Is Oncotype DX testing informative for breast cancers with low ER expression? A retrospective review from a biomarker testing referral center.

PURPOSE: It remains unclear whether patients with HER2-negative, low-estrogen receptor (ER-low)-positive early breast cancer (BC) benefit from Oncotype DX® (ODX) testing. METHODS: We conducted a retrospective review of cases referred for ODX testing over a seven-year period from a breast biomarker testing referral center (n = 854). For each case, we recorded the ODX Recurrence Score (RS) along with percentage of ER nuclear positivity and staining intensity on immunohistochemistry. Our criteria for ER-low was defined as ≤10% cells with nuclear positivity and/or weak intensity of staining. Slides from all ER-low cases were reviewed and the reported ODX ER gene scores were recorded. We randomly selected a comparator group of 56 patients with ER > 10% positivity and non-weak staining intensity (ER-high). RESULTS: We identified 27 cases (3.2%) that met our criteria for ER-low. Of these, 92.6% had a high RS (>25), and 7.4% had a RS of 25. All cases with ≤10% ER nuclear positivity had a high RS. Most ER-low cases (85.2%) had ODX quantitative ER gene scores in the negative range, whereas all (100%) ER-high cases had positive ER gene scores. CONCLUSION: ODX does not appear to add significant additional information to inform treatment decisions for most patients with ER-low BC. Incorporating weak ER staining intensity in addition to low percentage of nuclear positivity identifies about twice as many ER-low patients, although with reduced specificity for high RS. Our study supports the contention that most ER-low early BC should be regarded similarly to ER-negative BC.

ALDH1A3 promotes invasion and metastasis in triple-negative breast cancer by regulating the plasminogen activation pathway.

Aldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell marker that promotes metastasis. Triple-negative breast cancer (TNBC) progression has been linked to ALDH1A3-induced gene expression changes. To investigate the mechanism of ALDH1A3-mediated breast cancer metastasis, we assessed the effect of ALDH1A3 on the expression of proteases and the regulators of proteases that degrade the extracellular matrix, a process that is essential for invasion and metastasis. This revealed that ALDH1A3 regulates the plasminogen activation pathway; it increased the levels and activity of tissue plasminogen activator (tPA) and urokinase plasminogen activator (uPA). This resulted in a corresponding increase in the activity of serine protease plasmin, the enzymatic product of tPA and uPA. The ALDH1A3 product all-trans-retinoic acid similarly increased tPA and plasmin activity. The increased invasion of TNBC cells by ALDH1A3 was plasminogen-dependent. In patient tumours, ALDH1A3 and tPA are co-expressed and their combined expression correlated with the TNBC subtype, high tumour grade and recurrent metastatic disease. Knockdown of tPA in TNBC cells inhibited plasmin generation and lymph node metastasis. These results identify the ALDH1A3-tPA-plasmin axis as a key contributor to breast cancer progression.

Independent primary cutaneous and mammary apocrine carcinomas with neuroendocrine differentiation: Report of a case and literature review.

Cutaneous apocrine carcinomas share common features with their counterparts in the breast; hence, metastatic mammary carcinoma must be excluded before such lesions can be designated primary cutaneous neoplasms. Primary tumors from either source rarely exhibit neuroendocrine differentiation. We report a case of a 72-year-old female with a painless 1.2-cm scalp nodule. An incisional biopsy revealed dermal involvement by an invasive apocrine carcinoma juxtaposed to a benign apocrine cystic lesion. Immunohistochemically, the carcinoma expressed neuroendocrine proteins including synaptophysin, chromogranin, and CD56. A primary cutaneous apocrine carcinoma with neuroendocrine differentiation was favored, but additional investigations to exclude breast origin were recommended. These revealed a 1.1-cm nodule in the right breast, which proved to be an invasive ductal carcinoma, morphologically and immunophenotypically similar to the scalp lesion. This confounded the case, yet factors militating against metastatic breast carcinoma to skin included (a) the small size of the mammary tumor, (b) absence of other metastatic disease, and (c) juxtaposition of the scalp carcinoma to a putative benign precursor. Molecular studies were undertaken to resolve the diagnostic quandary. Single nucleotide polymorphism microarray analysis revealed distinct patterns of chromosomal copy number alterations in the two tumors, supporting the concept of synchronous and unusual primary neoplasms.

S100A10 Has a Critical Regulatory Function in Mammary Tumor Growth and Metastasis: Insights Using MMTV-PyMT Oncomice and Clinical Patient Sample Analysis.

