Breast cancer patient-derived organoids for the investigation of patient-specific tumour evolution.

BACKGROUND: A reliable preclinical model of patient-derived organoids (PDOs) was developed in a case study of a 69-year-old woman diagnosed with breast cancer (BC) to investigate the tumour evolution before and after neoadjuvant chemotherapy and surgery. The results were achieved due to the development of PDOs from tissues collected before (O-PRE) and after (O-POST) treatment. METHODS: PDO cultures were characterized by histology, immunohistochemistry (IHC), transmission electron microscopy (TEM), scanning electron microscopy (SEM), confocal microscopy, flow cytometry, real-time PCR, bulk RNA-seq, single-cell RNA sequencing (scRNA-seq) and drug screening. RESULTS: Both PDO cultures recapitulated the histological and molecular profiles of the original tissues, and they showed typical mammary gland organization, confirming their reliability as a personalized in vitro model. Compared with O-PRE, O-POST had a greater proliferation rate with a significant increase in the Ki67 proliferation index. Moreover O-POST exhibited a more stem-like and aggressive phenotype, with increases in the CD24low/CD44low and EPCAMlow/CD49fhigh cell populations characterized by increased tumour initiation potential and multipotency and metastatic potential in invasive lobular carcinoma. Analysis of ErbB receptor expression indicated a decrease in HER-2 expression coupled with an increase in EGFR expression in O-POST. In this context, deregulation of the PI3K/Akt signalling pathway was assessed by transcriptomic analysis, confirming the altered transcriptional profile. Finally, transcriptomic single-cell analysis identified 11 cell type clusters, highlighting the selection of the luminal component and the decrease in the number of Epithelial-mesenchymal transition cell types in O-POST. CONCLUSION: Neoadjuvant treatment contributed to the enrichment of cell populations with luminal phenotypes that were more resistant to chemotherapy in O-POST. PDOs represent an excellent 3D cell model for assessing disease evolution.

Inhibition of the Wnt Signalling Pathway: An Avenue to Control Breast Cancer Aggressiveness.

Breast cancer (BC) is the most common tumour in women. Although the introduction of novel therapeutic approaches in clinical practice has dramatically improved the clinical outcome of BC patients, this malignant disease remains the second leading cause of cancer-related death worldwide. The wingless/integrated (Wnt) signalling pathway represents a crucial molecular node relevantly implicated in the regulation of normal somatic stem cells as well as cancer stem cell (CSC) traits and the epithelial-mesenchymal transition cell program. Accordingly, Wnt signalling is heavily dysregulated in BC, and the altered expression of different Wnt genes is significantly associated with cancer-related aggressive behaviours. For all these reasons, Wnt signalling represents a promising therapeutic target currently under clinical investigation to achieve cancer eradication by eliminating CSCs, considered by most to be responsible for tumour initiation, relapse, and drug resistance. In this review, we summarized the current knowledge on the Wnt signalling pathway in BC and have presented evidence implicating the suitability of Wnt targeting in an attempt to improve the outcome of patients without affecting the normal somatic stem cell population.

Intratumor lactate levels reflect HER2 addiction status in HER2-positive breast cancer.

