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Autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy is an effective treatment for approximately 40% of relapsed/refractory large B cell lymphomas (LBCL), and early identification of patients at risk for relapse or progression after CAR T-cell therapy represents a clinical need.The authors conducted a single-center prospective study on 47 relapsed/refractory LBCL receiving CAR T-cell therapy to evaluate the prognostic value of baseline and after infusionDeep variation of standardized uptake value (SUV)PET parameters and association of DS and variation of SUVThis is a single-center prospective study on 47 lymphoma patients receiving commercial chimeric antigen receptor T-cell therapy aimed to evaluate the prognostic value of baseline and after infusion

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.

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Supplementary Figure S4. Event-free survival (EFS) probability curve for STAT1. The curve depicts the predicted EFS probability together with the observed pCR responses in the bottom part. Each segment on the abscissa indicates single patientachieving (in green) or not achieving (in red) pCR.

Purpose:To investigate the potential of circulating-miRNAs (ct-miRNA) as noninvasive biomarkers to predict the efficacy of single/dual HER2-targeted therapy in the NeoALTTO study.Experimental Design:Patients with plasma samples at baseline (T0) and/or after 2 weeks (T1) of treatment were randomized into training (n = 183) and testing (n = 246) sets. RT-PCR–based high-throughput miRNA profiling was employed in the training set. After normalization, ct-miRNAs associated with pathologic complete response (pCR) were identified by univariate analysis. Multivariate logistic regression models were implemented to generate treatment-specific signatures at T0 and T1, which were evaluated by RT-PCR in the testing set. Event-free survival (EFS) according to ct-miRNA signatures was estimated by Kaplan–Meier method and Cox regression model.Results:In the training set, starting from 51 ct-miRNAs associated with pCR, six signatures with statistically significant predictive capability in terms of area under the ROC curve (AUC) were identified. Four signatures were confirmed in the testing set: lapatinib at T0 and T1 [AUC 0.86; 95% confidence interval (CI), 0.73–0.98 and 0.71 (0.55–0.86)], respectively; trastuzumab at T1 (0.81; 0.70–0.92); lapatinib + trastuzumab at T1 (0.67; 0.51–0.83). These signatures were confirmed predictive after adjusting for known variables, including estrogen receptor status. ct-miRNA signatures failed to correlate with EFS. However, the levels of ct-miR-140-5p, included in the trastuzumab signature, were associated with EFS (HR 0.43; 95% CI, 0.22–0.84).Conclusions:ct-miRNAs discriminate patients with and without pCR after neoadjuvant lapatinib- and/or trastuzumab-based therapy. ct-miRNAs at week two could be valuable to identify patients responsive to trastuzumab, to avoid unnecessary combination with other anti-HER2 agents, and finally to assist deescalating treatment strategies.

Table S3. Multivariate logistic model in the study cohort (n=226; 80 pCR and 64 events).

Supplementary Figure S5. Patient-level heatmap of the 27 putative biomarkers assessed in the paper with annotation showing pCR (No; Yes), Event (No; Yes), TRT, Treatment (Combination; Single agent ), ER status (Negative; Positive), TRAR (Low; High), PAM50 subtype (HER2; Other)

Supplementary Figure S1. Correlation of immune-related metagenes, tumor-infiltrating immune cells, and expression of ERBB2, ESR1 and TRAR. The color indicates the direction of the correlation (yellow: negative-correlation; light blue: positive correlation) according to Spearman's correlation coefficient values.

Table S1. Association of clinico-pathological variables and the molecular classifiers TRAR and PAM50 with pathological complete response (pCR) and Event Free survival (EFS) in the study cohort (n=226; 80 pCR and 64 events).

Suppl figures without legends modified on feb 20th

Purpose:Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high).Patients and Methods:Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method.Results:A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1–6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40–5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12–5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45–1.22 (Pinteraction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74–0.86) to 0.83 (95% CI, 0.78–0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49–0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30–0.86).Conclusions:Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Including legends of Suppl Figures introduced on Feb 20th

Table S2. Association of tumor infiltrating immune cells with pathological complete response (pCR) and event free survival (EFS) in TRAR groups.

