaffinized by consecutive 10-min xylene washes before being rehydrated through 100% ethanol incubation for 10 min at room temperature. The tissue on the slides were then permeabilized and digested using the Abbott (Naperville, IL) tissue digestion kit containing pepsin, according to manufacturer’s instruction. The sections were washed with wash buffer after consecutive pretreatment and protease digestion. The probe was denatured at 74 °C for 10 min and immediately transferred to an ice bucket. The denatured probe was then added to the marked area on the slides and was sealed with rubber cement. The slides and the probe were co-denatured for 8 min in HYBrite heat plate (Vysis, Naperville, Illinois) at 85 °C for 8 min and then incubated for 16 to 24 h at 37 °C. Coverslips were removed and the slides were Post Washed in 2X SSC hybridization buffer for 2 min at 73 °C and transferred to 2X SSC at room temperature for 5 min. The slides were air dried in the dark for 1 h in an upright position. Lastly, the slides were counterstained with DAPI (4′, 6-diamidino-2-phenylindole) from Sigma Aldrich and were viewed with a Zeiss Axioscope fluorescence microscope and imaged with Applied Imaging (Pittsburgh, PA) Cytovision software.

FISH images were acquired using a Zeiss Axioscope fluorescence microscope equipped with a camera and multiple fluorescence filter sets with three color filter set DAPI, FITC and TRITC. The slides were screened with 100× oil objective lens through the DAPI filter to determine the cell area by observing the area of interest marked by the Pathologist. The centromeres were counted as green signals whereas the MYC signals were viewed as orange signals with the TRITC filter (Fig. 1). At least 50 cells were enumerated from a single slide when possible. The signal ratio of the centromere to MYC in normal cells was calculated. The normal control was 2:2; an increase in the ratio was considered abnormal or amplified. The results were expressed as number of MYC signals/number of chromosome 8 signals (MYC: CEP8) [18].

The statistical analysis software, IBM SPSS Statistics for Windows(Version 22. Armonk, NY: IBM Corp) was used to perform descriptive analysis of clinic-pathological variables and determine the association between MYC amplification (dependent variable) and prognostic clinic-pathological characteristics such as ER, PR, and HER2 positivity, molecular BCa subtype, stage, grade, and tumor size (independent variables). The Chi-square (χ²) test or T test, as appropriate, was used to examine bivariate association between MYC-amplification, CK 5/6 expression and clinic-pathologic variables. Age was categorized into two groups with a cutoff of 50 years. The logistic regression analysis or ANOVA, as appropriate, was used to examine independent association between MYC amplification and clinic-pathological variables. Results were reported as odds ratios (OR) along with calculated 95% confidence intervals (CI). Pearson’s correlation was used to determine the correlation between amplification ratio, tumor size, and age of diagnosis.. Kaplan-Meier (K-M) estimate of overall survival were plotted and log-rank test was performed to compare estimates among groups.

Clinicopathological characteristics of the study population are summarized in Table 1. Seventy paraffin-embedded archival tissues of BCa were studied. The average age at diagnosis was 56 years with a minimum and maximum age of 29 and 85 years, respectively. Approximately 37% of the cases were TNBCs; HER2-amplified tumors comprised 11.4% of the tumors in this study; and Luminal A and Luminal B tumors comprised 30% and 21% of the tumors, respectively. ER, PR, and HER2 were positive in 50%, 45.7%, and 18.6% of tumors, respectively. Most of the tumors were poorly differentiated (74%). The frequency of stage I, II, II, and IV tumors were 23.4%, 41.4%, 22.9%, and 5.7%, respectively.

Figure 1 demonstrates representative FISH images with a red labeled MYC probe and a green labeled chromosome 8 centromere probe. Normal, intermediate, and high MYC amplification tissues for these studies are presented. The normal MYC: CEP8 ratio is 1. The average MYC amplification ratio was 2.25. Thirty-six percent of the cases had high amplification ratio (MYC: CEP 8 ratio > 2). High MYC amplification scores were observed in HER2 (3.97 ± 1.99) and Luminal B (3.34 ± 2.11) subtypes, whereas low amplification was seen in TNBC (1.65 ± 0.94) and Luminal A (1.65 ± 0.93) subtypes as shown in Table 2. The mean MYC amplification ratio of HER2, Luminal A, and TNBC differ significantly; however, HER2 and Luminal B do not significantly differ from each other (p < 0.001).

In all, 20% of the samples were HER2 positive (13% HER2 subtype, 7% Luminal B). The average MYC amplification ratio for HER2 positive BCas was 4.36 (±2.24) and 1.77 (±0.97) for HER2 negative/not amplified BCa cases as presented in Table 3. The mean difference between Her2 + and Her2- tumors was statistically highly significant with p = < 0.001 as detailed in Table 3. Average MYC amplification scores did not differ significantly between ER+ and ER-, as well as PR+ and PR- tumors. Regression analysis also revealed an association between the HER2+ subtype (OR = 29.75 95% CI: 2.80–315.56; p < 0.0001) and Luminal B HER2 positive subtype (OR = 10.63 95% CI 2.16–52.15; p < 0.0001), as well as with HER2 immuno-histochemical expression (OR = 40.62 95% CI 4.82–342.39; p < 0.00001). However, no statistically significant association was found between MYC amplification and cancer grade, origin of tumor, tumor size, or age of diagnosis.

In addition, Table 4 shows the survival analysis data in relation to MYC amplification and clinic-pathological variables. Statistically significant differences in overall survival were found when stratifying by stage (p < 0.001), having metastatic disease (p < 0.001), and lymph node positivity (p = 0.039). However, no statistically significant difference in overall survival was observed among BCa subtypes in this study (Log rank test p = 0.201). The mean overall survival was 71.00 months for HER2 subtype (95% CI = 44.09–97.91), 94 months (95% CI = 84.56–103.44) for Luminal B, 67.71 months (95% CI = 52.05–83.36) for Luminal A, and 100.07 months (95% CI = 76.57–123.57) for TNBC subtype.

Table 5 demonstrates that differences in mean survival were not found when stratifying by MYC amplification (p = 0.265). .