S100A10 (p11) is a plasminogen receptor that regulates cellular plasmin generation by cancer cells. In the current study, we used the MMTV-PyMT mouse breast cancer model, patient tumor microarray, and immunohistochemical (IHC) analysis to investigate the role of p11 in oncogenesis. The genetic deletion of p11 resulted in significantly decreased tumor onset, growth rate, and spontaneous pulmonary metastatic burden in the PyMT/p11-KO (knock-out) mice. This phenotype was accompanied by substantial reduction in Ki67 positivity, macrophage infiltration, decreased vascular density in the primary tumors, and decrease in invasive carcinoma and pulmonary metastasis. Surprisingly, IHC analysis of wild-type MMTV-PyMT mice failed to detect p11 expression in the tumors or metastatic tumor cells and loss of p11 did not decrease plasmin generation in the PyMT tumors and cells. Furthermore, tumor cells expressing p11 displayed dramatically reduced lung metastasis when injected into p11-depleted mice, further strengthening the stromal role of p11 in tumor growth and metastasis. Transcriptome analysis of the PyMT tumors from p11-KO mice showed marked reduction in genes such as Areg, Muc1, and S100a8 involved in breast cancer development, progression, and inflammation. The PyMT/p11-KO tumors displayed a remarkable increase in inflammatory cytokines such as interleukin (Il)-6, Il-10, and interferon (Ifn)-γ. Gene expression profiling and IHC of primary breast cancer samples showed that p11 mRNA and protein levels were significantly higher in tumor tissues compared to normal mammary tissue. P11 mRNA expression was significantly associated with poor patient prognosis and significantly elevated in high grade, triple negative (TN) tumors, and tumors with high proliferative index. This is the first study examining the crucial role of p11 in breast tumor development and metastasis, thus emphasizing its potential as a diagnostic and prognostic biomarker in breast cancer.

Prolactin and androgen R1881 induce pro-survival carboxypeptidase-D and EDD E3 ligase in triple-negative and HER2+ breast cancer.

Plasma membrane carboxypeptidase-D (CPD) hydrolyzes C-terminal arginine (Arg) from extracellular substrates, and Arg is converted into nitric oxide (NO) in the cell. CPD is upregulated by prolactin (PRL) and androgens in breast cancer (BCa) cells, increasing NO production to promote cell survival. EDD E3 ubiquitin ligase, upregulated by PRL/androgens, is implicated in TORC1 signaling. This study investigated CPD and EDD in triple-negative (TNBC) and HER2+ BCa. Kaplan-Meier analysis showed a negative correlation between CPD or EDD mRNA expression in TNBC patients and relapse-free survival. Immunohistochemistry showed that benign and malignant breast tissues stained abundantly for the PRL receptor (PRLR) and androgen receptor (AR). CPD and EDD staining were elevated in TNBC and HER2+ tumors as compared to benign tissues. In TNBC/HER2+ cell lines, CPD and EDD protein expression were upregulated by PRL or synthetic androgen methyltrienolone (R1881) at 3-6 h. PRL/R1881-induced CPD in TNBC and HER2+ cells increased intracellular NO production, which was abolished by PRLR antagonist ∆1-9-G129R-hPRL and AR antagonist flutamide. In turn, treatment with NO increased viability and decreased apoptosis in Arg-deprived TNBC cells. Cell viability and apoptosis were also affected in HER2+ cells with CPD knockdown. Lastly, EDD knockdown decreased PRL/R1881-induced phosphorylation of initiation factor 4E binding protein-1 and decreased 4E release in TNBC cells. In summary, PRL/R1881-induced CPD promotes TNBC/HER2+ cell survival through production of NO, and EDD promotes TNBC cell survival by TORC1 activation. This study implicates CPD and EDD as useful therapeutic targets for TNBC/HER2+ tumors, and suggests that PRLR and AR blockade are also beneficial to these patients.

Prolactin-inducible EDD E3 ubiquitin ligase promotes TORC1 signalling, anti-apoptotic protein expression, and drug resistance in breast cancer cells.