Supplementary Figure S3. Interaction between tumor infiltrating immune cells and treatment arm in the study cohort (n=226; 80 pCR). Red diamond and line indicate Odds Ratio (OR) and its 95% confidence interval (CI) in the combination arm; blue diamond and line indicate OR and its 95% CI in single agent arm (trastuzumab or lapatinib).

Supplementary Figure S2. Interaction between immune-related metagenes and treatment arm in the study cohort (n=226; 80 pCR). Red diamond and line indicate Odds Ratio (OR) and its 95% confidence interval (CI) in the combination arm; blue diamond and line indicate OR and its 95% CI in single agent arm (trastuzumab or lapatinib).

Background: Despite great efforts to identify tumor-related prognostic biomarkers in early-stage Human Epidermal growth factor Receptor 2 (HER2)-positive Breast Cancer (HER2+ BC), reliable determinants of long-term clinical outcomes are lacking. HER2+ BC is a lipogenic malignancy, and HER2-driven fatty acid (FA) biosynthesis plays a crucial role in sustaining cancer cell growth, proliferation, and metastatization. Recently, FA uptake, a process mediated by the transmembrane transporter CD36, has emerged as a new determinant of acquired resistance to anti-HER2 treatments in preclinical studies. Methods: The phase III NeoALTTO (NCT00553358) trial randomized 455 HER2+ BC patients to receive lapatinib, trastuzumab, or the combination of both drugs with weekly paclitaxel, followed by surgery, adjuvant fluorouracil, epirubicin, and cyclophosphamide and the same anti-HER2 therapy received before surgery. In the present analysis, we tested the hypothesis that higher intratumor CD36 mRNA levels could be associated with worse Event Free Survival (EFS) in a cohort of NeoALTTO patients for whom global gene expression analysis of baseline tumor samples through ClariomS platform was available. Results: A total of 180 patients were included (trastuzumab arm: n=66; lapatinib arm: n=65; combination arm: n=49). Higher baseline CD36 expression levels were associated with significantly worse EFS in patients treated with trastuzumab-based therapy, at both univariate (continuous scale, HR:1.73, 95% CI 1.20-2.49) and multivariable (HR:1.72, 95% CI 1.20-2.46) analysis, but not in patients treated with lapatinib- or trastuzumab plus lapatinib (HR:1.01; 95% CI: 0.69-1.47; p=0.98 and HR:1.02; 95% CI:0.54-1.95; p=0.94, respectively). CD36 levels did not predict pathological Complete Response (pCR) probability, while combining CD36 expression (based on median levels) with pCR status identified two subsets of patients with especially bad or good prognosis (7-year EFS rates in patients with high CD36/no pCR: 47%, 95% CIs: 0.26-0.66 vs. 79%, 95% CIs: 0.62-0.88 in patients with low CD36/any pCR status (Yes/No) OR high CD36/pCR; Log-Rank test p= 0.006). Conclusions: This is the first study to show that high CD36 expression is independently predictive of worse long-term clinical outcomes in HER2+ BC patients treated with neoadjuvant trastuzumab-based therapy. Along with recent preclinical data highlighting CD36 role in tumor resistance to anti-HER2 therapies, our findings point to CD36 as a novel promising target for HER2+ BC treatment. Citation Format: Francesca Ligorio, Serena Di Cosimo, Paolo Verderio, Chiara Maura Ciniselli, Sara Pizzamiglio, Lorenzo Castagnoli, Tiziana Triulzi, Elda Tagliabue, Sarra El-Abed, Miguel Izquierdo, Evandro de Azambuja, Paolo Nuciforo, Jens Huober, Luca Moscetti, Wolfgang Janni, M Coccia-Portugal, Paola Corsetto, Antonino Belfiore, Daniele Lorenzini, Maria Grazia Daidone, Andrea VIngiani, Serenella M Pupa, Giancarlo Pruneri, Claudio Vernieri. High CD36 expression predicts worse event free survival in HER2-positive breast cancer patients treated with neoadjuvant trastuzumab-based therapy: An exploratory analysis of the NeoALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P2-13-12.