Despite different molecular tumor profiles indicate that human epidermal growth factor receptor 2 (HER2) messenger RNA (mRNA) levels mirror HER2 addiction and trastuzumab benefit in HER2-positive breast cancer (BC), the identification of noninvasive clinical predictors of trastuzumab sensitivity remains an unmet clinical need. In the current study, we investigated whether intratumor lactate levels reflect HER2 addiction and, in turn, trastuzumab susceptibility. Accordingly, the gene expression profiles of transgenic murine BC cell lines expressing the human d16HER2 variant (HER2-addicted) or human full-length HER2 (WTHER2; HER2-nonaddicted) revealed a significant enrichment of glycolysis-related gene pathways in HER2-addicted cells. We studied the metabolic content of 22 human HER2-positive BC by quantitative nuclear magnetic resonance spectroscopy and found that those cases with higher lactate levels were characterized by higher HER2 transcript levels. Moreover, gene expression analyses of HER2-positive BC samples from a TCGA data set revealed a significant enrichment in glycolysis-related pathways in high/HER2-addicted tumors. These data were confirmed by metabolic analyses of human HER2-positive BC cell lines with high or low HER2 transcript levels, which revealed significantly more active glycolytic metabolism in high HER2 transcript than in low HER2 transcript cells. Overall, our results provide evidence for noninvasive intratumor lactate detection as a potential metabolic biomarker of HER2 addiction and trastuzumab response suggesting the possibility to use in vivo imaging to assess lactate levels and, in turn, select HER2-positive BC patients who are more likely to benefit from anti-HER2 treatments.

Impact of systemic and tumor lipid metabolism on everolimus efficacy in advanced pancreatic neuroendocrine tumors (pNETs).

The mTOR inhibitor everolimus is effective against advanced pancreatic neuroendocrine tumors (pNETs). However, it can cause metabolic adverse events, such as hyperglycemia, hypertriglyceridemia and hypercholesterolemia. In this work we aimed at evaluating the impact of systemic and tumor lipid metabolism on everolimus efficacy. We carried out a monocentric, retrospective study to correlate plasma triglyceride and cholesterol levels with the progression free survival (PFS) of advanced pNET patients treated with everolimus. In formalin fixed, paraffin embedded (FFPE) tumor specimens, we also assessed by mRNA quantification and immunohistochemistry the expression of acetyl-CoA carboxylase 1 (ACC1) and fatty acid synthase (FASN), two enzymes crucially involved in fatty acid biosynthesis, and we analyzed their impact on PFS. We evaluated 58 consecutive pNET patients who started everolimus between December 2006 and January 2015. Patients with higher plasma triglycerides during the first 3 months of treatment had an increased risk of disease progression (aHR 3.08, 95% CIs 1.15-8.21; p = 0.025). In 23 FFPE tumor specimens amenable for IHC evaluations, we found a positive correlation between ACC1 and FASN at both mRNA (r = 0.87, p = 0.00045) and protein (r = 0.68, p = 0.0004) level. Patients with higher ACC1 protein expression in metastatic lesions had significantly lower PFS when compared to patients with lower ACC1 levels (5.5 vs. 36 months; aHR 4.49, 95% CIs 1.08-18.72; p = 0.039). In conclusion, systemic and tumor lipid metabolism are associated with the PFS of everolimus-treated patients with advanced pNETs; based on these findings, dietary and pharmacological interventions targeting lipid metabolism could improve everolimus efficacy in this patient population.

HSPH1 inhibition downregulates Bcl-6 and c-Myc and hampers the growth of human aggressive B-cell non-Hodgkin lymphoma.

We have shown that human B-cell non-Hodgkin lymphomas (B-NHLs) express heat shock protein (HSP)H1/105 in function of their aggressiveness. Here, we now clarify its role as a functional B-NHL target by testing the hypothesis that it promotes the stabilization of key lymphoma oncoproteins. HSPH1 silencing in 4 models of aggressive B-NHLs was paralleled by Bcl-6 and c-Myc downregulation. In vitro and in vivo analysis of HSPH1-silenced Namalwa cells showed that this effect was associated with a significant growth delay and the loss of tumorigenicity when 10(4) cells were injected into mice. Interestingly, we found that HSPH1 physically interacts with c-Myc and Bcl-6 in both Namalwa cells and primary aggressive B-NHLs. Accordingly, expression of HSPH1 and either c-Myc or Bcl-6 positively correlated in these diseases. Our study indicates that HSPH1 concurrently favors the expression of 2 key lymphoma oncoproteins, thus confirming its candidacy as a valuable therapeutic target of aggressive B-NHLs.

Impact of in vitro SARS-CoV-2 infection on breast cancer cells.

