Extracellular Matrix Features Discriminate Aggressive HER2-Positive Breast Cancer Patients Who Benefit from Trastuzumab Treatment.

We previously identified an extracellular matrix (ECM) gene expression pattern in breast cancer (BC), called ECM3, characterized by a high expression of genes encoding structural ECM proteins. Since ECM is reportedly implicated in response to therapy of BCs, the aim of this work is to investigate the prognostic and predictive value of ECM3 molecular classification in HER2-positive BCs. ECM3 resulted in a robust cluster that identified a subset of 25-37% of HER2-positive tumors with molecular aggressive features. ECM3 was significantly associated with worse prognosis in two datasets of HER2-positive BCs untreated with adjuvant therapy. Analyses carried out on two of our cohorts of patients treated or not with adjuvant trastuzumab showed association of ECM3 with worse prognosis only in patients not treated with trastuzumab. Moreover, investigating a dataset that includes gene profile data of tumors treated with neoadjuvant trastuzumab plus chemotherapy or chemotherapy alone, ECM3 was associated with increased pathological complete response if treated with trastuzumab. In the in vivo experiments, increased diffusion and trastuzumab activity were found in tumors derived from injection of HER2-positive cells with Matrigel that creates an ECM-rich tumor environment. Taken together, these results indicate that HER2-positive BCs classified as ECM3 have an aggressive phenotype but they are sensitive to trastuzumab treatment.

Wound Healing Fluid Reflects the Inflammatory Nature and Aggressiveness of Breast Tumors.

Wound healing fluid that originates from breast surgery increases the aggressiveness of cancer cells that remain after the surgery. We determined the effects of the extent of surgery and tumor-driven remodeling of the surrounding microenvironment on the ability of wound-healing to promote breast cancer progression. In our analysis of a panel of 34 cytokines, chemokines, and growth factors in wound healing fluid, obtained from 27 breast carcinoma patients after surgery, the levels of several small molecules were associated with the extent of cellular damage that was induced by surgery. In addition, the composition of the resulting wound healing fluid was associated with molecular features of the removed tumor. Specifically, IP-10, IL-6, G-CSF, osteopontin, MIP-1a, MIP-1b, and MCP1-MCAF were higher in more aggressive tumors. Altogether, our findings indicate that the release of factors that are induced by removal of the primary tumor and subsequent wound healing is influenced by the extent of damage due to surgery and the reactive stroma that is derived from the continuously evolving network of interactions between neoplastic cells and the microenvironment, based on the molecular characteristics of breast carcinoma cells.

The PDGFRß/ERK1/2 pathway regulates CDCP1 expression in triple-negative breast cancer.

BACKGROUND: CDCP1, a transmembrane protein with tumor pro-metastatic activity, was recently identified as a prognostic marker in TNBC, the most aggressive breast cancer subtype still lacking an effective molecular targeted therapy. The mechanisms driving CDCP1 over-expression are not fully understood, although several stimuli derived from tumor microenvironment, such as factors present in Wound Healing Fluids (WHFs), reportedly increase CDCP1 levels. METHODS: The expression of CDCP1, PDGFRß and ERK1/2cell was tested by Western blot after stimulation of MDA-MB-231 cells with PDGF-BB and, similarly, in presence or not of ERK1/2 inhibitor in a panel of TNBC cell lines. Knock-down of PDGFRß was established in MDA-MB-231 cells to detect CDCP1 upon WHF treatment. Immunohistochemical staining was used to detect the expression of CDCP1 and PDGFRß in TNBC clinical samples. RESULTS: We discovered that PDGF-BB-mediated activation of PDGFRß increases CDCP1 protein expression through the downstream activation of ERK1/2. Inhibition of ERK1/2 activity reduced per se CDCP1 expression, evidence strengthening its role in CDCP1 expression regulation. Knock-down of PDGFRß in TNBC cells impaired CDCP1 increase induced by WHF treatment, highlighting the role if this receptor as a central player of the WHF-mediated CDCP1 induction. A significant association between CDCP1 and PDGFRß immunohistochemical staining was observed in TNBC specimens, independently of CDCP1 gene gain, thus corroborating the relevance of the PDGF-BB/PDGFRß axis in the modulation of CDCP1 expression. CONCLUSION: We have identified PDGF-BB/PDGFRß-mediated pathway as a novel player in the regulation of CDCP1 in TNCBs through ERK1/2 activation. Our results provide the basis for the potential use of PDGFRß and ERK1/2 inhibitors in targeting the aggressive features of CDCP1-positive TNBCs.

