Search

Abstract Background During targeted treatment, HER2-positive breast cancers invariably lose HER2 DNA amplification. In contrast, and interestingly, HER2 proteins may be either lost or gained. To longitudinally and systematically appreciate complex/discordant changes in HER2 DNA/protein stoichiometry, HER2 DNA copy numbers and soluble blood proteins (aHER2/sHER2) were tested in parallel, non-invasively (by liquid biopsy), and in two-dimensions, hence HER2-2D. Methods aHER2 and sHER2 were assessed by digital PCR and ELISA before and after standard-of-care treatment of advanced HER2-positive breast cancer patients (n=37) with the antibody-drug conjugate (ADC) Trastuzumab-emtansine (T-DM1). Results As expected, aHER2 was invariably suppressed by T-DM1, but this loss was surprisingly mirrored by sHER2 gain, sometimes of considerable entity, in most (30/37; 81%) patients. This unorthodox split in HER2 oncogenic dosage was supported by reciprocal aHER2/sHER2 kinetics in two representative cases, and an immunohistochemistry-high status despite copy-number-neutrality in 4/5 available post-T-DM1 tumor re-biopsies from sHER2-gain patients. Moreover, sHER2 was preferentially released by dying breast cancer cell lines treated in vitro by T-DM1. Finally, sHER2 gain was associated with a longer PFS than sHER2 loss (mean PFS 282 vs 133 days, 95% CI [210-354] vs [56-209], log-rank test p=0.047), particularly when cases (n=11) developing circulating HER2-bypass alterations during T-DM1 treatment were excluded (mean PFS 349 vs 139 days, 95% CI [255-444] vs [45-232], log-rank test p=0.009). Conclusions HER2 gain is adaptively selected in tumor tissues and recapitulated in blood by sHER2 gain. Possibly, an increased oncogenic dosage is beneficial to the tumor during anti-HER2 treatment with naked antibodies, but favorable to the host during treatment with a strongly cytotoxic ADC such as T-DM1. In the latter case, HER2-gain tumors may be kept transiently in check until alternative oncogenic drivers, revealed by liquid biopsy, bypass HER2. Whichever the interpretation, HER2-2D might help to tailor/prioritize anti-HER2 treatments, particularly ADCs active on aHER2-low/sHER2-low tumors. Trial registration NCT05735392 retrospectively registered on January 31, 2023 https://www.clinicaltrials.gov/search?term=NCT05735392

Abstract Background Breast Cancer (BC) can be classified, due to its heterogeneity, into multiple subtypes that differ for prognosis and clinical management. Notably, triple negative breast cancer (TNBC) – the most aggressive BC form – is refractory to endocrine and most of the target therapies. In this view, taxane-based therapy still represents the elective strategy for the treatment of this tumor. However, due variability in patients’ response, management of TNBC still represents an unmet medical need. Telomeric Binding Factor 2 (TRF2), a key regulator of telomere integrity that is over-expressed in several tumors, including TNBC, has been recently found to plays a role in regulating autophagy, a degradative process that is involved in drug detoxification. Based on these considerations, we pointed, here, at investigating if TRF2, regulating autophagy, can affect tumor sensitivity to therapy. Methods Human TNBC cell lines, over-expressing or not TRF2, were subjected to treatment with different taxanes and drug efficacy was tested in terms of autophagic response and cell proliferation. Autophagy was evaluated first biochemically, by measuring the levels of LC3, and then by immunofluorescence analysis of LC3-puncta positive cells. Concerning the proliferation, cells were subjected to colony formation assays associated with western blot and FACS analyses. The obtained results were then confirmed also in mouse models. Finally, the clinical relevance of our findings was established by retrospective analysis on a cohort of TNBC patients subjected to taxane-based neoadjuvant chemotherapy. Results This study demonstrated that TRF2, inhibiting autophagy, is able to increase the sensitivity of TNBC cells to taxanes. The data, first obtained in in vitro models, were then recapitulated in preclinical mouse models and in a cohort of TNBC patients, definitively demonstrating that TRF2 over-expression enhances the efficacy of taxane-based neoadjuvant therapy in reducing tumor growth and its recurrence upon surgical intervention. Conclusions Based on our finding it is possible to conclude that TRF2, already known for its role in promoting tumor formation and progression, might represents an Achilles’ heel for cancer. In this view, TRF2 might be exploited as a putative biomarker to predict the response of TNBC patients to taxane-based neoadjuvant chemotherapy.