The pandemic of coronavirus disease 19 (COVID-19), caused by severe respiratory syndrome coronavirus 2 (SARS-CoV-2), had severe repercussions for breast cancer patients. Increasing evidence indicates that SARS-CoV-2 infection may directly impact breast cancer biology, but the effects of SARS-CoV-2 on breast tumor cells are still unknown. Here, we analyzed the molecular events occurring in the MCF7, MDA-MB-231 and HCC1937 breast cancer cell lines, representative of the luminal A, basal B/claudin-low and basal A subtypes, respectively, upon SARS-CoV-2 infection. Viral replication was monitored over time, and gene expression profiling was conducted. We found that MCF7 cells were the most permissive to viral replication. Treatment of MCF7 cells with Tamoxifen reduced the SARS-CoV-2 replication rate, suggesting an involvement of the estrogen receptor in sustaining virus replication in malignant cells. Interestingly, a metagene signature based on genes upregulated by SARS-CoV-2 infection in all three cell lines distinguished a subgroup of premenopausal luminal A breast cancer patients with a poor prognosis. As SARS-CoV-2 still spreads among the population, it is essential to understand the impact of SARS-CoV-2 infection on breast cancer, particularly in premenopausal patients diagnosed with the luminal A subtype, and to assess the long-term impact of COVID-19 on breast cancer outcomes.

Cigarette smoke sustains immunosuppressive microenvironment inducing M2 macrophage polarization and viability in lung cancer settings.

BACKGROUND: It is amply demonstrated that cigarette smoke (CS) has a high impact on lung tumor progression worsening lung cancer patient prognosis and response to therapies. Alteration of immune cell types and functions in smokers' lungs have been strictly related with smoke detrimental effects. However, the role of CS in dictating an inflammatory or immunosuppressive lung microenvironment still needs to be elucidated. Here, we investigated the effect of in vitro exposure to cigarette smoke extract (CSE) focusing on macrophages. METHODS: Immortalized murine macrophages RAW 264.7 cells were cultured in the presence of CS extract and their polarization has been assessed by Real-time PCR and cytofluorimetric analysis, viability has been assessed by SRB assay and 3D-cultures and activation by exposure to Poly(I:C). Moreover, interaction with Lewis lung carcinoma (LLC1) murine cell models in the presence of CS extract were analyzed by confocal microscopy. RESULTS: Obtained results indicate that CS induces macrophages polarization towards the M2 phenotype and M2-phenotype macrophages are resistant to the CS toxic activity. Moreover, CS impairs TLR3-mediated M2-M1 phenotype shift thus contributing to the M2 enrichment in lung smokers. CONCLUSIONS: These findings indicate that, in lung cancer microenvironment of smokers, CS can contribute to the M2-phenotype macrophages prevalence by different mechanisms, ultimately, driving an anti-inflammatory, likely immunosuppressive, microenvironment in lung cancer smokers.

Serological identification of HSP105 as a novel non-Hodgkin lymphoma therapeutic target.

We reported that the clinical efficacy of dendritic cell-based vaccination is strongly associated with immunologic responses in relapsed B-cell non-Hodgkin lymphoma (B-NHL) patients. We have now investigated whether postvaccination antibodies from responders recognize novel shared NHL-restricted antigens. Immunohistochemistry and flow cytometry showed that they cross-react with allogeneic B-NHLs at significantly higher levels than their matched prevaccination samples or nonresponders' antibodies. Western blot analysis of DOHH-2 lymphoma proteome revealed a sharp band migrating at approximately 100 to 110 kDa only with postvaccine repertoires from responders. Mass spectrometry identified heat shock protein-105 (HSP105) in that molecular weight interval. Flow cytometry and immunohistochemistry disclosed HSP105 on the cell membrane and in the cytoplasm of B-NHL cell lines and 97 diagnostic specimens. A direct correlation between HSP105 expression and lymphoma aggressiveness was also apparent. Treatment of aggressive human B-NHL cell lines with an anti-HSP105 antibody had no direct effects on cell cycle or apoptosis but significantly reduced the tumor burden in xenotransplanted immunodeficient mice. In vivo antilymphoma activity of HSP105 engagement was associated with a significant local increase of Granzyme B(+) killer cells that very likely contributed to the tumor-restricted necrosis. Our study adds HSP105 to the list of nononcogenes that can be exploited as antilymphoma targets.

