Pedram Argani

Heterogeneity of Breast Cancer Metastases: Comparison of Therapeutic Target Expression and Promoter Methylation Between Primary Tumors and Their Multifocal Metastases

Purpose: A comprehensive comparison of biomarker expression between patients primary breast carcinoma (PBC) and their metastatic breast carcinomas (MBC) has not been done. Experimental Design: We did rapid autopsies (postmortem intervals, 1-4 hours) on 10 consenting patients who died of MBC. We constructed single-patient tissue microarrays from the patients archived PBC and multiple different MBCs harvested at autopsy, which were immunohistochemically labeled for multiple biomarkers. Methylation of multiple gene promoters was assessed quantitatively on dissected PBC and MBC samples. Results: Extensive heterogeneity was observed between PBC and their paired MBC, as well as among multiple MBC from the same patient. Estrogen and progesterone receptors tended to be uniformly down-regulated in metastases. E-cadherin was down-regulated in a subset of the MBC of one case. Variable overexpression in MBC compared with the PBC was observed for cyclooxygenase-2 (five cases), epidermal growth factor receptor (EGFR; four cases), MET (four cases), and mesothelin (four cases). No case strongly overexpressed HER-2/neu by immunohistochemistry, but eight cases showed variable protein expression ranging from negative to equivocal (2+) in different MBC. In one case, variable low-level HER-2/neu gene amplification was found. EGFR and MET overexpression were restricted to the four basal-type cancers. EGFR protein overexpression did not correlate with EGFR gene amplification. Multigene promoter hypermethylation of RASSF1a, HIN1, cyclin D2, Twist, estrogen receptor α, APC1, and RARβ was overall very similar in the PBC and all MBCs in all cases. Conclusions: Therapeutic targets identified in the PBC or even some MBC may not reflect targets present in all metastatic sites.

Telomere Shortening Occurs in Subsets of Normal Breast Epithelium as well as in Situ and Invasive Carcinoma

In the setting of inactivated DNA damage-sensitive checkpoints, critically shortened telomeres promote chromosomal instability and the types of widespread cytogenetic alterations that characterize most human carcinomas. Using a direct telomere fluorescence in situ hybridization technique, we analyzed 114 invasive breast carcinomas, 29 carcinoma in situ lesions, 10 benign proliferative lesions, and different normal epithelial components of the male and female breast. We found marked telomere shortening in the majority (52.5%) of invasive carcinomas; smaller subsets of invasive carcinoma demonstrated moderate telomere shortening (17.5%) or normal telomere lengths (21%), while a small subgroup (5%) contained elongated telomeres. Strikingly, the majority (78%) of ductal carcinoma in situ demonstrated markedly or moderately shortened telomeres. Surprisingly, unlike all other normal epithelia studied to date, moderate telomere shortening was observed in benign secretory cells in approximately 50% of histologically-normal terminal duct lobular units (from which most breast cancer is thought to arise), while such shortening was not seen in myoepithelial cells or normal large lactiferous ducts of the female breast or male breast ducts (from which breast cancer infrequently arises). We postulate that such shortening is the result of hormonally driven, physiological proliferation, and may delineate a population of epithelial cells at risk for subsequent malignant transformation.

Very high frequency of hypermethylated genes in breast cancer metastasis to the bone, brain, and lung.

Purpose: Most often it is not the primary tumor, but metastasis to distant organs that results in the death of breast cancer patients. To characterize molecular alterations in breast cancer metastasis, we investigated the frequency of hypermethylation of five genes (Cyclin D2, RAR-β, Twist, RASSF1A, and HIN-1) in metastasis to four common sites: lymph node, bone, brain, and lung. Experimental Design: Methylation-specific PCR for the five genes was performed on DNA extracted from archival paraffin-embedded specimens of paired primary breast cancer and its lymph nodes (LN) metastasis (n = 25 each); in independent samples of metastasis to the bone (n = 12), brain (n = 8), and lung (n = 10); and in normal bone, brain, and lung (n = 22). Results: No hypermethylation was detected in the five genes in the normal host tissues. In paired samples, LN metastasis had a trend of higher prevalence of methylation compared with the primary breast carcinoma for all five genes with significance for HIN-1 (P = 0.04). Compared with the primary breast carcinomas, all five genes had higher methylation frequencies in the bone, brain, and lung metastasis, with HIN-1 and RAR-β methylation being significantly higher (P < 0.01) in each group. Loss of expression of all five genes correlated, with a few exceptions, to hypermethylation of their promoter sequences in metastatic carcinoma cells microdissected from LNs. Conclusion: The frequent presence of hypermethylated genes in locoregional and distant metastasis could render them particularly susceptible to therapy targeted toward gene reactivation combining demethylating agents, histone deacetylase inhibitors, and/or differentiating agents.