Previously, we identified a prolactin (PRL)-inducible gene encoding EDD E3 ubiquitin ligase in human breast cancer (BCa) cells. We reported that EDD binds the mTOR (TORC1)-associated α4 phosphoprotein-PP2Ac protein phosphatase complex that regulates initiation of translation and cell cycle progression, and that EDD targets PP2Ac for proteasomal degradation. The present study showed that EDD immunostaining was low in benign human breast tissues, but increased progressively in ductal carcinoma in-situ, low-grade, and high-grade BCa, and in triple-negative BCa (TNBC). EDD mRNA and protein levels varied in human BCa cell lines. In high-EDD expressing MCF-7 and T47D cells, siRNA knockdown of EDD arrested cells in the G2-phase of the cell cycle, decreased cell viability, and increased apoptosis. EDD siRNA-induced apoptosis in MCF-7 cells correlated with significantly increased levels of pro-apoptotic Bim and Bak mRNAs and proteins (P < 0.05, n = 3-6), and increased levels of pro-apoptotic Bax and MOAP-1 proteins (P < 0.001, n = 3-6), leading to increased cleavage of caspase-7 and caspase substrate poly-ADP-ribose polymerase-1 (PARP-1), as compared to control cells. Loss of EDD in MCF-7 cells decreased PRL-induced phosphorylation of eukaryotic initiation factor 4E-binding protein-1, a mediator of TORC1 signaling, resulting in decreased binding of 4E to γ-aminophenyl-m7GTP agarose in Cap-binding assays. In low-EDD expressing MDA-MB-436 TNBC cell line, gain of EDD following pCMV-Tag2B.EDD transfection increased cell resistance to chemotherapeutic drugs cisplatin and doxorubicin, TORC1 inhibitor rapamycin, and TORC1/TORC2 inhibitor INK128, as compared to controls. In contrast, loss of EDD in MCF-7 cells increased cell sensitivity to cisplatin, doxorubicin, rapamycin, and selective estrogen receptor modulator tamoxifen. In summary, EDD levels increase with BCa progression in vivo. PRL-inducible EDD in BCa cells promotes TORC1 signaling, anti-apoptotic protein expression, and drug resistance in vitro. These findings implicate EDD as a potential therapeutic target and support PRL receptor blockade as an additional therapy for BCa.

Prolactin/androgen-inducible carboxypeptidase-D increases with nitrotyrosine and Ki67 for breast cancer progression in vivo, and upregulates progression markers VEGF-C and Runx2 in vitro.

PURPOSE: Carboxypeptidase-D (CPD) cleaves C-terminal arginine (Arg) to produce nitric oxide (NO). Upregulation of CPD and NO by 17ß-estradiol, prolactin (PRL), and androgen increases survival of human breast cancer (BCa) cells in vitro. To demonstrate similar events in vivo, CPD, nitrotyrosine (NT, hallmark of NO action), androgen receptor (AR), prolactin receptor (PRLR), and phospho-Stat5a (for activated PRLR) levels were evaluated in benign and malignant human breast tissues, and correlated with cell proliferation (Ki67) and BCa progression (Cullin-3) biomarkers. METHODS: Paraffin-embedded breast tissues were analyzed by immunohistochemistry (IHC). BCa progression markers in human MCF-7 and T47D BCa cell lines treated with NO donor SIN-1 or PRL, ±CPD inhibitors were analyzed by RT-qPCR and immunoblotting. RESULTS: IHC showed progressive increases in CPD, NT, Ki67, and Cullin-3 from low levels in benign tissues to high levels in ductal carcinoma in situ, low-grade, high-grade, and triple-negative BCa. CPD and NT staining were closely associated, implicating CPD in NO production. Phospho-Stat5a increased significantly from benign to high-grade BCa and was mostly nuclear. AR and PRLR were abundant in benign breast and BCa, including triple-negative tumors. SIN-1 and PRL increased VEGF-C and Runx2, but not Cullin-3, in BCa cell lines. PRL induction of VEGF-C and Runx2 was inhibited partly by CPD inhibitors, implicating NO, produced by PRL-regulated CPD, in BCa progression. CONCLUSIONS: The CPD-Arg-NO pathway contributes to BCa progression in vitro and in vivo. PRL/androgen activation of the pathway support combined AR and PRLR blockade as an additional therapy for BCa.

Well-differentiated invasive breast cancers with equivocal HER2 immunohistochemistry: what is the yield of routine reflex in-situ hybridization testing?