The Emerging Role of the Microbiota in Breast Cancer Progression.

Emerging evidence suggests a profound association between the microbiota composition in the gastrointestinal tract and breast cancer progression. The gut microbiota plays a crucial role in modulating the immune response, releasing metabolites, and modulating estrogen levels, all of which have implications for breast cancer growth. However, recent research has unveiled a novel aspect of the relationship between the microbiota and breast cancer, focusing on microbes residing within the mammary tissue, which was once considered sterile. These localized microbial communities have been found to change in the presence of a tumor as compared to healthy mammary tissue, unraveling their potential contribution to tumor progression. Studies have identified specific bacterial species that are enriched within breast tumors and have highlighted the mechanisms by which even these microbes influence cancer progression through immune modulation, direct carcinogenic activity, and effects on cellular pathways involved in cell proliferation or apoptosis. This review aims to provide an overview of the current knowledge on the mechanisms of crosstalk between the gut/mammary microbiota and breast cancer. Understanding this intricate interplay holds promise for developing innovative therapeutic approaches.

HER2 Copy Number and Resistance Mechanisms in Patients with HER2-positive Advanced Gastric Cancer Receiving Initial Trastuzumab-based Therapy in JACOB Trial.

PURPOSE: In JACOB trial, pertuzumab added to trastuzumab-chemotherapy did not significantly improve survival of patients with HER2-positive metastatic gastric cancer, despite 3.3 months increase versus placebo. HER2 copy-number variation (CNV) and AMNESIA panel encompassing primary resistance alterations (KRAS/PIK3CA/MET mutations, KRAS/EGFR/MET amplifications) may improve patients' selection for HER2 inhibition. EXPERIMENTAL DESIGN: In a post hoc analysis of JACOB on 327 samples successfully sequenced by next-generation sequencing (NGS; Oncomine Focus DNA), HER2 CNV, HER2 expression by IHC, and AMNESIA were correlated with overall response rate (ORR), progression-free survival (PFS), and overall survival (OS) by univariable/multivariable models. RESULTS: Median HER2 CNV was 4.7 (interquartile range, 2.2-16.9). HER2 CNV-high versus low using the median as cutoff was associated with longer median PFS (10.5 vs. 6.4 months; HR = 0.48; 95% confidence interval: 0.38-0.62; P < 0.001) and OS (20.3 vs. 13.0 months; HR = 0.54; 0.42-0.72; P < 0.001). Combining HER2 CNV and IHC improved discriminative ability, with better outcomes restricted to HER2-high/HER2 3+ subgroup. AMNESIA positivity was found in 51 (16%), with unadjusted HR = 1.35 (0.98-1.86) for PFS; 1.43 (1.00-2.03) for OS.In multivariable models, only HER2 CNV status remained significant for PFS (P < 0.001) and OS (P = 0.004). Higher ORR was significantly associated with IHC 3+ [61% vs. 34% in 2+; OR = 3.11 (1.89-5.17)] and HER2-high [59% vs. 43% in HER2-low; OR = 1.84 (1.16-2.94)], with highest OR in the top CNV quartile. These biomarkers were not associated with treatment effect of pertuzumab. CONCLUSIONS: HER2 CNV-high assessed by NGS may be associated with better ORR, PFS, and OS in a JACOB subgroup, especially if combined with HER2 3+. The negative prognostic role of AMNESIA requires further clinical validation.

Fatty acid synthase as a new therapeutic target for HER2-positive gastric cancer.