Most Basal-like Breast Carcinomas Demonstrate the Same Rb−/p16+ Immunophenotype as the HPV-related Poorly Differentiated Squamous Cell Carcinomas Which They Resemble Morphologically

Basal-like carcinomas (BLCs) of the breast share discriminatory morphologic features with poorly differentiated high-risk human papilloma virus (HPV)-related squamous cell carcinomas of the oropharynx, penis, and vulva. Because HPV E7 protein inactivates the retinoblastoma (Rb) protein, diffuse p16 expression is a surrogate marker for these high-risk HPV-related carcinomas. HPV E6 protein also inactivates p53, further compromising the G1-S cell cycle checkpoint. The Rb/p16/p53 immunohistochemical profile of BLC of the breast has not been well characterized. Tissue microarrays containing 71 invasive ductal carcinomas (IDCs) of the breast were immunolabeled for p16, Rb, p53, and Ki-67. The cases included 4 distinct groups of IDCs having surrogate immunohistochemical profiles corresponding to categories defined by gene expression profiling (17 luminal A, 7 luminal B, 14 HER-2+, and 21 BLC), along with 12 unclassifiable triple negative carcinomas (UTNCs). Twenty-five of the 71 IDC were Rb negative/p16 diffuse positive (Rb−/p16+). These included 15 of 21 BLC and 9 of 12 UTNC, but only 1 of 14 HER-2 positive cases and none of the 17 luminal A or 7 luminal B cases (P<0.01, BLC or UTNC vs. others). Six of the Rb−/p16+ IDC also had a significant ductal carcinoma in situ component. The ductal carcinoma in situ in 4 of these 6 cases showed the same Rb−/p16+ phenotype as the associated IDC. BLC and UTNC had the highest Ki-67 indices of the 5 groups, even when matched for grade. The Rb−/p16+ phenotype and the Rb−/p16+/p53 overexpressing phenotype correlated with increased proliferation within the BLC group. In conclusion, BLC and UTNC, but not HER-2, luminal A, or luminal B carcinomas, frequently demonstrate an Rb−/p16+ phenotype, similar to the HPV-related squamous cell carcinomas that BLC resemble morphologically. This subset may represent a more homogenous group than BLC as defined currently.

Epithelial cell adhesion molecule (EpCAM) is overexpressed in breast cancer metastases

EpCAM (CD326) has diverse roles in cell adhesion and proliferation, and is known to be overexpressed in primary breast carcinomas (PBCs). While clinical and preclinical data suggest a role for EpCAM in metastases, the only prior study of EpCAM expression in breast cancer metastases suggested that EpCAM expression is decreased after first-line chemotherapy. This study evaluates EpCAM expression in metastatic breast carcinoma (MBC) versus matched PBC . Rapid autopsies were performed on 17 patients with widely metastatic breast cancer. Single patient tissue microarrays (TMAs) were constructed from archived PBC and post-mortem MBCs. In total, 169 spots from 17 PBCs and 895 spots from 195 MBCs were labeled for EpCAM by immunohistochemistry (IHC). Expression was scored as intensity (1–3) multiplied by percent membrane labeling (0–100%) and was subclassified as low (0–100), moderate (101–200), or high (201–300) labeling. PBCs exhibited exclusively low-moderate EpCAM labeling. EpCAM labeling was present in all metastases and was significantly increased in MBCs of 14 of 17 patients (P value range <0.05 to <0.0001, t test). In the remaining three patients, EpCAM labeling was nonsignificantly increased in 1 and unchanged in 2. High EpCAM labeling was verified using a different antibody for IHC, as well as in a separate series of surgically resected metastases compared to unmatched surgically resected primary breast cancers. In conclusion, EpCAM is highly expressed in MBCs compared to matched PBCs, verifying that it is a promising therapeutic target.