Reprogramming the lung microenvironment by inhaled immunotherapy fosters immune destruction of tumor.

Due to their constant exposure to inhaled antigens, lungs represent a particularly immunosuppressive environment that limits excessive immune responses; however, cancer cells can exploit this unique environment for their growth. We previously described the ability of aerosolized CpG-ODN combined with Poly(I:C) (TLR9 and TLR3 agonists, respectively) to promote antitumor immunity in a B16 melanoma lung metastasis model. Here, we explored the possibility of improving the therapeutic efficacy of TLR9/TLR3 agonist combinations by including in the inhalant either an antibody directed to both Ly6G and Ly6C markers to locally deplete myeloid-derived suppressive cells (MDSCs) or IFNα to directly activate the natural killer (NK) and macrophage innate immune cells in the lung. Addition of nebulized anti-MDSC antibody RB6-8C5 to aerosolized CpG-ODN/Poly(I:C) resulted in reduced mRNA levels of immunsuppressive molecules (IL10, Arg-1, and Nos2), increased activation of resident NK cells and improved treatment outcome, with a significant reduction in established B16 melanoma lung metastases compared to treatment with CpG-ODN/Poly(I:C) alone. Likewise, addition of aerosolized IFNα led to increased mRNA levels of proinflammatory cytokines (IL15 and IFNγ) in the lung and recruitment of highly activated NK cells, with no evident signs of toxicity and with a significantly improved antitumor effect as compared with aerosolized CpG-ODN/Poly(I:C). Combining both IFNα and RB6-8C5 with CpG-ODN/Poly(I:C) did not produce an additive effect compared to IFNα + CpG-ODN/Poly(I:C) or RB6-8C5 + CpG-ODN/Poly(I:C). Our results indicate that the inhalation therapy is a feasible and non-invasive strategy to deliver immunodulatory molecules, including antibodies and cytokines that reprogram the lung tumor microenvironment to foster immune destruction of tumors.

CDCP1 is a novel marker of the most aggressive human triple-negative breast cancers.

CDCP1, a transmembrane noncatalytic receptor, the expression of which has been associated with a poor prognosis in certain epithelial cancers, was found to be expressed in highly aggressive triple-negative breast cancer (TNBC) cell models, in which it promoted aggressive activities-ie, migration, invasion, anchorage-independent tumor growth, and the formation of vascular-like structures in vitro. By immunohistochemical (IHC) analysis of 100 human TNBC specimens, CDCP1 was overexpressed in 57% of samples, 38% of which exhibited a gain in CDCP1 copy number by fluorescence in situ hybridization (FISH). CDCP1 positivity was significantly associated between FISH and IHC. CDCP1 expression and gains in CDCP1 copy number synergized with nodal (N) status in determining disease-free and distant disease-free survival. The hazard ratios (HRs) of the synergies between CDCP1 positivity by IHC and FISH and lymph node positivity in predicting relapse did not differ significantly, indicating that CDCP1 overexpression in human primary TNBCs, regardless of being driven by gains in CDCP1, is for a critical factor in the progression of N-positive TNBCs. Thus, CDCP1 is a novel marker of the most aggressive N-positive TNBCs and a potential therapeutic target.

Whole-transcriptome analysis links trastuzumab sensitivity of breast tumors to both HER2 dependence and immune cell infiltration.

