Sandra J. Shin

XBP1 promotes triple-negative breast cancer by controlling the HIF1α pathway

Cancer cells induce a set of adaptive response pathways to survive in the face of stressors due to inadequate vascularization. One such adaptive pathway is the unfolded protein (UPR) or endoplasmic reticulum (ER) stress response mediated in part by the ER-localized transmembrane sensor IRE1 (ref. 2) and its substrate XBP1 (ref. 3). Previous studies report UPR activation in various human tumours, but the role of XBP1 in cancer progression in mammary epithelial cells is largely unknown. Triple-negative breast cancer (TNBC)—a form of breast cancer in which tumour cells do not express the genes for oestrogen receptor, progesterone receptor and HER2 (also called ERBB2 or NEU)—is a highly aggressive malignancy with limited treatment options. Here we report that XBP1 is activated in TNBC and has a pivotal role in the tumorigenicity and progression of this human breast cancer subtype. In breast cancer cell line models, depletion of XBP1 inhibited tumour growth and tumour relapse and reduced the CD44highCD24low population. Hypoxia-inducing factor 1α (HIF1α) is known to be hyperactivated in TNBCs. Genome-wide mapping of the XBP1 transcriptional regulatory network revealed that XBP1 drives TNBC tumorigenicity by assembling a transcriptional complex with HIF1α that regulates the expression of HIF1α targets via the recruitment of RNA polymerase II. Analysis of independent cohorts of patients with TNBC revealed a specific XBP1 gene expression signature that was highly correlated with HIF1α and hypoxia-driven signatures and that strongly associated with poor prognosis. Our findings reveal a key function for the XBP1 branch of the UPR in TNBC and indicate that targeting this pathway may offer alternative treatment strategies for this aggressive subtype of breast cancer.

Molecular profiling pleomorphic lobular carcinomas of the breast: evidence for a common molecular genetic pathway with classic lobular carcinomas

Pleomorphic lobular carcinomas (PLC) of the breast display histological features associated with classic invasive lobular carcinoma (ILC), yet they also exhibit more conspicuous nuclear atypia and pleomorphism, and an aggressive clinical behaviour. From a breast cancer progression perspective, it is unclear whether PLC is a variant of ILC or is a high‐grade invasive ductal carcinoma (IDC) that has lost E‐cadherin. The molecular features of 26 PLC were studied using immunohistochemistry [oestrogen receptor (ER), progesterone receptor (PR), HER2, p53 and E‐cadherin], 0.9 Mb resolution, microarray‐based comparative genomic hybridization (aCGH), fluorescent (FISH) and chromogenic (CISH) in situ hybridization and loss of heterozygosity. Comparative analysis was performed with aCGH data from PLC with classic ILC (16 cases) and high grade IDC (35 cases). PLCs were frequently ER‐ and PR‐positive, E‐cadherin‐negative and occasionally HER2‐ and p53‐positive. Recurrent copy number changes identified by aCGH included gains on 1q, 8q, 11q, 12q, 16p and 17q and losses on 8p, 11q, 13q, 16q and Xq. Highly recurrent 1q+ (100% of cases), 16p+ (93%), 11q− (53%) and 16q− (93%) and evidence of the der(1;16)/der(16)t(1;16) rearrangement, as detected by FISH, suggested that PLC had a ‘lobular genotype’. Focal amplifications were evident at 8p12‐p11, 8q24, 11q13.1‐q14.1, 12q14, 17q12 and 20q13. Loss of BRCA2 was detected in 40% of PLC by LOH. Comparative analysis of aCGH data suggested the molecular features of PLC (ER/PR‐positive, E‐cadherin‐negative, 1q+, 11q−, 16p+ and 16q−) were more closely related to those of ILC than IDC, implicating an overlapping developmental pathway for these lobular tumour types. Molecular alterations found in PLC that are more typical of high‐grade IDC than ILC (p53 and HER2 positivity, 8q+, 17q24‐q25+, 13q− and amplification of 8q24, 12q14, 17q12 and 20q13) are likely to drive the high‐grade and more aggressive biology of PLC. Copyright

Prolyl hydroxylation by EglN2 destabilizes FOXO3a by blocking its interaction with the USP9x deubiquitinase

The three EglN prolyl hydroxylases (EglN1, EglN2, and EglN3) regulate the stability of the HIF transcription factor. We recently showed that loss of EglN2, however, also leads to down-regulation of Cyclin D1 and decreased cell proliferation in a HIF-independent manner. Here we report that EglN2 can hydroxylate FOXO3a on two specific prolyl residues in vitro and in vivo. Hydroxylation of these sites prevents the binding of USP9x deubiquitinase, thereby promoting the proteasomal degradation of FOXO3a. FOXO transcription factors can repress Cyclin D1 transcription. Failure to hydroxylate FOXO3a promotes its accumulation in cells, which in turn suppresses Cyclin D1 expression. These findings provide new insights into post-transcriptional control of FOXO3a and provide a new avenue for pharmacologically altering Cyclin D1 activity.