Neural crest transcription factor Sox10 is preferentially expressed in triple-negative and metaplastic breast carcinomas

The transcription factor Sox10 mediates the differentiation of neural crest-derived cells, and Sox10 labeling by immunohistochemistry (IHC) is used clinically primarily to support the diagnosis of melanoma. Sox10 expression by IHC has been previously documented in benign breast myoepithelial cells but not in breast carcinomas. Here, we report the first systematic study of Sox10 expression in invasive ductal carcinomas subclassified by IHC-defined molecular subtype (100 cases), as well as in 24 cases of ductal carcinoma in situ and 44 mammary fibroepithelial neoplasms. Tissue microarrays containing 168 primary breast tumors were subjected to IHC for Sox10. The extent of nuclear Sox10 labeling was scored by percentage labeling as follows: 0 (0%), 1+ (1%-25%), 2+ (25%-50%), 3+ (50%-75%), and 4+ (>75%). Overall, 40 (40%) of 100 invasive breast carcinomas demonstrated Sox10 immunoreactivity, which was seen primarily in the basal-like, unclassified triple-negative, and metaplastic carcinomas. Sox10 labeling was seen in 66% (38/58) of the basal-like, unclassified triple-negative, and metaplastic carcinomas as compared with 5% (2/42) of the luminal A, luminal B, and Her-2 carcinomas (P < .00001). Sox10 labeling was seen in 1 (4%) of 24 cases of ductal carcinoma in situ, which was negative for estrogen receptor/progesterone receptor. No labeling was seen in the stromal component of phyllodes tumors or fibroadenomas. These findings show that breast carcinoma must be considered in the differential diagnosis of melanoma for an S100-positive, Sox10-positive metastatic malignant neoplasm. Sox10 expression in the basal-like, unclassified triple-negative, and metaplastic carcinomas types supports the concept that these neoplasms show myoepithelial differentiation.

Relationship between molecular subtype of invasive breast carcinoma and expression of gross cystic disease fluid protein 15 and mammaglobin.

We investigated the expression of gross cystic disease fluid protein 15 (GCDFP) and mammaglobin (MGB) by immunohistochemical analysis in 71 invasive breast carcinomas (IBCs) subtyped into luminal (A and B), HER2, basal-like carcinoma (BLC), and unclassified triple-negative carcinoma (UTNC) by established surrogate immunohistochemical profiles. GCDFP and MGB were less likely to be expressed in BLC than in HER2 cancers (P = .000021 and P = .013, respectively) or luminal cancers (P = .00002 and P = .00008, respectively). However, the difference in GCDFP or MGB expression between HER2 and luminal cancers was not significant (P = 1.0 and P = .671, respectively). Our results suggest that luminal cancers demonstrate similar degrees of apocrine differentiation as HER2 cancers. Most BLCs and UTNCs are negative for MGB and GCDFP. Correlation with clinical findings may be needed to exclude the possibility of a metastasis to the breast when BLCs or UTNCs are encountered in a limited sample such as a core biopsy sample.

The immune microenvironment of breast ductal carcinoma in situ

The host immune response has a key role in breast cancer progression and response to therapy. However, relative to primary invasive breast cancers, the immune milieu of breast ductal carcinoma in situ (DCIS) is less understood. Here, we profile tumor infiltrating lymphocytes and expression of the immune checkpoint ligand programmed death ligand 1 (PD-L1) in 27 cases of DCIS with known estrogen receptor (ER), progesterone receptor, and human epidermal growth factor 2 (HER-2) expression using tissue microarrays. Twenty-four cases were pure DCIS and three had associated invasive ductal carcinoma. Tumors were stained by immunohistochemistry for PD-L1, as well as the lymphocyte markers CD3, CD4, CD8, FoxP3, and CD20. The expression of PD-L1 by DCIS carcinoma cells and tumor infiltrating lymphocytes was determined, and the average tumor infiltrating lymphocytes per high power field were manually scored. None of the DCIS cells expressed PD-L1, but 81% of DCIS lesions contained PD-L1+ tumor infiltrating lymphocytes. DCIS with moderate-diffuse tumor infiltrating lymphocytes was more likely to have PD-L1+ tumor infiltrating lymphocytes (P=0.004). Tumor infiltrating lymphocytes with high levels of PD-L1 expression (>50% cells) were seen only in triple-negative DCIS (P=0.0008), and PD-L1−tumor infiltrating lymphocytes were seen only in ER+/HER-2−DCIS (P=0.12). The presence of PD-L1+ tumor infiltrating lymphocytes was associated with a younger mean patient age (P=0.01). Further characterization of the DCIS immune microenvironment may identify useful targets for immune-based therapy and breast cancer prevention.