While results thus far demonstrate the clinical benefit of trastuzumab, some patients do not respond to this therapy. To identify a molecular predictor of trastuzumab benefit, we conducted whole-transcriptome analysis of primary HER2+ breast carcinomas obtained from patients treated with trastuzumab-containing therapies and correlated the molecular portrait with treatment benefit. The estimated association between gene expression and relapse-free survival allowed development of a trastuzumab risk model (TRAR), with ERBB2 and ESR1 expression as core elements, able to identify patients with high and low risk of relapse. Application of the TRAR model to 24 HER2+ core biopsies from patients treated with neo-adjuvant trastuzumab indicated that it is predictive of trastuzumab response. Examination of TRAR in available whole-transcriptome datasets indicated that this model stratifies patients according to response to trastuzumab-based neo-adjuvant treatment but not to chemotherapy alone. Pathway analysis revealed that TRAR-low tumors expressed genes of the immune response, with higher numbers of CD8-positive cells detected immunohistochemically compared to TRAR-high tumors. The TRAR model identifies tumors that benefit from trastuzumab-based treatment as those most enriched in CD8-positive immune infiltrating cells and with high ERBB2 and low ESR1 mRNA levels, indicating the requirement for both features in achieving trastuzumab response.

Fhit Nuclear Import Following EGF Stimulation Sustains Proliferation of Breast Cancer Cells.

The tumor-suppressor protein fragile histidine triad (Fhit) exerts its functions in the cytoplasm, although some reports suggest that it may also act in the nucleus. We previously showed that cytosolic Fhit protein levels in cancer cell lines stimulated to proliferate were reduced by proteasomal degradation. Here, we demonstrate that Fhit is physiologically present in the nucleus of breast cancer cell lines and tissues at a low level and that proliferative stimulation increases nuclear levels. Breast cancer cells expressing the FhitY114F mutant, which do not undergo proteasomal degradation, contained mutated Fhit in the nucleus, while cells treated with a proteasome inhibitor accumulated nuclear Fhit during proliferation. Thus, Fhit nuclear shuttling and proteasome degradation phenomena occur independently. When Fhit was coupled to a nuclear localization sequence, the proliferation rate of the transfected cells increased together with levels of proliferation pathway mediators cyclin D1, phospho-MAPK, and phospho-STAT3. Fhit nuclear translocation upon mitogenic stimulation may represent a new regulatory mechanism that allows rapid restoration of Fhit cytoplasmic levels and promotes the proliferation cascade activated by mitogenic stimulation.

Circulating biomarkers in advanced colorectal cancer patients randomly assigned to three bevacizumab-based regimens.

The need to identify biomarkers for bevacizumab-based treatment in advanced colorectal cancer is imperative. The aim of this study was to investigate the prognostic role of circulating VEGF, PDGF, SDF-1, osteopontin and CEA in patients randomly assigned to three bevacizumab-based regimens. Plasma samples from 50 patients treated at a single Institution were analysed using the multiplex assay BioPlex™ 2200 (Bio-Rad Laboratories, Inc, Berkeley, CA, USA) at baseline, before first three cycles and subsequently every three cycles until disease progression. Prognostic analyses of baseline values were performed using multivariable Cox models, including disease extension >10 cm or ≤10 cm (measured as the sum of the diameters for all target lesions) as adjustment factor. The association between progression-free and overall survival and biomarkers modulation during treatment was studied using multivariable Cox models, which included summary statistics synthesizing during-treatment modulation together with disease extension. The biomarkers significantly associated with disease extension were baseline CEA (p = 0.012) and SDF-1 (p = 0.030). High values of VEGF and SDF-1 tended to be associated with worse prognosis, especially in terms of overall survival. The negative prognostic trend was more marked for baseline CEA as compared to other biomarkers; increasing values during treatment was significantly related to worse prognosis independently of disease extension (p = 0.007 and 0.016 for progression-free and overall survival, respectively). VEGF is related to bevacizumab pharmacodynamics and is associated to other angiogenic cytokines; some of the proposed biomarkers such as SDF-1 and CEA should be further validated for prognosis assessment and monitoring of bevacizumab-based treatment of advanced colorectal cancer.

Activated d16HER2 homodimers and SRC kinase mediate optimal efficacy for trastuzumab.

A splice isoform of the HER2 receptor that lacks exon 16 (d16HER2) is expressed in many HER2-positive breast tumors, where it has been linked with resistance to the HER2-targeting antibody trastuzumab, but the impact of d16HER2 on tumor pathobiology and therapeutic response remains uncertain. Here, we provide genetic evidence in transgenic mice that expression of d16HER2 is sufficient to accelerate mammary tumorigenesis and improve the response to trastuzumab. A comparative analysis of effector signaling pathways activated by d16HER2 and wild-type HER2 revealed that d16HER2 was optimally functional through a link to SRC activation (pSRC). Clinically, HER2-positive breast cancers from patients who received trastuzumab exhibited a positive correlation in d16HER2 and pSRC abundance, consistent with the mouse genetic results. Moreover, patients expressing high pSRC or an activated "d16HER2 metagene" were found to derive the greatest benefit from trastuzumab treatment. Overall, our results establish the d16HER2 signaling axis as a signature for decreased risk of relapse after trastuzumab treatment.