Intratumoral Heterogeneity of HER-2/neu in Invasive Mammary Carcinomas Using Fluorescence In-Situ Hybridization and Tissue Microarray

Fluorescence in-situ hybridization is increasingly being used to determine HER-2/neu status in patients with breast carcinoma. The possibility that intratumoral heterogeneity of HER-2/neu gene amplification may potentially contribute to inaccurate assessment of HER-2/neu status was investigated in routine cases of invasive mammary carcinomas. From 169 representative formalin-fixed, paraffin-embedded blocks of invasive duct mammary carcinomas with grade 3 architecture, 48 cases were analyzed by fluorescence in-situ hybridization and 59 analyses were performed. Intratumoral heterogeneity for HER-2/neu gene amplification was demonstrated in only 5 (16%) of 31 cases where morphologically similar areas of a single tumor were analyzed. We conclude from this study that intratumoral heterogeneity of HER-2/neu gene amplification is a demonstrable but relatively uncommon occurrence. For invasive mammary carcinomas, the accurate assessment of HER-2/neu status by fluorescence in-situ hybridization analysis is not significantly confounded by intratumoral heterogeneity of HER-2/neu gene amplification in individual tumors.

Diagnostic utility of MYC amplification and anti-MYC immunohistochemistry in atypical vascular lesions, primary or radiation-induced mammary angiosarcomas, and primary angiosarcomas of other sites

Breast cancer patients who receive radiation therapy or develop chronic lymphedema following axillary dissection can develop secondary mammary angiosarcomas (ASs) and, additionally, atypical vascular lesions (AVLs) in the former group. Recently, MYC amplification by fluorescence in situ hybridization (FISH) has been identified in secondary mammary AS but not in AVL and most primary mammary AS as well as AS of other sites. We studied MYC amplification and MYC protein expression in 7 radiation-induced AVLs, 9 secondary mammary ASs, 17 primary mammary ASs, and 20 primary ASs of other sites by FISH analysis and immunohistochemistry. All 9 secondary mammary ASs showed gene amplification and protein expression, whereas neither was found in any of 7 AVLs. No MYC amplification or protein expression was identified in any of the 17 primary mammary ASs. Among primary ASs of other sites, 1 cardiac AS and 1 skin AS showed gene amplification and protein expression. The remaining 18 did not show amplification (90%), but some demonstrated protein expression (39%). We conclude that MYC amplification by FISH is present in secondary mammary AS but not in AVL. We also found MYC amplification in 1 primary skin AS and 1 primary cardiac AS. There was 100% concordance between MYC amplification and protein expression in all AVL, primary mammary AS, and secondary mammary AS, whereas only 65% concordance was found in AS of other sites. MYC protein expression in AS can be helpful in certain diagnostic scenarios in the breast but not in other sites.

Cancer/testis antigen CT45: Analysis of mRNA and protein expression in human cancer

CT45 is a cancer/testis gene that we previously identified by massively parallel signature sequencing. Encoded by a multigene family on chromosome X, CT45 showed restricted mRNA expression to normal testis and various cancers. In this study, monoclonal antibodies were generated against recombinant CT45 protein, and CT45 protein expression in normal and tumor tissues was evaluated by immunohistochemical analysis. In adult normal tissue, CT45 expression was restricted to testicular germ cells, detected as a nuclear protein mainly at the stage of primary spermatocytes. In tumors, CT45 protein expression correlated with the mRNA levels detected by quantitative RT‐PCR, and most lung cancer and ovarian cancers with CT45 mRNA at levels >1% of testicular expression were CT45 protein‐positive. In positive cases, CT45 showed expression patterns that ranged from diffuse strong staining to heterogeneous and patchy expression. In lung cancer, CT45 expression was least frequent in adenocarcinoma, more frequent in squamous cell carcinoma and neuroendocrine tumors. Using tissue microarrays, 376 lung cancer, 219 ovarian cancer and 155 breast cancer were evaluated for CT45 protein expression. The expression frequency was highest in ovarian cancer (37%), followed by lung cancer (13%) and lowest in breast cancer (<5%). Given the focal nature of CT45 expression in many cases, these numbers represented the minimal frequency of expression in these tumor types. In summary, the expression frequency and characteristics of CT45 expression are similar to other CT cancer vaccine targets currently in clinical trials, e.g., NY‐ESO‐1 and MAGE‐A, suggesting CT45 as a potentially useful cancer target.

