Breast cancer staging by subtype in the Lower Mississippi Delta region States

IntroductionTo evaluate disparities in breast cancer stage by subtype (categorizations of breast cancer based upon molecular characteristics) in the Delta Regional Authority (Delta), an impoverished region across eight Lower Mississippi Delta Region (LMDR) states with a high proportion of Black residents and high breast cancer mortality rates.MethodsWe used population-based cancer registry data from seven of the eight LMDR states to explore breast cancer staging (early and late) differences by subtype between the Delta and non-Delta in the LMDR and between White and Black women within the Delta. Age-adjusted incidence rates and rate ratios were calculated to examine regional and racial differences. Multilevel negative binomial regression models were constructed to evaluate how individual-level and area-level factors affect rates of early- and late-stage breast cancers by subtype.ResultsFor all subtypes combined, there were no Delta/non-Delta differences in early and late stage breast cancers. Delta women had lower rates of hormone-receptor (HR+)/human epidermal growth factor 2 (HER2-) and higher rates of HR-/HER2- (the most aggressive subtype) early and late stage cancers, respectively, but these elevated rates were attenuated in multilevel models. Within the Delta, Black women had higher rates of late-stage breast cancer than White women for most subtypes; elevated late-stage rates of all subtypes combined remained in Black women in multilevel analysis (RR = 1.10; 95% CI = 1.04–1.15).ConclusionsBlack women in the Delta had higher rates of late-stage cancers across subtypes. Culturally competent interventions targeting risk-appropriate screening modalities should be scaled up in the Delta to improve early detection.     

C-FLIP(L) contributes to TRAIL resistance in HER2-positive breast cancer

Breast cancers with HER2 amplification have a poorer prognosis than the luminal phenotypes. TRAIL activates apoptosis upon binding its receptors in some but not all breast cancer cell lines. Herein, we investigated the expression pattern of c-FLIP(L) in a cohort of 251 invasive breast cancer tissues and explored its potential role in TRAIL resistance. C-FLIP(L) was relatively high-expressed in HER2-positive breast cancer in comparison with other molecular subtypes, co-expressed with TRAIL death receptors, and inversely correlated with the apoptosis index. Downregulation of c-FLIP(L) sensitized SKBR3 cells to TRAIL-induced apoptosis in a concentration- and time-dependent manner and enhanced the activities and cleavages of caspase-8 and caspase-3, without altering the surface expression of death receptors. Together, our results indicate that c-FLIP(L) promotes TRAIL resistance and inhibits caspase-3 and caspase-8 activation in HER2-positive breast cancer.     

Quantum dots-based double-color imaging of HER2 positive breast cancer invasion

It has been well recognized that human epidermal growth factor receptor 2 (HER2) level in breast cancer (BC) is closely related to the malignant biologic behaviors of the tumor, including invasion and metastasis. Yet, there has been a lack of directly observable evidence to support such notion. Here we report a quantum dots (QDs)-based double-color imaging technique to simultaneously show the HER2 level on BC cells and the type IV collagen in the tumor matrix. In benign breast tumor, the type IV collagen was intact. With the increasing of HER2 expression level, there has been a progressive decrease in type IV collagen around the cancer nest. At HER2 (3+) expression level, there has virtually been a total destruction of type IV collagen. Moreover, HER2 (3+) BC cells also show direct invasion into the blood vessels. This novel imaging method provides direct observable evidence to support the theory that the HER2 expression level is directly related to BC invasion.     

Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2 gene-amplified breast cancer cells with primary resistance to HER1/2-targeted therapies

Primary resistance of HER2 gene-amplified breast carcinomas (BC) to HER-targeted therapies can be explained in terms of overactive HER2-independent downstream pro-survival pathways. We here confirm that constitutive overexpression of Inhibitor of Apoptosis (IAP) survivin is indispensable for survival of HER2-positive BC cells with intrinsic cross-resistance to multiple HER1/2 inhibitors. The IC₅₀ values for the HER1/2 Tyrosine Kinase Inhibitors (TKIs) gefitinib, erlotinib and lapatinib were up to 40-fold higher in trastuzumab-unresponsive JIMT-1 cells than in trastuzumab-naïve SKBR3 cells. ELISA-based and immunoblotting assays demonstrated that trastuzumab-refractory JIMT-1 cells constitutively expressed ∼4 times more survivin protein than trastuzumab-responsive SKBR3 cells. In response to trastuzumab, JIMT-1 cells accumulated ∼10 times more survivin than SKBR3 cells. HER1/2 TKIs failed to down-regulate survivin expression in JIMT-1 cells whereas equimolar doses of HER1/HER2 TKIs drastically depleted survivin protein in SKBR3 cells. ELISA-based detection of histone-associated DNA fragments confirmed that trastuzumab-refractory JIMT-1 cells were intrinsically protected against the apoptotic effects of HER1/2 TKIs. Of note, when we knocked-down survivin expression using siRNA and then added trastuzumab, cell proliferation and colony formation were completely suppressed in JIMT-1 cells. Our current findings may be extremely helpful to design successful combinatorial strategies aimed to circumvent the occurrence of de novo resistance to HER2-directed drugs using survivin antagonists.     