PDGFRß and FGFR2 mediate endothelial cell differentiation capability of triple negative breast carcinoma cells.

Triple negative breast cancer (TNBC) is a very aggressive subgroup of breast carcinoma, still lacking specific markers for an effective targeted therapy and with a poorer prognosis compared to other breast cancer subtypes. In this study we investigated the possibility that TNBC cells contribute to the establishment of tumor vascular network by the process known as vasculogenic mimicry, through endothelial cell differentiation. Vascular-like functional properties of breast cancer cell lines were investigated in vitro by tube formation assay and in vivo by confocal microscopy, immunofluorescence or immunohistochemistry on frozen tumor sections. TNBCs express endothelial markers and acquire the ability to form vascular-like channels in vitro and in vivo, both in xenograft models and in human specimens, generating blood lacunae surrounded by tumor cells. Notably this feature is significantly associated with reduced disease free survival. The impairment of the main pathways involved in vessel formation, by treatment with inhibitors (i.e. Sunitinib and Bevacizumab) or by siRNA-mediating silencing, allowed the identification of PDGFRß and FGFR2 as relevant players in this phenomenon. Inhibition of these tyrosine kinase receptors negatively affects vascular lacunae formation and significantly inhibits TNBC growth in vivo. In summary, we demonstrated that TNBCs have the ability to form vascular-like channels in vitro and to generate blood lacunae lined by tumor cells in vivo. Moreover, this feature is associated with poor outcome, probably contributing to the aggressiveness of this breast cancer subgroup. Finally, PDGFRß and FGFR2-mediated pathways, identified as relevant in mediating this characteristic, potentially represent valid targets for a specific therapy of this breast cancer subgroup.

Potential role of HER2-overexpressing exosomes in countering trastuzumab-based therapy.

Exosomes are endosome-derived nanovesicles actively released into the extracellular environment and biological fluids, both under physiological and pathological conditions, by different cell types. We characterized exosomes constitutively secreted by HER2-overexpressing breast carcinoma cell lines and analyzed in vitro and in vivo their potential role in interfering with the therapeutic activity of the humanized antibody Trastuzumab and the dual tyrosine kinase inhibitor (TKI) Lapatinib anti-HER2 biodrugs. We show that exosomes released by the HER2-overexpressing tumor cell lines SKBR3 and BT474 express a full-length HER2 molecule that is also activated, although to a lesser extent than in the originating cells. Release of these exosomes was significantly modulated by the growth factors EGF and heregulin, two of the known HER2 receptor-activating ligands and naturally present in the surrounding tumor microenvironment. Exosomes secreted either in HER2-positive tumor cell-conditioned supernatants or in breast cancer patients' serum bound to Trastuzumab. Functional assays revealed that both xenogeneic and autologous HER2-positive nanovesicles, but not HER2-negative ones, inhibited Trastuzumab activity on SKBR3 cell proliferation. By contrast, Lapatinib activity on SKBR3 cell proliferation was unaffected by the presence of autologous exosomes. Together, these findings point to the role of HER2-positive exosomes in modulating sensitivity to Trastuzumab, and, consequently, to HER2-driven tumor aggressiveness.

Activity and resistance of trastuzumab according to different clinical settings.