PURPOSE: Trastuzumab is an HER2-specific agent approved as the gold-standard therapy for advanced HER2-positive (HER2+) gastric cancer (GC), but the high rate and rapid appearance of resistance limit its clinical efficacy, resulting in the need to identify new vulnerabilities. Defining the drivers influencing HER2+ cancer stem cell (CSC) maintenance/survival could represent a clinically useful strategy to counteract tumor growth and therapy resistance. Accumulating evidence show that targeting crucial metabolic hubs, as the fatty acid synthase (FASN), may be clinically relevant. METHODS: FASN protein and transcript expression were examined by WB and FACS and by qRT-PCR and GEP analyses, respectively, in trastuzumab-sensitive and trastuzumab-resistant HER2+ GC cell lines cultured in adherent (2D) or gastrosphere promoting (3D) conditions. Molecular data were analyzed in silico in public HER2+ GC datasets. The effectiveness of the FASN inhibitor TVB3166 to overcome anti-HER2 therapy resistance was tested in vitro in gastrospheres forming efficiency bioassays and in vivo in mice bearing trastuzumab-resistant GC cells. RESULTS: We compared the transcriptome profiles of HER2+ GC cells cultured in 2D versus 3D conditions finding a significant enrichment of FASN in 3D cultures. FASN upregulation significantly correlated with high stemness score and poor prognosis in HER2+ GC cases. TVB3166 treatment significantly decreased GCSCs in all cell targets. HER2 and FASN cotargeting significantly decreased the capability to form gastrospheres versus monotherapy and reduced the in vivo growth of trastuzumab-resistant GC cells. CONCLUSION: Our findings indicate that cotargeting HER2 and FASN increase the benefit of anti-HER2 therapy representing a new opportunity for metabolically combating trastuzumab-resistant HER2+ GC.

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.

BCL6 and the Notch pathway: a signaling axis leading to a novel druggable biotarget in triple negative breast cancer.

BACKGROUND: The transcriptional repressor B-cell lymphoma 6 (BCL6) is dysregulated in several neoplasms, but its role in triple negative breast cancer (TNBC), a highly aggressive subtype which lacks effective treatment, is unclear. The presence of intratumoral cancer stem cells (CSCs) is a main cause of tumor relapse. The Notch signaling pathway is crucial for regulating CSC self-renewal and promoting breast cancer (BC) development and resistance to anticancer therapies. Here, we investigated signaling cascades of BCL6 in the CSC compartment of TNBCs, and the mechanisms that govern its activity, mainly through Notch signaling. METHODS: Gene expression, somatic copy number alterations and clinical data from the Cancer Genome Atlas and METABRIC were accessed through the Xena and cbioportal browsers. Public transcriptome profiles from TNBC datasets were retrieved from the Gene Expression Omnibus. Mammosphere formation efficiency was calculated after BCL6 knockdown via transient siRNA transfection, stable silencing or pharmacological inhibition. The effects exhibited via BCL6 inhibition in putative TNBC stem-like cells were evaluated by immunofluorescence and qRT-PCR analyses. Chromatin immunoprecipitation experiments were performed to validate a putative BCL6 responsive element located in the first intron of the Numb gene and to define the circuit of corepressors engaged by BCL6 following its inhibition. Immunoprecipitation assays were carried out to investigate a novel interaction at the basis of BCL6 control of CSC activity in TNBC. RESULTS: In silico analyses of benchmarked public datasets revealed a significant enrichment of BCL6 in cancer stemness related pathways, particularly of Notch signaling in TNBC. In vitro stable inhibition of BCL6 significantly reduced tumor cell growth and, accordingly, we found that the mammosphere formation efficiency of BCL6 silenced cells was significantly impaired by pharmacological inhibition of Notch signaling. BCL6 was found to be expressed at significantly higher levels in TNBC mammospheres than in their adherent counterparts, and loss of BCL6 function significantly decreased mammosphere formation with preferential targeting of CD44-positive versus ALDH-positive stem-like cells. Functional interplay between BCL6 and the chromatin remodeling factor EZH2 triggered the BCL6/Notch stemness signaling axis via inhibition of Numb transcription. CONCLUSIONS: Our results may be instrumental for the prospective design of combination treatment strategies that selectively target novel TNBC-associated biomarker(s) whose activity is implicated in the regulation of cancer stemness (such as BCL6) and molecules in developmentally conserved signaling pathways (such as Notch) to achieve long-lasting tumor control and improve patient outcomes.