Metastatic triple-negative breast cancers at first relapse have fewer tumor-infiltrating lymphocytes than their matched primary breast tumors: a pilot study

Tumor-infiltrating lymphocytes (TILs) convey clinically relevant information for primary breast cancers (PBCs). However, limited data characterizing the immunobiology of metastatic breast cancers (MBCs) are available. Here, we examine TILs in surgically resected MBCs relative to their matched PBCs. Tissue microarrays of PBCs and MBCs were labeled for CD3 (total T cells), CD4 (helper T cells), CD8 (cytotoxic T cells), FoxP3 (regulatory T cells), and CD20 (B cells) to characterize TILs. Expression of estrogen receptor (ER), progesterone receptor (PR), and human epidermal growth factor-2 (HER-2) classified the tumors as luminal (ER(+)/PR(+)/HER-2(-)), triple negative (ER(-)/PR(-)/HER-2(-)), or HER-2(+) (ER(-)/PR(-)/HER-2(+)). These analyses reveal 5 novel findings. First, MBCs overall contained fewer TILs (mean, 35.1 CD3(+) TILs/high-power field [hpf]) than their matched PBCs (mean, 23.6 CD3(+) TILS/hpf), with fewer CD20(+) cells than CD3(+) cells in PBC and MBC (P = .0247). Second, the number of CD3(+), CD8(+), CD4(+), and FoxP3 TILs was decreased in triple-negative MBCs relative to matched PBCs, whereas only CD8(+) TILs were decreased in luminal MBCs relative to matched PBCs. Third, triple-negative MBCs contain fewer TILS (mean, 16 CD3(+) TILs/hpf) than luminal MBCs (mean, 21.7 CD3(+) TILs/hpf). Fourth, brain metastases contained fewer TILs relative to MBC from other sites. Finally, in this series, a CD8(+)/FoxP3(+) T-cell ratio of 3 or greater in PBCs was associated with improved overall survival from diagnosis, whereas a CD8(+)/FoxP3(+) T-cell ratio less than 3 in MBCs at first relapse was associated with improved overall survival. These findings suggest that evaluating the immunologic microenvironment of both PBCs and MBCs may yield important clinical information to guide breast cancer prognosis and therapy.

The growth response to androgen receptor signaling in ERα-negative human breast cells is dependent on p21 and mediated by MAPK activation

IntroductionAlthough a high frequency of androgen receptor (AR) expression in human breast cancers has been described, exploiting this knowledge for therapy has been challenging. This is in part because androgens can either inhibit or stimulate cell proliferation in pre-clinical models of breast cancer. In addition, many breast cancers co-express other steroid hormone receptors that can affect AR signaling, further obfuscating the effects of androgens on breast cancer cells.MethodsTo create better-defined models of AR signaling in human breast epithelial cells, we took estrogen receptor (ER)-α-negative and progesterone receptor (PR)-negative human breast epithelial cell lines, both cancerous and non-cancerous, and engineered them to express AR, thus allowing the unambiguous study of AR signaling. We cloned a full-length cDNA of human AR, and expressed this transgene in MCF-10A non-tumorigenic human breast epithelial cells and MDA-MB-231 human breast-cancer cells. We characterized the responses to AR ligand binding using various assays, and used isogenic MCF-10A p21 knock-out cell lines expressing AR to demonstrate the requirement for p21 in mediating the proliferative responses to AR signaling in human breast epithelial cells.ResultsWe found that hyperactivation of the mitogen-activated protein kinase (MAPK) pathway from both AR and epidermal growth factor receptor (EGFR) signaling resulted in a growth-inhibitory response, whereas MAPK signaling from either AR or EGFR activation resulted in cellular proliferation. Additionally, p21 gene knock-out studies confirmed that AR signaling/activation of the MAPK pathway is dependent on p21.ConclusionsThese studies present a new model for the analysis of AR signaling in human breast epithelial cells lacking ERα/PR expression, providing an experimental system without the potential confounding effects of ERα/PR crosstalk. Using this system, we provide a mechanistic explanation for previous observations ascribing a dual role for AR signaling in human breast cancer cells. As previous reports have shown that approximately 40% of breast cancers can lack p21 expression, our data also identify potential new caveats for exploiting AR as a target for breast cancer therapy.