AIMS: The 2013 American Society of Clinical Oncology and the College of American Pathologists (ASCO/CAP) HER2 guidelines recommend testing all invasive breast cancers for HER2, typically with immunohistochemistry (IHC) followed by in-situ hybridization (ISH) when IHC is equivocal. As well-differentiated breast cancers are rarely HER2-positive, we assessed the value of routine reflex HER2 ISH testing for this subset of breast cancers. METHODS AND RESULTS: We collected HER2 IHC 2+ cases and fluorescence in-situ hybridization (FISH) data from primary breast cancers with well-differentiated tumour types (grade 1 ductal carcinomas, classic lobular carcinomas, tubular, cribriform and pure mucinous carcinomas) at our centre from 2010 to 2015. Haematoxylin and eosin (H&E) and IHC slides were reviewed to confirm tumour type, grade and IHC score based on ASCO/CAP 2013 guidelines and their recent revisions. Of 4633 invasive carcinomas, 1133 had a well-differentiated tumour type; 177 of these were HER2 IHC equivocal, three of which were low-level amplified by FISH (0.3% of all well-differentiated tumours). One amplified case was classic invasive lobular carcinoma and two were invasive ductal carcinomas, grade 1. One amplified case had chromosome 17 monosomy, and one was rescored as HER2 IHC 1+ upon review. 'Basolateral' staining was noted in one amplified case and in 65 of 174 (37.4%) non-amplified cases. This incomplete membranous staining pattern was observed in the majority of invasive ductal carcinomas that were rescored as 1+ according to the revised 2013 guidelines. CONCLUSIONS: The rate of HER2 amplification among well-differentiated breast cancers is very low. Basolateral staining in well-differentiated tumours may be overinterpreted as HER2 IHC 2+, but is rarely associated with HER2 amplification.

Loss of expression of DNA mismatch repair proteins is rare in pancreatic and small intestinal neuroendocrine tumors.

CONTEXT: Two recent studies have identified a high rate of microsatellite instability (MSI) in pancreatic neuroendocrine tumors (pNETs). Microsatellite instability is rare in small intestinal neuroendocrine tumors (NETs). It is unclear why there is discordance in the frequency of MSI in the 2 studies of pNETs and why this mechanism is comparatively rare in small intestinal tumors. Loss of expression of DNA mismatch repair (MMR) proteins, which is known to correlate strongly with MSI, is not well studied in pancreatic or small intestinal NETs. OBJECTIVE: To determine if there is loss of expression of MMR protein expression in pancreatic or small intestinal NETs. DESIGN: Sixty-nine patients (31 male, 38 female; mean age, 59.2 years) were identified who had a resection for a primary pancreatic (n  =  35) or primary small intestinal (n  =  34) NET during an 18-year period. Immunohistochemical stains for MLH1, MSH2, MSH6, and PMS2 were applied to archived tissue from all cases. All pNETs with adequate tissue (n  =  32) were also assessed by MSI analysis. RESULTS: There was preserved expression of MLH1, MSH2, MSH6, and PMS2 in all 35 pNETs. Of 32 pNETs tested by polymerase chain reaction, 28 were microsatellite stable and DNA did not amplify in 4. In 34 small intestinal NETs, 2 cases had indeterminate MLH1 and 1 case had indeterminate PMS2 expression. The remainder had intact MMR protein expression. CONCLUSION: Defects in DNA MMR proteins are rare in pancreatic and small intestinal NETs, raising doubt that MSI plays a significant role in the pathogenesis of these tumors.

Immunohistochemical expression and prognostic value of ER, PR and HER2/neu in pancreatic and small intestinal neuroendocrine tumors.

BACKGROUND AND AIMS: There has been limited study of estrogen and progesterone receptor (ER/PR) expression in gastrointestinal neuroendocrine tumors (GINETs) despite emerging evidence of hormone receptor regulation of pancreatic islet cells. Beta cells express PR and progesterone has been implicated in the pathogenesis of gestational diabetes. There is conflicting information regarding HER2/neu protein overexpression in GINETs. Investigation of ER, PR and HER2/neu expression in GINETs is therefore warranted. METHODS: A pathology database search identified 77 patients with primary pancreatic (40) or small intestinal (37) NETs diagnosed from 1991 to 2009. Ki67, ER, PR and HER2/neu were assessed via immunohistochemistry. ER and PR were interpreted as negative (0), 1+ (Allred score 3-7/8) or 2+ (Allred score 8/8), and HER2/neu was assessed according to ASCO/CAP guidelines for breast carcinoma. Clinical correlation and survival outcomes were ascertained by a retrospective clinical chart review. RESULTS: 2+ PR staining was observed more often in pancreatic compared to small intestinal cases (55 vs. 8%; p < 0.001). All small intestinal NETs with 2+ PR were duodenal primaries. Cases with 2+ PR presented significantly less often with nodal or distant metastases compared to cases with 0/1+ PR (13 vs. 61.5%; p < 0.001) and had significantly improved disease-free survival (median 155 vs. 38 months; p = 0.037). Only one case demonstrated 2+ ER staining and all were negative for HER2/neu. CONCLUSION: GINETs with strong (2+) PR expression are associated with pancreatic/duodenal origin, lower stage disease, and more favorable clinical prognosis. Further study is needed to determine the clinical utility of PR expression in GINETs.