Abstract Background Molecular Tumor Boards (MTB) operating in real-world have generated limited consensus on good practices for accrual, actionable alteration mapping, and outcome metrics. These topics are addressed herein in 124 MTB patients, all real-world accrued at progression, and lacking approved therapy options. Methods Actionable genomic alterations identified by tumor DNA (tDNA) and circulating tumor DNA (ctDNA) profiling were mapped by customized OncoKB criteria to reflect diagnostic/therapeutic indications as approved in Europe. Alterations were considered non-SoC when mapped at either OncoKB level 3, regardless of tDNA/ctDNA origin, or at OncoKB levels 1/2, provided they were undetectable in matched tDNA, and had not been exploited in previous therapy lines. Results Altogether, actionable alterations were detected in 54/124 (43.5%) MTB patients, but only in 39 cases (31%) were these alterations (25 from tDNA, 14 from ctDNA) actionable/unexploited, e.g. they had not resulted in the assignment of pre-MTB treatments. Interestingly, actionable and actionable/unexploited alterations both decreased (37.5% and 22.7% respectively) in a subset of 88 MTB patients profiled by tDNA-only, but increased considerably (77.7% and 66.7%) in 18 distinct patients undergoing combined tDNA/ctDNA testing, approaching the potential treatment opportunities (76.9%) in 147 treatment-naïve patients undergoing routine tDNA profiling for the first time. Non-SoC therapy was MTB-recommended to all 39 patients with actionable/unexploited alterations, but only 22 (56%) accessed the applicable drug, mainly due to clinical deterioration, lengthy drug-gathering procedures, and geographical distance from recruiting clinical trials. Partial response and stable disease were recorded in 8 and 7 of 19 evaluable patients, respectively. The time to progression (TTP) ratio (MTB-recommended treatment vs last pre-MTB treatment) exceeded the conventional Von Hoff 1.3 cut-off in 9/19 cases, high absolute TTP and Von Hoff values coinciding in 3 cases. Retrospectively, 8 patients receiving post-MTB treatment(s) as per physician’s choice were noted to have a much longer overall survival from MTB accrual than 11 patients who had received no further treatment (35.09 vs 6.67 months, p = 0.006). Conclusions MTB-recommended/non-SoC treatments are effective, including those assigned by ctDNA-only alterations. However, real-world MTBs may inadvertently recruit patients electively susceptible to diverse and/or multiple treatments.

Abstract Background Metastatic breast cancer (MBC) is an incurable disease and its treatment focuses on prolonging patients’ (pts) overall survival (OS) and improving their quality of life. Eribulin is a microtubule inhibitor that increases OS in pre-treated MBC pts. The most common adverse events (AEs) are asthenia, neutropenia and peripheral neuropathy (PN). Methods PAINTER is a single arm, phase IV study, aimed at evaluating the tolerability of eribulin in MBC pts. Secondary objectives were the description of treatment efficacy and safety, the assessment of the incidence and severity of PN and its association with genetic polymorphisms. Genomic DNA was isolated from blood samples and 15 Single Nucleotide Polymorphisms (SNPs) were genotyped by Taqman specific assays. The association between PN and SNPs were evaluated by Fisher exact test. Results Starting from May 2014 until June 2018 180 pts were enrolled in this study by 20 Italian centers. 170 of these pts could be evaluated for efficacy and toxicity and 159 for polymorphisms analysis. The median age of pts was 60 years old and the biological subtypes were luminal type (64.7%), Her2 positive (18.3%) and triple negative (17%). Pts were pretreated with a median of 5 lines for MBC. The median follow up of this study was 15.4 months with a median number of 4.5 cycles administered (minimum–maximum 1–23). The median overall survival was 12 months. 48.8% of pts experienced a dose reduction, mainly for neutropenia (23.9%) and liver toxicity (12%). 65 pts (38.2%) reported at least one severe toxicity. Neutropenia and neurotoxicity were the most frequent severe AEs (15.3% and 14.7%, respectively); other reported toxicities were osteo-muscular, abdominal or tumor site pain (19.4%), liver toxicity (6.6%), pulmonary toxicity (6.5%) and dermatological toxicity (3.6%). Among the 15 evaluated SNPs, an association with PN was found for rs2233335 and rs7214723. Conclusions Eribulin is a well-tolerated treatment option in MBC. Schedule and dosage modifications were common, but toxicity rarely led to treatment discontinuation. SNPs rs2233335 (G/T and T/T) in the NDRG1 gene and rs7214723 (CC and CT) in the CAMKK1 gene were associated with PN. These findings, if validated, could allow a tailored treatment with eribulin in cancer patients. Trial registration: ClinicalTrials.gov ID: NCT02864030.