The HER2 amplicon in breast cancer: Topoisomerase IIA and beyond

HER2 gene amplification is observed in about 15% of breast cancers. The subgroup of HER2-positive breast cancers appears to be heterogeneous and presents complex patterns of gene amplification at the locus on chromosome 17q12-21. The molecular variations within the chromosome 17q amplicon and their clinical implications remain largely unknown. Besides the well-known TOP2A gene encoding Topoisomerase IIA, other genes might also be amplified and could play functional roles in breast cancer development and progression. This review will focus on the current knowledge concerning the HER2 amplicon heterogeneity, its clinical and biological impact and the pitfalls associated with the evaluation of gene amplifications at this locus, with particular attention to TOP2A and the link between TOP2A and anthracycline benefit. In addition it will discuss the clinical and biological implications of the amplification of ten other genes at this locus (MED1, STARD3, GRB7, THRA, RARA, IGFPB4, CCR7, KRT20, KRT19 and GAST) in breast cancer.     

Detection of HER2/neu gene amplification in archival paraffin-embedded breast cancer tissues by fluorescence in situ hybridization

We evaluated HER2/neu gene amplification by fluorescence in situ hybridization (FISH) in archival paraffin-embedded breast cancer tissues. Tumors from 63 human invasive breast cancers were categorized into two groups depending on whether the paraffin-embedded tissue blocks had been stored for more or less than 12 months duration. These were subjected to routine and modified FISH protocols. As microwave oven formalin fixation of tissues was carried out in the majority of the older archived specimens, the effect of this fixation method was also analyzed. FISH signals were obtained in all 13 archival specimens stored for less than 12 months. However, in 50 specimens stored for more than 12 months duration, the procedure was successful in only 10 specimens (20%), for which the pretreatment procedure had to be individually optimized for each specimen. There was no significant difference in the detection of FISH signals between microwave oven and routinely fixed specimens.     

Basal/HER2 breast carcinomas

High rates of inherent primary resistance to the humanized monoclonal antibody trastuzumab (Herceptin) are frequent among HER2 gene-amplified breast carcinomas in both metastatic and adjuvant settings. The clinical efficacy of trastuzumab is highly correlated with its ability to specifically and efficiently target HER2-driven populations of breast cancer stem cells (CSCs). Intriguingly, many of the possible mechanisms by which cancer cells escape trastuzumab involve many of the same biomarkers that have been implicated in the biology of CS-like tumor-initiating cells. In the traditional, one-way hierarchy of CSCs in which all cancer cells descend from special self-renewing CSCs, HER2-positive CSCs can occur solely by self-renewal. Therefore, by targeting CSC self-renewal and resistance, trastuzumab is expected to induce tumor shrinkage and further reduce breast cancer recurrence rates when used alongside traditional therapies. In a new, alternate model, more differentiated non-stem cancer cells can revert to trastuzumab-refractory, CS-like cells via the activation of intrinsic or microenvironmental paths-to-stemness, such as the epithelial-to-mesenchymal transition (EMT). Alternatively, stochastic transitions of trastuzumab-responsive CSCs might also give rise to non-CSC cellular states that lack major attributes of CSCs and, therefore, can remain “hidden” from trastuzumab activity. Here, we hypothesize that a better understanding of the CSC/non-CSC social structure within HER2-overexpressing breast carcinomas is critical for trastuzumab-based treatment decisions in the clinic. First, we decipher the biological significance of CSC features and the EMT on the molecular effects and efficacy of trastuzumab in HER2-positive breast cancer cells. Second, we reinterpret the genetic heterogeneity that differentiates trastuzumab-responders from non-responders in terms of CSC cellular states. Finally, we propose that novel predictive approaches aimed at better forecasting early tumor responses to trastuzumab should identify biological determinants that causally underlie the intrinsic flexibility of HER2-positive CSCs to “enter” into or “exit” from trastuzumab-sensitive states. An accurate integration of CSC cellular states and EMT-related biomarkers with the currently available breast cancer molecular taxonomy may significantly improve our ability to make a priori decisions about whether patients belonging to HER2 subtypes differentially enriched with a “mesenchymal transition signature” (e.g., luminal/HER2 vs. basal/HER2) would distinctly benefit from trastuzumab-based therapy ab initio.     