Importance. Neoadjuvant therapy with dual HER2 blockade improved pathological complete response (pCR) rate. Nevertheless, it would be desirable to identify patients exquisitely responsive to single agent trastuzumab to minimize or avoid overtreatment. Objective. To evaluate the predictive and prognostic value of basal primary tumor miRNA expression profile within the trastuzumab arm of the NeoALTTO study.Design, Setting and Participants. RNA samples from baseline biopsies were randomized into training (n =45) and testing (n =47) sets. After normalization, miRNAs associated with Event-free survival (EFS) and pCR were identified by univariate analysis. Multivariate models were implemented to generate specific signatures which were first confirmed, and then analyzed according to other clinical and pathological variables.Main outcomes and measures. Association between miRNA expression and pCR and/or EFS.Results. We identified a prognostic signature including hsa-miR-153-3p (HR 1.831, 95%CI: 1.34-2.50) and hsa-miR-219a-5p (HR 0.629, 95%CI: 0.50 - 0.78). For two additional miRNAs (miR-215-5p and miR-30c-2-3p), we found a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Besides, a two-miRNA signature was predictive of pCR (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53 - 0.92, and hsa-miR-382-3p, OR: 1.39, 95%CI: 1.01 -1.91). Notably, the performance of this predictive miRNA signature resembled that of the genomic classifiers PAM50 and TRAR, and did not improve when the extended models were fitted.Conclusions and relevance. Analysis of primary tumor tissue miRNAs holds the potential of a parsimonious tool to identify patients with differential clinical outcomes after trastuzumab based neoadjuvant therapy. Trial registration. ClinicalTrials.gov Identifier: NCT00553358. Table 1.MiRNAs associated with EFS. Results of the univariate Cox regression model- training set.miRNAsHR95% CIhsa-miR-12000.671(0.512; 0.879)ahsa-miR-1238-3p0.669(0.543; 0.826)ahsa-miR-12650.839(0.705; 0.997)ahsa-miR-129-5p0.785(0.628; 0.980)ahsa-miR-153-3p1.412(1.026; 1.943)ahsa-miR-15390.826(0.697; 0.979)ahsa-miR-1908-5p0.796(0.648; 0.978)ahsa-miR-205-5p0.737(0.553; 0.982)hsa-miR-219a-5p0.787(0.627; 0.989)ahsa-miR-25-5p0.566(0.391; 0.819)ahsa-miR-3000.581(0.399; 0.845)ahsa-miR-382-3p0.791(0.636; 0.985)ahsa-miR-4920.774(0.635; 0.944)ahsa-miR-519a-3p0.834(0.696; 0.998)ahsa-miR-551b-5p0.666(0.452; 0.981)ahsa-miR-5830.727(0.549; 0.963)ahsa-miR-6001.373(1.03; 1.829)ahsa-miR-6260.819(0.673; 0.998)ahsa-miR-651-5p0.611(0.382; 0.977)hsa-miR-7610.845(0.721; 0.991)ahsa-miR-891b0.658(0.479; 0.904)ahsa-miR-92a-2-5p0.787(0.637; 0.971)ahsa-miR-937-3p0.748(0.578; 0.967)aHR, Hazard Ratio; CI, Confidence Intervala microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. Table 2.MiRNAs associated with pCR. Results of the univariate logistic model - training set.miRNAsOR95% CIhsa-miR-132-3p0.410(0.169; 0.991)ahsa-miR-23b-5p0.520(0.316; 0.856)ahsa-miR-31-3p0.566(0.351; 0.912)ahsa-miR-31-5p0.508(0.291; 0.887)ahsa-miR-330-3p0.501(0.251; 0.999)hsa-miR-34b-3p0.673(0.474; 0.956)ahsa-miR-382-3p1.392(1.006; 1.925)ahsa-miR-548j-5p1.702(1.090; 2.658)aOR, Odds Ratio; CI, Confidence Interval. a microRNAs retained its statistically significance (at 10%) also when normalized for the overall mean. 2 Citation Format: Marilena Valeria Iorio, Sara Pizzamiglio, Giulia Cosentino, Chiara M Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan Ellard, David Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo. The future of HER2-positive breast cancer patients might be written in miRNAs: An exploratory analysis from the NeoALTTO study [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P5-13-26.