Prostate-specific membrane antigen expression in tumor-associated vasculature of breast cancers

Prostate‐specific membrane antigen (PSMA) has been found to be expressed in the tumor‐associated neovasculature of multiple solid tumor types including breast cancers. However, thus far, the number of cases studied from some tumor types has been limited. In this study, we set out to assess PSMA expression in the tumor‐associated vasculature associated with invasive breast carcinomas in a sizable cohort of patients. One hundred and six patients with AJCC stage 0‐IV breast cancer were identified. Ninety‐two of these patients had primary breast cancer [invasive breast carcinoma with or without co‐existing ductal carcinoma in situ (DCIS) (74) or DCIS alone (18)]. In addition, 14 patients with breast cancer metastases to the brain were identified. Immunohistochemical staining for PSMA and CD31 was performed on parallel representative tumor sections in each case. Tumor‐associated vascular endothelial cell PSMA immunoreactivity was semi‐quantitatively assessed based on two parameters: overall percent of endothelial positivity and staining intensity. PSMA expression for tumor‐associated vascular endothelial cells was scored 0 if there was no detectable PSMA expression, 1 if PSMA staining was detectable in 5–50%, and 2 if PSMA expression was positive in >50% of microvessels. CD 31 staining was concurrently reviewed to confirm the presence of vasculature in each case. Tumor‐associated vasculature was PSMA‐positive in 68/92 (74%) of primary breast cancers and in 14/14 (100%) of breast cancers metastatic to brain. PSMA was not detected in normal breast tissue or carcinoma cells. All but 2 cases (98%) showed absence of PSMA expression in normal breast tissue‐associated vasculature. The 10‐year overall survival was 88.7% (95% CI = 80.0%, 93.8%) in patients without brain metastases. When overall survival (OS) was stratified based on PSMA score group, patients with PSMA scores of 0, 1, and 2 had 10‐year OS of 95.8%, 96.0%, and 79.7%, respectively (p = 0.12). When PSMA scores of 0 and 1 were compared with 2, there was a statistically significant difference in OS (96.0% vs 79.7%, respectively, p = 0.05). Patients with a PSMA score of 2 had a significantly higher median tumor size compared with patients in the lower PSMA score groups (p = 0.04). Patients with higher nuclear grade were more likely to have a PSMA score of 2 compared with patients with lower nuclear grade (p < 0.0001). Patients with a PSMA score of 2 had a significantly higher median Ki‐67 proliferation index compared with patients in the lower PSMA score groups (p < 0.0001). Patients with estrogen receptor (ER)‐negative tumors were more likely to have a PSMA score of 2 compared with patients with ER‐positive tumors (p < 0.0001). Patients with progesterone receptor (PR)‐negative tumors were more likely to have a PSMA score of 2 compared with patients with PR‐positive tumors (p = 0.03). No significant association was observed between PSMA score group status and lymph node involvement (p = 0.95). Too little variability was present in Human epidermal growth factor receptor‐2 (Her2/neu) amplified tumors to correlate with PSMA score group status. To date, this is the first detailed assessment of PSMA expression in the tumor‐associated vasculature of primary and metastatic breast carcinomas. Further studies are needed to evaluate whether PSMA has diagnostic and/or potential therapeutic value.