Trastuzumab, a humanized monoclonal antibody directed against HER2, has shown efficacy in breast cancers; however many patients do not respond to this reagent. Here, we discuss the potential mechanisms of trastuzumab efficacy and resistance in different clinical settings as a step toward optimizing the appropriate application of this antibody. The three major antitumor mechanisms of trastuzumab, i.e., inhibition of proliferation, antibody-dependent cell cytotoxicity (ADCC) and inhibition of DNA repair, appear to be differentially operative in different clinical settings. ADCC appears to be the prevalent mechanism in trastuzumab neoadjuvant monotherapy, whereas in neoadjuvant, adjuvant or metastatic settings in which trastuzumab is combined with chemotherapy, the relative role of ADCC is probably small, considering the compromising effects of chemotherapy on the immune cells that mediate this mechanism. In neoadjuvant and adjuvant settings involving concomitant use of trastuzumab and chemotherapy, the primary mechanism at play is presumably inhibition of DNA repair by the antibody, while in sequential protocols, the antibody acts mostly by exerting cytostatic activity through inhibition of HER2-mediated tumor cell proliferation. According to the ability of the antibody to induce cytotoxic or cytostatic antitumor effects depending on the clinical setting, different criteria, i.e., RECIST for cytotoxic effect, OS, and DFS for cytostatic, must be considered in accurately estimating antibody efficacy. Moreover, since trastuzumab resistance likely depends directly on the mechanisms responsible for its antitumor activity, resistance mechanisms must also be considered with respect to the different clinical settings.

The HER2 World: Better Treatment Selection for Better Outcome.

Understanding the mechanisms of trastuzumab efficacy and resistance is a step toward optimizing treatment outcome in HER2-positive breast carcinoma patients. Preclinical studies have indicated different trastuzumab antitumor mechanisms, that is, cytostatic inhibition of tumor proliferation, antibody-dependent cell cytotoxicity, and inhibition of HER2-mediated DNA repair. Clinical studies point to the clinical setting dependence of these mechanisms, with antibody-dependent cell cytotoxicity predominating when trastuzumab is used as monotherapy in neoadjuvant and metastatic settings, whereas inhibition of DNA repair predominates in neoadjuvant and adjuvant settings involving concomitant trastuzumab and chemotherapy; in sequential protocols, the antibody appears to act primarily through cytostatic activity by inhibiting HER2-mediated cell proliferation. Because the mechanisms of resistance to trastuzumab likely depend directly on those of its antitumor activity, resistance mechanisms must also be considered with respect to the different clinical settings. Moreover, the response to this reagent should be assessed according to its ability to induce tumor cytotoxic or cytostatic activity.

Increased overall survival independent of RECIST response in metastatic breast cancer patients continuing trastuzumab treatment: evidence from a retrospective study.

Recent studies have reported the potential clinical utility for metastatic breast cancer (MBC) patients of continuing trastuzumab beyond progression. Based on those results, here the authors have examined the benefits of trastuzumab-continuation by specifically evaluating RECIST responses upon first line trastuzumab-treatment as a potential predictive marker for therapeutic effect of trastuzumab-continuation beyond metastatic disease progression. The authors carried out a retrospective analysis of 272 HER2 positive MBC patients under trastuzumab treatment at 22 different oncology Italian centers during the years of 2000 and 2001 who progressed under first line trastuzumab-treatment. The primary end point of the study was the survival from the date of first documented progression upon first line trastuzumab treatment of disease. Data analysis involved the use of matching on propensity score to balance variables between treated and untreated subjects and to reduce bias. Of the 272 HER2-positive MBC patients, 154 (56.6%) continued treatment. 79 (51.3%) of those 154 patients showed responses based on RECIST criteria during first-line trastuzumab-treatment. Of the 118 patients that suspended trastuzumab, RECIST responses had been observed in 44 (37.3%). Cox proportional hazards analysis of progressed patients, matched using propensity score, showed that discontinuation of trastuzumab at metastatic disease progression was a risk factor for significantly reduced overall survival in both responder (HR = 2.23; 95% CI = 1.03-4.82) and non-responder groups (HR = 3.53, 95% CI = 1.73-7.21), with no significant differences in the two estimated HRs (P-value of the likelihood-ratio test = 0.690). Continued trastuzumab treatment after disease progression has clinically and statistically significant effects in both RECIST responder and non-responder MBC patients.

Shed HER2 extracellular domain in HER2-mediated tumor growth and in trastuzumab susceptibility.