Background: Dual HER2-inhibition combined with neoadjuvant chemotherapy allows increased pathological complete response (pCR) rate. However, with the addition of new agents, there is a growing need to select patients to minimise overtreatment. Herein, we evaluated the 41-gene classifier TRAR to predict pCR to anti-HER2 therapies in the NeoALTTO trial. Patients and methods: Gene expression data were obtained using RNA from 226 pretreatment tumour biopsies. Logistic regression analysis and the area under the receiver operating characteristic (ROC) curve (AUC) were used to evaluate TRAR predictive and discriminatory capabilities. Results: TRAR levels were associated with pCR (odds ratio, OR: 0.25, 95% confidence interval, CI: 0.15–0.42). The ROC analysis showed AUC values of 0.73 (95% CI: 0.67–0.80) overall; 0.70 (0.59–0.81) and 0.71 (0.62–0.80) for positive and negative oestrogen receptor cases and 0.74 (0.60–0.88), 0.76 (0.65–0.87) and 0.71 (0.59–0.83) for trastuzumab, lapatinib and combined treatment arms, respectively. TRAR provided reliable predictive information beyond established clinicopathological variables (OR: 0.26, 95% CI: 0.14–0.47). Furthermore, addition of TRAR to these variables provided greater predictive capability than the addition of PAM50: AUC 0.78 (0.72–0.84) versus 0.74 (0.67–0.81), p = 0.04. Conclusion: TRAR represents a promising tool to refine the ability to identify patients sensitive to anti-HER2 (including trastuzumab-only)-based therapy and eligible for de-escalated treatment strategies., SCOPUS: ar.j, info:eu-repo/semantics/published

The implementation of trastuzumab has revolutionized the clinical management of HER2 positive breast cancers. Unfortunately, 50% of patients are resistant to the treatment. Researchers have already designed alternative anti-HER2 agents, such as pertuzumab and lapatinib. Still, predicting which patients will benefit from the therapy would prevent overtreatment and avoid unnecessary risks of side effects. MiRNAs are small non-coding RNAs involved in post-transcriptional gene regulation, and participate in almost all biological processes, including cancer. Since aberrant miRNA levels can be detected both at tissue level and in the circulation, they are good candidates as predictive and prognostic biomarkers. This study had access to tumor tissue samples from the phase III NeoALTTO trial, aimed at evaluating the efficacy of a HER2 dual blockade with trastuzumab and Lapatinib vs single blocking, in concomitance with chemotherapy, in a pre-operative setting. The primary end-point of the study was pathologic complete response (pCR); the secondary end-point event-free survival (EFS). Focusing on the trastuzumab arm, we identified both a predictive signature (hsa-miR-31-3p, OR 0.70, 95%CI: 0.53-0.92 and hsa-miR-382-3p, OR 1.39, 95%CI: 1.01-1.91) with an AUC value of 0.73 (95%CI: 0.60-0.87), and a prognostic signature (miR-153-3p, HR 1.83, 95%CI: 1.34-2.50 and miR-219a-5p, HR 0.629, 95%CI: 0.51-0.79) leading to a C-statistics of 0.730 (95%CI: 0.63-0.83). Moreover, we identified 2 miRNAs (miR-215-5p and miR-30c-2-3p) associated to EFS with a statistically significant interaction term with pCR (p.interaction: 0.017 and 0.038, respectively). Aiming at deepening the understanding of resistance mechanism, we modulated the expression of miR-31-3p and miR-382-3p in vitro, upon trastuzumab treatment in HER2 positive breast cancer cell lines. Given that miR-31-3p negatively correlates with pCR, we transfected it in HER2-addicted SKBr3 cells to appreciate a possible gain of resistance to trastuzumab; conversely, miR-382-3p positively correlates with pCR and was transfected in HER2 non-addicted HCC1954 cells. Western blot analysis of HER2 signaling pathway highlighted that overexpression of miR-31-3p was able to counteract the reduction of phosphorylated HER2 levels induced by trastuzumab treatment in SKBR3 cells compared to control. Interestingly, miR-31-3p upmodulation increased the proliferation of both treated and non-treated SKBr3 cell in a 3D setting. Conversely, miR-382-3p overexpression in HCC1954 cells only slightly increased responsiveness to trastuzumab in the 3D setting, compared to control. Trastuzumab efficacy also relies on the immune system reaction, thus, in vivo experiments will likely provide further insights into the mechanism of action of these miRNAs. Citation Format: Giulia Cosentino, Sara Pizzamiglio, Chiara M. Ciniselli, Loris De Cecco, Alessandra Cataldo, Ilaria Plantamura, Tiziana Triulzi, Sarra El-abed, Yingbo Wang, Mohammed Bajji, Paolo Nuciforo, Jens Huober, Susan L. Ellard, David L. Rimm, Andrea Gombos, Mariagrazia Daidone, Paolo Verderio, Elda Tagliabue, Serena Di Cosimo, Marilena V. Iorio. miRNA and response to trastuzumab [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1229.