Abstract Background Breast cancer (BC) presents important physical and psychological challenges that should be appropriately addressed through continuous, integrated and individualized rehabilitation programs after treatment. In this study, we aimed to collect more information on the rehabilitation patterns and utilization of healthcare services by women with BC. Methods We retrospectively analyzed data from two archives of the Lazio Regional Health System Database to assess rehabilitation patterns in women diagnosed with BC in the Lazio region (Italy) in 2008. Results A total of 5538 women diagnosed with BC were considered in the present study. Most patients (81.7%) received outpatient rehabilitative care, consisting mainly of pathology-related interventions and, more rarely, disability-related interventions (mainly motor rehabilitation and rarely cognitive or psychological therapy). Few patients followed an inpatient (1.3%) or an intensive outpatient rehabilitation program (1.0%). Conclusion Most patients do not receive adequate rehabilitation care during the first year after diagnosis. More information and better rehabilitation services should be provided to help patients with BC access rehabilitation programs. The study also suggests the importance of psychosocial and cognitive interventions, which is a major unmet need in women with BC.

Abstract Background NEPA is an oral fixed-dose combination of netupitant, a new highly selective neurokinin-1 receptor antagonist, and palonosetron. This study was conducted to evaluate whether the efficacy of NEPA against chemotherapy-induced nausea and vomiting (CINV) in cycle 1 would be maintained over subsequent chemotherapy cycles in breast cancer patients receiving adjuvant anthracycline plus cyclophosphamide (AC). The study also describes the relationship between efficacy on day 1 through 5 (overall period) and control of CINV on day 6 through 21 (very late period) in each cycle. Methods In this multicentre, phase II study, patients received both NEPA and dexamethasone (12 mg intravenously) just before chemotherapy. The primary efficacy endpoint was overall complete response (CR; no emesis and no rescue medication use) in cycle 1. Sustained efficacy was evaluated during the subsequent cycles by calculating the rate of CR in cycles 2–4 and by assessing the probability of sustained CR over multiple cycles. The impact of both overall CR and risk factors for CINV on the control of very late events (vomiting and moderate-to-severe nausea) were also examined. Results Of the 149 patients enrolled in the study, 139 were evaluable for a total of 552 cycles; 97.8% completed all 4 cycles. The proportion of patients with an overall CR was 70.5% (90% CI, 64.1 to 76.9) in cycle 1, and this was maintained in subsequent cycles. The cumulative percentage of patients with a sustained CR over 4 cycles was 53%. NEPA was well tolerated across cycles. In each cycle, patients with CR experienced a significantly better control of very late CINV events than those who experienced no CR. Among the patients with CR, the only predictor for increased likelihood of developing very late CINV was pre-chemotherapy (anticipatory) nausea (adjusted odds ratio = 0.65–0.50 for no CINV events on cycles 3 and 4). Conclusion The high anti-emetic efficacy seen with the NEPA regimen in the first cycle was maintained over multiple cycles of adjuvant AC for breast cancer. Preliminary evidence also suggests that patients achieving a CR during the overall period gain high protection even against very late CINV events in each chemotherapy cycle. Trial registration This trial was retrospectively registered at Clinicaltrials.gov identifier ( NCT03862144 ) on 05/Mar/2019.

Abstract RANKL/RANK/OPG system consists of three essential signaling molecules: i) the receptor activator of nuclear factor (NF)-kB-ligand (RANKL), ii) the receptor activator of NF-kB (RANK), and iii) the soluble decoy receptor osteoprotegerin (OPG). Although this system is critical for the regulation of osteoclast differentiation/activation and calcium release from the skeleton, different studies have elucidated its specific role in mammary gland physiology and hormone-driven epithelial proliferation during pregnancy. Of note, several data suggest that progesterone induces mammary RANKL expression in mice and humans. In turn, RANKL controls cell proliferation in breast epithelium under physiological conditions typically associated with higher serum progesterone levels, such as luteal phase of the menstrual cycle and pregnancy. Hence, RANKL/RANK system can be regarded as a major downstream mediator of progesterone-driven mammary epithelial cells proliferation, potentially contributing to breast cancer initiation and progression. Expression of RANKL, RANK, and OPG has been detected in breast cancer cell lines and in human primary breast cancers. To date, dysregulation of RANKL/RANK/OPG system at the skeletal level has been widely documented in the context of metastatic bone disease. In fact, RANKL inhibition through the RANKL-blocking human monoclonal antibody denosumab represents a well-established therapeutic option to prevent skeletal-related events in metastatic bone disease and adjuvant therapy-induced bone loss in breast cancer. On the other hand, the exact role of OPG in breast tumorigenesis is still unclear. This review focuses on molecular mechanisms linking RANKL/RANK/OPG system to mammary tumorigenesis, highlighting pre-clinical and clinical evidence for the potential efficacy of RANKL inhibition as a prevention strategy and adjuvant therapy in breast cancer settings.