Predictive Role of CD36 Expression in HER2-Positive Breast Cancer Patients Receiving Neoadjuvant Trastuzumab.

BACKGROUND: Despite huge efforts to identify biomarkers associated with long-term clinical outcomes in patients with early-stage HER2-positive breast cancer (HER2+ BC) treated with (neo)adjuvant anti-HER2 therapy, no reliable predictors have been identified so far. Fatty acid uptake, a process mediated by the transmembrane transporter CD36, has recently emerged as a potential determinant of resistance to anti-HER2 treatments in preclinical HER2+ BC models. METHODS: Here, we investigated the association between baseline intratumor CD36 gene expression and event-free survival in 180 patients enrolled in the phase III trial Neoadjuvant Lapatinib and/or Trastuzumab Treatment Optimization (NeoALTTO), which randomly assigned stage II-III HER2+ BC patients to receive neoadjuvant lapatinib, trastuzumab, or lapatinib-trastuzumab in combination with chemotherapy. To this aim, we selected NeoALTTO trial patients for whom pretreatment whole transcriptomic data were available. The main study results were validated in an independent cohort of patients enrolled in the neoadjuvant phase II trial NeoSphere. RESULTS: In 180 NeoALTTO patients, high intratumor CD36 expression was independently associated with worse event-free survival in patients treated with trastuzumab-based therapy (hazard ratio [HR] = 1.72, 95% confidence interval [CI] = 1.20 to 2.46), but not with lapatinib-based (HR = 1.02, 95% CI = 0.68 to 1.53) or trastuzumab-lapatinib-based (HR = 1.08, 95% CI = 0.60 to 1.94) therapy. Among 331 NeoSphere patients evaluated, high CD36 expression was independently associated with worse patient disease-free survival in both the whole study cohort (HR = 1.197, 95% CI = 1.002 to 1.428) and patients receiving trastuzumab-based neoadjuvant therapy (HR = 1.282, 95% CI = 1.049 to 1.568). CONCLUSIONS: High CD36 expression predicts worse clinical outcomes in early-stage HER2+ BC treated with trastuzumab-based neoadjuvant therapy.

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.

Vaccination with autologous tumor-loaded dendritic cells induces clinical and immunologic responses in indolent B-cell lymphoma patients with relapsed and measurable disease: a pilot study.

Eighteen relapsed patients with measurable indolent non-Hodgkin lymphoma (NHL) were vaccinated with dendritic cells (DCs) loaded with killed autologous tumor cells. Six patients had objective clinical responses including 3 continuous complete responses (CRs) and 3 partial responses (PRs), with a median follow up of 50.5 months. Eight patients had stable disease, whereas 4 had progressive disease. Clinical responses were significantly associated with a reduction in CD4(+)CD25(+)FOXP3(+) regulatory T cells, an increase in CD3(-)CD56(dim)CD16(+) natural killer (NK) cells, and maturation of lymphocytes to the effector memory stage in either postvaccination peripheral blood or tumor specimen samples. In partial responding patients, vaccination significantly boosted the IFN-gamma-producing T-cell response to autologous tumor challenge. In one HLA-A*0201(+) patient who achieved CR, IL-4 release by circulating T cells in response to tumor-specific IgH-encoded peptides was also documented. Immunohistochemical analysis of tumor biopsies using biotin-conjugated autologous serum samples revealed a tumor-restricted humoral response only in the postvaccination serum from responding patients. Collectively these results demonstrate that vaccination with tumor-loaded DCs may induce both T- and B-cell responses and produces clinical benefits in indolent NHL patients with measurable disease. This study is registered with the Istituto Superiore di Sanità: http://www.iss.it with protocol number 7578-PRE 21-801.

