Quadruple Negative Breast Cancers (QNBC) Demonstrate Subtype Consistency among Primary and Recurrent or Metastatic Breast Cancer

Abstract

Purpose: Despite the availability of current standards of care treatments for triple negative breast cancer (TNBC), many patients still die from this disease. Quadruple negative tumors, which are TNBC tumors that lack androgen receptor (AR), represent a more aggressive subtype of TNBC; however, the molecular features are not well understood.

Methods: Immunohistochemistry of estrogen receptor (ER), progesterone receptor (PR), HER2, and AR was determined in 244 primary and 630 recurrent/metastatic site biopsies. Expression was correlated with a panel of 25 cancer-related genes and proteins by IHC and in situ hybridization (ISH).

Results: We observed that 80.2% (65 of 81) of primary TNBC tumors and 75.7% (159 of 210) of recurrent/metastatic TNBC tumors are QNBC. Bivariate fit analysis demonstrated that QNBC (n = 224) significantly (P < .03) correlated with younger aged patients at initial biopsy compared to AR positive TNBC patients (n = 51). In paired primary tissue samples and primary to recurrent/metastatic samples, at least 70% Luminal, HER2 enriched, and QNBC subtype did not change molecular profile. But, TNBC seems to be the "unstable" subtype. Within the total cohort, discordance in molecular profiles was identified in both synchronous (20%) and asynchronous (21%) intra-individual analyses. Irrespective of sample type, (Synchronous or Asynchronous), QNBC demonstrated higher concordant than TNBC. IHC and ISH results of the cancer related genes, demonstrated that gene/protein expression differ by molecular profile: TNBC (HR-/HER2-, AR+) and QNBC (HR-/HER2-, AR-). IHC in metastatic tumors, showed that the percentage of tumors positive of EGFR were higher, while PTEN and TLE3 were lower in QNBC compared to TNBC.

Conclusion: Standard treatment of Breast Cancer (BC) relies on reliable assessment by IHC analysis of ER, PR, and HER2. Our analyses suggest that the heterogeneity of TNBC is at least partially associated with the presence or absence of AR expression, suggesting that QNBC should be considered as a clinically relevant BC subtype. IHC analysis of AR appears to be a practical assay to determine the most aggressive TNBC subtypes and identifies tumors that could benefit from available targeted therapies.

Figure 1

Median and Mean age for QNBC patients is lower than TNBC AR positive patients: Bivariate fit analysis of patients age and AR status determined that AR negative patients were diagnosed at younger ages compares to AR-positive patients (P < .05). QNBC is significantly correlated with younger aged patients compared to AR positive TNBC patients.

Figure 2

QNBC tumors have decreased discordance. Comparison of the frequency and type of change in ER, PR, HER2, AR status between paired patient profiles. Status at first profile A-D vs. status at 2nd profile (designated by color as shown above). Samples that lack IHC expression of biomarkers were excluded from the analysis. A. This represents comparison between paired patient first biopsy to second biopsy profile at primary site. The number of paired primary biopsies were 55(total), Luminal A/B (n = 39), Her2 type (n = 0), TNBC (n = 3) and QNBC (n = 13). Paired primary tissue samples did not display significant differences in ER, PR, HER2, or AR status for Luminal A/B (79%). The number of AR positive TNBC, those have paired primary biopsies were low. However, within the AR positive TNBC tumors, 66% had an alteration in biomarker status, resulting in a change in molecular profile at second biopsy. Furthermore, in the AR negative QNBC tumors (69%) of the tumors remained QNBC at the second biopsy. B. This illustrates comparison between paired patient primary – recurrent/metastatic samples of all subtypes .Number of paired primary to metastatic/ recurrent biopsies were n = 91 (total), Luminal A/B (n = 47), Her2 type (n = 5), TNBC (n = 4) and QNBC (n = 35).The discordance rate was observed in all subtypes (Luminal A/B (24%, HER2 enriched (0%), and QNBC (23%)), with TNBCs displaying the most discordance (50%). C. This illustrates comparison between paired patient first biopsy and second biopsy profile in metastatic/recurrent site. We further evaluated 195 (total) paired recurrent/metastatic samples based on subtype at first biopsy and second biopsy, Luminal A/B (n = 118), Her2 type (n = 14), TNBC (n = 12) and QNBC (n = 51). We observed that as the cancer progressed to recurrent/metastatic disease, the initial biomarker phenotype often exhibited a more aggressive phenotype, with an increase in QNBC subtype at second profile status. For example, 16% of AR positive TNBC tumors 78% of QNBC tumors not exhibiting any change.

Figure 3

QNBC tumors demonstrate increased concordance in asynchronous and synchronous biopsies. Illustration of the concordance and discordance of paired ER, PR, HER2, and AR status in paired patient tissue samples. Samples that lack IHC expression of biomarkers were excluded from the analysis. The paired samples were then classified based on collection dates as synchronous (taken within 6 months of initial biopsy) or asynchronous (taken more than 6 months of initial biopsy). The number of paired samples were 133 (synchronous) and 192 (asynchronous). Both synchronous and asynchronous paired samples displayed consistent concordant molecular profiles, 80% and 79%, respectively. Despite the concordance of intra-individual samples, discordance of biomarker statuses was illustrated for synchronous (20%) and asynchronous (21%) stratification. The paired synchronous and asynchronous samples were then categorized by TNBC and QNBC (synchronous QNBC (n = 40), synchronous TNBC (n = 8), asynchronous QNBC (n = 55), asynchronous TNBC (n = 13). Within the TNBC subset, in both the asynchronous and synchronous categories, there was a 62% discordance of paired samples. Contrarily, synchronous and asynchronous QNBC tumors only displayed 23% and 24% discordance, respectively.

Figure 4

Gene and protein expression in TNBC compared to QNBC tumors in metastatic or recurrent site. The expression multiple cancer-related genes in metastatic TNBC and QNBC tumors was determined by IHC and ISH. EGFR, PTEN and TLE3, demonstrated statistically significance between TNBC (n = 39) and QNBC (n = 159) patients. The number of samples positive or negative for a gene was obtained and the total number of samples that have the IHC for that gene was obtained. The percentage of patients positive for each protein was calculated and statistical significance was obtained using Fisher's exact test (2×2 contingency, two-tailed test). *** (P < .0001), **(P < .001), *(P < .05).