Abstract Background This is a retrospective cross sectional study aimed to verify whether Multiplex Ligation-dependent Probe Amplification (MLPA), a quantitative molecular assay, may represent a valuable reflex test in breast cancer with equivocal HER2 expression by immunohistochemistry and HER2 gene signals/nucleus (s/n) ranging between 4.0 and 5.9 by in situ hybridization. Methods A series of 170 breast carcinomas scored as 2+ for HER2 expression by immunohistochemistry, were selected from our files and analyzed in parallel by silver in situ hybridization and by MLPA. According to ASCO-CAP 2013 guidelines, 54/170 tumors, displaying 4.0–5.9 HER2 gene s/n, were defined as low amplified (ratio ≥ 2) or equivocal (ratio 2.0. These data were further confirmed in the external validation set. Interestingly, the 54 low amplified/equivocal breast carcinomas presented a frequency of hormonal receptor positivity significantly higher than that observed in the amplified tumors and similar to the non-amplified one (p = 0.016 for estrogen receptor and p = 0.001 for progesterone receptor). Conclusions To avoid to offer patients an ineffective therapy, HER2 status should be studied more thoroughly in low amplified and equivocal cases which can have lower response rates and shorter time to progression to trastuzumab. In this context, our data indicate that MLPA may be a reliable, objective supporting test in selecting HER2 positive breast cancer patients.

Abstract Background Cancer is a mosaic of tumor cell subpopulations, where only a minority is responsible for disease recurrence and cancer invasiveness. We focused on one of the most aggressive circulating tumor cells (CTCs) which, from the primitive tumor, spreads to the central nervous system (CNS), evaluating the expression of prognostic and putative cancer stem cell markers in breast cancer (BC) leptomeningeal metastasis (LM). Methods Flow cytometry immunophenotypic analysis of cerebrospinal fluid (CSF) samples (4.5 ml) was performed in 13 consecutive cases of BCLM. Syndecan-1 (CD138), MUC-1 (CD227) CD45, CD34, and the putative cancer stem cell markers CD15, CD24, CD44, and CD133 surface expression were evaluated on CSF floating tumor cells. The tumor-associated leukocyte population was also characterized. Results Despite a low absolute cell number (8 cell/μl, range 1–86), the flow cytometry characterization was successfully conducted in all the samples. Syndecan-1 and MUC-1 overexpression was documented on BC cells in all the samples analyzed; CD44, CD24, CD15, and CD133 in 77%, 75%, 70%, and 45% of cases, respectively. A strong syndecan-1 and MUC-1 expression was also documented by immunohistochemistry on primary breast cancer tissues, performed in four patients. The CSF tumor population was flanked by T lymphocytes, with a different immunophenotype between the CSF and peripheral blood samples (P ≤ 0.02). Conclusions Flow cytometry can be successfully employed for solid tumor LM characterization even in CSF samples with low cell count. This in vivo study documents that CSF floating BC cells overexpress prognostic and putative cancer stem cell biomarkers related to tumor invasiveness, potentially representing a molecular target for circulating tumor cell detection and LM treatment monitoring, as well as a primary target for innovative treatment strategies. The T lymphocyte infiltration, documented in all CSF samples, suggests a possible involvement of the CNS lymphatic system in both lymphoid and cancer cell migration into and out of the meninges, supporting the extension of a new form of cellular immunotherapy to LM. Due to the small number of cases, validation on large cohorts of patients are warranted to confirm these findings and to evaluate the impact and value of these results for diagnosis and management of LM.

Abstract The United States Food and Drug Administration (FDA) recently approved the clinical use of two comprehensive ‘mid-size’ Next Generation Sequencing (NGS) panels calling actionable genomic aberrations in cancer. This is the first endorsement, by a regulatory body, of a new standard of care in oncology. Herein, we argue that besides its many practice-changing implications, this approval tears down the conceptual walls dividing system biology from clinical practice, diagnosis from research, prevention from therapy, cancer genetics from cancer genomics, and computational biology from empirical therapy assignment.