Do pre-diagnostic drinking habits influence breast cancer survival?

AIMS AND BACKGROUND: Alcohol consumption increases the risk of developing breast cancer and may also be associated with late diagnosis, recurrence, distant metastases and death. Many studies have examined the role of alcohol as a risk factor for the development of breast cancer, but very few studies have addressed the role of alcohol as a prognostic factor for survival among women diagnosed with breast cancer. The aim of this study was to investigate the survival of women with breast cancer in relation to pre-diagnostic alcohol intake and other factors known to influence prognosis. METHODS: We analyzed data for 264 women in the EUROCARE and ORDET studies who were diagnosed with breast cancer from 1987 up to 31 December 2001 and for whom information was available on follow-up, stage at diagnosis, HER-2 and hormone receptor status, and pre-diagnostic dietary alcohol intake, categorized as zero (0 g/day, non-drinkers), moderate (up to 13 g/day, about 1 serving) and high (>13 g/day). Ten-year relative survival was estimated using the maximum-likelihood approach. The excess risk of death within 10 years of diagnosis was modeled by level of alcohol intake, adjusting separately for age, stage, body mass index and tumor subtype. RESULTS: Ten-year relative survival was lower in women who drank more than 13 g/day (65%; 95% CI, 47-78) than in non-drinkers (88%; 95% CI, 75-95). The excess risk of death within 10 years was significantly higher in women who drank more than 13 g/day than non-drinkers (relative excess risk, 4.13; 95% CI, 1.69-10.10) and was not altered by adjustment for other prognostic factors. The excess risk within 10 years was higher for women with a body mass index of 25 kg/m2 or higher (relative excess risk, 2.20; 95% CI, 1.01-4.70) and higher for those with more advanced disease. CONCLUSIONS: Women who drank more than 13 g alcohol per day had lower survival than non-drinkers. The excess risk of death within 10 years of diagnosis was unaffected by other known risk factors. High alcohol consumption may be an adverse prognostic factor for breast cancer.

HER2 Signaling and Breast Cancer Stem Cells: The Bridge behind HER2-Positive Breast Cancer Aggressiveness and Therapy Refractoriness.

HER2 overexpression/amplification occurs in 15-20% of breast cancers (BCs) and identifies a highly aggressive BC subtype. Recent clinical progress has increased the cure rates of limited-stage HER2-positive BC and significantly prolonged overall survival in patients with advanced disease; however, drug resistance and tumor recurrence remain major concerns. Therefore, there is an urgent need to increase knowledge regarding HER2 biology and implement available treatments. Cancer stem cells (CSCs) represent a subset of malignant cells capable of unlimited self-renewal and differentiation and are mainly considered to contribute to tumor onset, aggressiveness, metastasis, and treatment resistance. Seminal studies have highlighted the key role of altered HER2 signaling in the maintenance/enrichment of breast CSCs (BCSCs) and elucidated its bidirectional communication with stemness-related pathways, such as the Notch and Wingless/ß-catenin cascades. d16HER2, a splice variant of full-length HER2 mRNA, has been identified as one of the most oncogenic HER2 isoform significantly implicated in tumorigenesis, epithelial-mesenchymal transition (EMT)/stemness and the response to targeted therapy. In addition, expression of a heterogeneous collection of HER2 truncated carboxy-terminal fragments (CTFs), collectively known as p95HER2, identifies a peculiar subgroup of HER2-positive BC with poor prognosis, with the p95HER2 variants being able to regulate CSC features. This review provides a comprehensive overview of the current evidence regarding HER2-/d16HER2-/p95HER2-positive BCSCs in the context of the signaling pathways governing their properties and describes the future prospects for targeting these components to achieve long-lasting tumor control.