Urinary proteome alterations in HER2 enriched breast cancer revealed by multipronged quantitative proteomics

Globally, breast cancer is the second most common cancer among women. Although biomarker discoveries through various proteomic approaches of tissue and serum samples have been studied in breast cancer, urinary proteome alterations in breast cancer are least studied. Urine being a noninvasive biofluid and a significant source of proteins, it has the potential in early diagnosis of breast cancer. This study used complementary quantitative gel‐based and gel‐free proteomic approaches to find a panel of urinary protein markers that could discriminate HER2 enriched (HE) subtype breast cancer from the healthy controls. A total of 183 differentially expressed proteins were identified using three complementary approaches, namely 2D‐DIGE, iTRAQ, and sequential window acquisition of all theoretical mass spectra. The differentially expressed proteins were subjected to various bioinformatics analyses for deciphering the biological context of these proteins using protein analysis through evolutionary relationships, database for annotation, visualization and integrated discovery, and STRING. Multivariate statistical analysis was undertaken to identify the set of most significant proteins, which could discriminate HE breast cancer from healthy controls. Immunoblotting and MRM‐based validation in a separate cohort testified a panel of 21 proteins such as zinc‐alpha2‐glycoprotein, A2GL, retinol‐binding protein 4, annexin A1, SAP3, SRC8, gelsolin, kininogen 1, CO9, clusterin, ceruloplasmin, and α1‐antitrypsin could be a panel of candidate markers that could discriminate HE breast cancer from healthy controls.     

Identification of quinones as HER2 inhibitors for the treatment of trastuzumab resistant breast cancer

HER2 overexpression is associated with aggressive breast cancer with high recurrence rate and poor patient prognosis. Treatment of HER2 overexpressing patients with the HER2 targeting therapy trastuzumab results in acquired resistance within a year. The HER2/EGFR dual kinase inhibitor lapatinib was shown to inhibit some trastuzumab resistant breast cancer cell lines and is currently in clinical trials. Our group has found two new quinone compounds that show excellent inhibition of breast tumor cells expressing HER2 or the trastuzumab resistant HER2 oncogenic isoform, HER2Δ16. Compound 4 ((1R,2S,3S)-1,2,3,5,8-pentahydroxy-1,2,3,4-tetrahydroanthracene-9,10-dione) and compound 5 (5,8-dihydroxy-2,3-bis(hydroxymethyl)naphthalene-1,4-dione) showed sub-micromolar inhibition potency against these cell lines. These compounds also inhibit auto-phosphorylation of the Y1248 and Y1068 residues of HER2 and EGFR, respectively.     

HER2/neu expression correlates with vasculogenic mimicry in invasive breast carcinoma

Vasculogenic mimicry (VM) refers to the condition in which tumour cells mimic endothelial cells to form extracellular matrix‐rich tubular channels. VM is more extensive in more aggressive tumours. The human epidermal growth factor receptor 2 (HER2) gene is amplified in 20–30% of human breast cancers and has been implicated in mediating aggressive tumour growth and metastasis. However, thus far, there have been no data on the role of HER2 in VM formation. Immunohistochemical and histochemical double‐staining methods were performed to display VM in breast cancer specimens. Transfection in MCF7 cells was performed and clones were selected by G418. The three‐dimensional Matrigel culture was used to evaluate VM formation in the breast cancer cell line. According to statistical analysis, VM was related to the presence of a positive nodal status and advanced clinical stage. The positive rate of VM increased with increased HER2 expression. In addition, cases with HER2 3+ expression showed significantly greater VM channel count than those in other cases. The exogenous HER2 overexpression in MCF‐7 cells induced vessel‐like VM structures on the Matrigel and increased the VM mediator vascular endothelial (VE) cadherin. Our data provide evidence for a clinically relevant association between HER2 and VM in human invasive breast cancer. HER2 overexpression possibly induces VM through the up‐regulation of VE cadherin. Understanding the key molecular events may provide therapeutic intervention strategies for HER2+ breast cancer.     