MYB-NFIB gene fusion in adenoid cystic carcinoma of the breast with special focus paid to the solid variant with basaloid features

Adenoid cystic carcinomas (ACCs) from various anatomical sites harbor a translocation t(6;9)(q22-23;p23-24), resulting in MYB-NFIB gene fusion. This gene fusion is not well studied in mammary ACCs, and there are no studies examining this abnormality in solid variant of ACC with basaloid features (SBACC), a high-grade variant thought to behave more aggressively than ACCs with conventional histologic growth. Our aim was to investigate the frequency of MYB-NFIB gene fusion in mammary ACCs with a focus paid to SBACC. MYB rearrangement and MYB-NFIB fusion were assessed by fluorescence in situ hybridization and reverse-transcription polymerase chain reaction, respectively. Histologic features and the presence of MYB rearrangement were correlated with clinical outcome. MYB rearrangement was present in 7 (22.6%) of 31 mammary ACCs (5/15 [33.3%] ACCs with conventional growth; 2/16 [12.5%] SBACCs). One patient with conventional ACC developed distant metastasis, and no patients had axillary lymph node involvement by ACC (mean follow-up, 34 months; range, 12-84 months). Two patients with SBACC had axillary lymph node involvement at initial surgery, and 2 additional patients experienced disease recurrence (1 local, 1 distant; mean follow-up, 50 months; range, 9-192 months). MYB-NFIB fusion status did not correlate with clinical outcome in studied patients. We confirm that MYB-NFIB gene fusion is observed in mammary ACCs and that a subset lacks this abnormality. This study is the first to confirm the presence of MYB rearrangement in SBACC. Additional validation with long-term follow-up is needed to determine the relationship, if any, between MYB-NFIB gene fusion and clinical outcome.

Diagnostic utility of the monoclonal antibody A103 in fine-needle aspiration biopsies of the adrenal.

Fine-needle aspiration (FNA) of the adrenal is a useful modality for the evaluation of primary and metastatic neoplasms. Until now, however, few reliable markers existed for the positive identification of adrenal cortical cells. Originally studied as a melanoma marker, Melan-A, as detected by the murine monoclonal antibody, A103, has gained recent attention as a marker for steroid-producing cells. Formalin-fixed, paraffin-embedded cell blocks from 24 adrenal FNA specimens were stained for cytokeratins (AE1/AE3) and Melan-A (A103). Seven of 8 cases containing normal, hyperplastic, and neoplastic adrenal cortical cells were positive for A103. Among 16 cases of metastatic carcinoma, tumor cells in 14 samples were positive for cytokeratins but negative for A103. The A103 monoclonal antibody is a sensitive marker for the identification of normal, hyperplastic, and neoplastic adrenal cortical cells in cell blocks of adrenal FNA specimens. With the exception of melanoma, A103 reactivity is restricted to adrenal cortical and other steroid-producing cells. A103 should be used routinely for the evaluation of FNA specimens of adrenal mass lesions.

Florid lobular carcinoma in situ: molecular profiling and comparison to classic lobular carcinoma in situ and pleomorphic lobular carcinoma in situ.

We evaluated genomic alterations and biomarker expression in 20 florid lobular carcinomas in situ using array-based comparative genomic hybridization and immunohistochemical analysis. The genetic characteristics of florid lobular carcinoma in situ were compared with 20 classic lobular carcinomas in situ and 21 pleomorphic lobular carcinomas in situ (which included 8 apocrine variants), from our previously published data performed on a similar array-based comparative genomic hybridization platform. All 20 florid lobular carcinoma in situ cases were E-cadherin negative, and 92% were positive for estrogen receptor. Cyclin D1 expression correlated significantly negatively with estrogen receptor expression and was higher in cases with cyclin D1 (CCND1) gene amplification. Compared with classic lobular carcinoma in situ, florid lobular carcinoma in situ displayed significantly more fraction genome alteration (mean, 0.109 versus 0.072; P=.007), fraction genome loss (mean, 0.06 versus 0.03; P=.007), numbers of breakpoints (mean, 11.55 versus 6.95; P=.002), numbers of chromosome with breakpoints (mean, 5.85 versus 3.8; P=.004), and higher numbers of amplifications (mean, 2.10 versus 0.25; P=.03). Interestingly, florid lobular carcinoma in situ had the same genetic complexity as apocrine pleomorphic lobular carcinoma in situ. Our study demonstrated that florid lobular carcinoma in situ shares the cytologic features, E-cadherin loss, and the lobular genetic signature of 1q gain and 16q loss found in classic lobular carcinoma in situ. However, this variant demonstrates more genomic alterations than classic lobular carcinoma in situ and shares the same genetic complexity as apocrine pleomorphic lobular carcinoma in situ. Our data support the conclusion that florid lobular carcinoma in situ is genetically more advanced compared with the indolent phenotype of classic lobular carcinoma in situ. This may explain the greater frequency of concurrent invasive carcinoma in florid lobular carcinoma in situ compared with classic lobular carcinoma in situ.