The question of the serum HER2 extracellular domain (HER2/ECD) measurement for prediction of response to the anti-HER2 antibody Trastuzumab is still an open and current matter of clinical debate. To elucidate the involvement of shed HER2/ECD in HER2-driven tumor progression and in guiding therapy of individual patients, we examined biological effects exerted by elevated HER2/ECD in cancer growth and in response to Trastuzumab. To this purpose SKOV3 tumor cells were stably transfected to release a recombinant HER2/ECD molecule (rECD). Transfectants releasing high levels of 110-kDa rECD, identical in size to native HER2/ECD (nECD), grew significantly slower than did controls, which constitutively released only basal levels of nECD. While transmembrane HER2 and HER1 were expressed at equal levels by both controls and transfected cells, activation of these molecules and of downstream ERK2 and Akt was significantly reduced only in rECD transfectants. Surface plasmon resonance analysis revealed heterodimerization of the rECD with HER1, -2, and -3. In cell growth bioassays in vitro, shed HER2 significantly blocked HER2-driven tumor cell proliferation. In mice, high levels of circulating rECD significantly impaired HER2-driven SKOV3 tumor growth but not that of HER2-negative tumor cells. In vitro and in mice, Trastuzumab significantly inhibited tumor growth due to the rECD-facilitated accumulation of the antibody on tumor cells. Globally our findings sustain the biological relevance of elevated HER2/ECD levels in the outcome of HER2-disease and in the susceptibility to Trastuzumab-based therapy.

HER2 as a target for breast cancer therapy.

IMPORTANCE OF THE FIELD: Differential levels of HER2 expression in normal versus HER2-overexpressing breast carcinomas, together with the demonstration of a key role for HER2 in tumor progression, make HER2 an ideal target for specific therapeutic approaches. AREAS COVERED IN THIS REVIEW: This review considers the clinical value of trastuzumab and lapatinib, the two HER2-targeted therapies approved for clinical practice. References were chosen by searching the PubMed and MEDLINE datasets using as search term: 'HER2', in association with 'prognosis', 'response', 'trastuzumab', 'lapatinib' and 'resistance'. WHAT THE READER WILL GAIN: This review deals with HER2 as a target for breast carcinoma treatment, focusing on anti-HER2 therapies used in clinical practice, their merits and shortcomings. TAKE HOME MESSAGE: The benefit of anti-HER2 therapies demonstrated in clinical trials indicates that HER2 is, to date, one of the most promising molecules for targeted therapy. Nevertheless, since tumor cells utilizing alternative growth signaling pathways through transmembrane receptors as well as intracellular signaling transduction molecules can bypass HER2 blockade, a future ambitious aim is the successful combination of anti-HER2 strategies with drugs directed to molecules that contribute to anti-HER2 resistance.

Ascites regression and survival increase in mice bearing advanced-stage human ovarian carcinomas and repeatedly treated intraperitoneally with CpG-ODN.

Tumor cell growth, even in advanced stages of ovarian cancer, is nearly always restricted to the peritoneal cavity; therefore, repeated intraperitoneal injections of oligodeoxynucleotides containing dinucleotides with unmethylated CpG motifs (CpG-ODN) recruiting and activating innate effector cells throughout the abdominal cavity to the tumor site might control tumor cell growth and ascites formation. After a single CpG-ODN treatment, in IGROV-1 ovarian tumor ascites-bearing athymic mice, the number of tumor cells declined rapidly and markedly, and ascites volumes declined shortly after treatment (5 h), increasing thereafter at a slower rate than in controls. When administered every 7 days for 4 weeks, CpG-ODN had only a marginal effect on survival time, whereas administration 5 days/wk for 3 or 4 weeks led to a significantly increased survival time as compared with controls (P<0.005) and completely controlled ascites growth without apparent toxicity, although a disorganization of lymphoid organs was observed. Bio-plex assay of cytokine levels in peritoneal fluid of ascites-bearing mice after CpG-ODN treatment revealed an increase in interleukin (IL)-6, IL-10, IL-12, and interferon-gamma at 24 hours, which returned to control mice levels at 48 to 96 hours, whereas the high levels of angiogenic factors remained unchanged. Depletion of natural killer or monocytes/macrophages only slightly influenced the CpG-ODN-induced reduction of ascites tumor cells, indicating that the antitumor activity might not be related to a specific cell/cytokine but rather to the repertoire of cells and cytokines accumulated in the peritoneal cavity. Thus, our data suggest a relevant role for repeated activation of cells and cytokines of innate immunity in the therapy of ovarian cancer patients with malignant ascites.