Overcoming trastuzumab resistance in HER2-positive breast cancer using combination therapy

Human epidermal growth factor receptor 2 (HER2)-positive breast cancer (BC) comprises around 20–30% of all BC subtypes and is correlated with poor prognosis. For many years, trastuzumab, a monoclonal antibody, has been used to inhibit the HER2 activity. Though, the main resistance to trastuzumab has challenged the use of this drug in the management of HER2-positive BC. Therefore, the determination of resistance mechanisms and the incorporation of new agents may lead to the development of a better blockade of the HER family receptor signaling. During the last few years, some therapeutic drugs have been developed for treating patients with trastuzumab-resistant HER2-positive BC that have more effective influences in the management of this condition. In this regard, the present study aimed at reviewing the mechanisms of trastuzumab resistance and the innovative therapies that have been investigated in trastuzumab-resistant HER2-positive BC subjects.     

Proteomic profiling of cancer stem cells derived from primary tumors of HER2/Neu transgenic mice

Human epidermal growth factor receptor 2 (HER2) overexpression leads to mammary tumorigenesis and its elevated levels lead to increase in cancer stem cells (CSCs), invasion, and metastasis. CSCs are resistant to radiation/chemotherapeutic drugs and are believed to be responsible for recurrence/relapse of cancer. CSCs are isolated using flow cytometry based sorting, although reliable, this technology hinders the convenient identification of molecular targets of CSCs. Therefore to understand the molecular players of increased CSC through HER2 overexpression and to develop meaningful targets for combination therapy, we isolated and characterized breast CSCs through convenient tumorsphere culture. We identified the altered protein expression in CSC as compared to non‐CSC using LC‐MS/MS and confirmed those results using qRT‐PCR and Western blotting. Ferritin heavy chain 1 (FTH1) was identified as a candidate gene, which is involved in iron metabolism and iron depletion significantly decreased the self‐renewal of CSCs. We further performed in silico analysis of altered genes in tumorsphere and identified a set of genes (PTMA, S100A4, S100A6, TNXRD1, COX‐1, COX‐2, KRT14, and FTH1), representing possible molecular targets, which in combination showed a promise to be used as prognostic markers for breast cancer.     

The impact of HER2-directed targeted therapy on HER2-positive DCIS of the breast

BackgroundIn invasive breast cancer, HER2 is a well-established negative prognostic factor. However, its significance on the prognosis of ductal carcinoma in situ (DCIS) of the breast is unclear. As a result, the impact of HER2-directed therapy on HER2-positive DCIS is unknown and is currently the subject of ongoing clinical trials. In this study, we aim to determine the possible impact of HER 2-directed targeted therapy on survival outcomes for HER2-positive DCIS patients.Materials and methodsThe National Cancer Data Base (NCDB) was used to retrieve patients with biopsy-proven DCIS diagnosed from 2004–2015. Patients were divided into two groups based on the adjuvant therapy they received: systemic HER2-directed targeted therapy or no systemic therapy. Statistics included multivariable logistic regression to determine factors predictive of receiving systemic therapy, Kaplan-Meier analysis to evaluate overall survival (OS), and Cox proportional hazards modeling to determine variables associated with OS.ResultsAltogether, 1927 patients met inclusion criteria; 430 (22.3%) received HER2-directed targeted therapy; 1497 (77.7%) did not. Patients who received HER2-directed targeted therapy had a higher 5-year OS compared to patients that did not (97.7% vs. 95.8%, p = 0.043). This survival benefit remained on multivariable analysis. Factors associated with worse OS on multivariable analysis included Charlson-Deyo Comorbidity Score ≥ 2 and no receipt of hormonal therapy.ConclusionIn this large study evaluating HER2-positive DCIS patients, the receipt of HER2-directed targeted therapy was associated with an improvement in OS. The results of currently ongoing clinical trials are needed to confirm this finding.     

MiR-221 promotes trastuzumab-resistance and metastasis in HER2-positive breast cancers by targeting PTEN

HER2-overexpressing breast cancers are characterized by frequent distant metastasis and often develop resistance after short-term effective treatment with the monoclonal antibody drug, trastuzumab. Here, we found that the oncogenic miRNA, miR-221, inhibited apoptosis, induced trastuzumab resistance and promoted metastasis of HER2-positive breast cancers. The tumor suppressor PTEN was identified as a miR-221 target; overexpression of PTEN abrogated the aforementioned miR-221-induced malignant phenotypes of the cells. These findings indicate that miR-221 may promote trastuzumab resistance and metastasis of HER2-positive breast cancers by targeting PTEN, suggesting its role as a potential biomarker for progression and poor prognosis, and as a novel target for trastuzumab-combined treatment of breast cancers. [BMB Reports 2014; 47(5): 268-273].