Hormone receptor status influences the impact of body mass index and hyperglycemia on the risk of tumor relapse in early-stage HER2-positive breast cancer patients.

BACKGROUND: High body mass index (BMI) has been associated with worse clinical outcomes in patients with early-stage breast cancer (BC), and its negative effects could be mediated by hyperglycemia/diabetes. However, the prognostic impact of high BMI in early-stage HER2-positive (HER2+) BC patients remains controversial. METHODS: We conducted a retrospective study to investigate the impact of baseline BMI or glycemia on relapse-free survival (RFS) and overall survival (OS) in patients with surgically resected, stage I-III HER2+ BC treated with standard-of-care, trastuzumab-containing adjuvant biochemotherapy. The optimal BMI and glycemia cut-off values for RFS were identified through maximally selected rank statistics. Cox regression models were used to assess the impact of BMI, glycemia and other relevant variables on clinical outcomes. RESULTS: Among 505 patients included in the study, a BMI cut-off of 27.77 kg/m2 was identified as the best threshold to discriminate between patients with low BMI (n = 390; 77.2%) or high BMI (n = 115; 22.8%). At multivariable analysis, higher BMI was associated with significantly worse RFS [hazard ratio 2.26; 95% confidence interval (CI): 1.08-4.74, p = 0.031] and worse OS (hazard ratio 2.25, 95% CI 1.03-4.94, p = 0.043) in the whole patient population. The negative impact of high BMI was only observed in patients with hormone receptor (HR)-negative/HER2+ BC (hazard ratio 2.29; 95% CI: 1.01-5.20; p = 0.047), but not in patients with HR-positive (HR+)/HER2+ BC (hazard ratio 1.36; 95% CI: 0.61-3.07, p = 0.452). By contrast, hyperglycemia (⩾109 mg/dl) at baseline was associated with a trend toward significantly worse RFS at multivariable analysis only in patients with HR+/HER2+ BC (hazard ratio 2.52; 95% CI: 0.89-7.1; p = 0.080). CONCLUSIONS: High BMI is associated with worse clinical outcomes in early-stage HR-/HER2+ BC patients treated with trastuzumab-containing adjuvant biochemotherapy, while baseline hyperglycemia could be a predictor of worse RFS in HR+/HER2+ BC patients. Prospective studies are needed to investigate if modifying patient BMI/glycemia during treatment can improve clinical outcomes.

Targeting lipid metabolism is an emerging strategy to enhance the efficacy of anti-HER2 therapies in HER2-positive breast cancer.

De novo or acquired resistance of cancer cells to currently available Human Epidermal Growth Factor Receptor 2 (HER2) inhibitors represents a clinical challenge. Several resistance mechanisms have been identified in recent years, with lipid metabolism reprogramming, a well-established hallmark of cancer, representing the last frontier of preclinical and clinical research in this field. Fatty Acid Synthase (FASN), the key enzyme required for fatty acids (FAs) biosynthesis, is frequently overexpressed/activated in HER2-positive (HER2+) breast cancer (BC), and it crucially sustains HER2+ BC cell growth, proliferation and survival. After the synthesis of new, selective and well tolerated FASN inhibitors, clinical trials have been initiated to test if these compounds are able to re-sensitize cancer cells with acquired resistance to HER2 inhibition. More recently, the upregulation of FA uptake by cancer cells has emerged as a potentially new and targetable mechanism of resistance to anti-HER2 therapies in HER2+ BC, thus opening a new era in the field of targeting metabolic reprogramming in clinical setting. Here, we review the available preclinical and clinical evidence supporting the inhibition of FA biosynthesis and uptake in combination with anti-HER2 therapies in patients with HER2+ BC, and we discuss ongoing clinical trials that are investigating these combination approaches.