11508 Background: An ISG, GEIS, FSG and PSG randomized trial on 3 cycles of neoadjuvant epirubicine+ifosfamide (EI) versus a histology-tailored (HT) regimen in selected localized high-risk STS showed some superiority of EI in all histologies with the exception of Myxoid Liposarcoma (MLPS) where EI and HT regimens seemed equivalent (J Clin Oncol 2020; 38:2178-86). This MLPS cohort was expanded with the aim to assess the non-inferiority of the HT regimen compared to EI. Methods: This was a multicenter European randomized trial comparing EI versus a HT regimen. Patients (pts) had localized high-risk (grade = 3; size >5 cm; deeply seated) undifferentiated pleomorphic sarcoma, leiomyosarcoma, malignant peripheral nerve sheath tumor, synovial sarcoma or MLPS of extremities or trunk wall. Primary end-point was Disease Free Survival (DFS). Secondary end-point was Overall Survival (OS). The MLPS cohort was expanded after the results of the 3rd interim analysis (Lancet Oncol 2017; 18:812-22) in order to reject the hypothesis that HT regimen trabectedin is associated with a HR of relapse = 1.25 with a non-inferiority design. To this aim, a Bayesian monitoring approach was used until the probability that the true HR is higher than 1.25 was greater than 80% or smaller than 5%. Results: From May 2011 to June 2020, 101 pts affected by high-risk MLPS were randomized, 56 to EI and 45 to HT regimen. The median follow-up was 66 months (IQ range 37-89). Median size was 107 mm (IQ range 84-143), 108 mm (IQ range 86-150) in the EI and 106 mm (IQ range75-135) in the HT arm. The DFS and OS probabilities at 60 months were 0.86 and 0.73 (HR:0.60; 95%CI: 0.24-1.46; log rank p = 0.26 for DFS) and 0.88 and 0.90 (HR:1.20; 95%CI:0.37-3.93; log rank p = 0.77 for OS), in the HT and EI arm, respectively. 5-yr observed and Sarculator-predicted OS were 0.89 (95% CI 0.82-0.97) and 0.80 in all patients (p = 0.020), 0.90 (95% CI 0.81-1.00) and 0.79 in the EI arm (p = 0.049) and 0.88 (95% CI 0.77-1.00) and 0.81 in the HT arm (p = 0.204) respectively. Conclusions: In the expanded cohort of MLPS, the HT neoadjuvant therapy trabectedin was not inferior to EI. While survival in both arms was better than predicted by Sarculator, it is left to understand whether this patient population, who had on average a lower Sarculator-predicted risk of death compared with the rest of the trial population, may benefit from a neoadjuvant therapy. Clinical trial information: NCT01710176.

Purpose: Little is known about the efficacy of HER2-targeted therapy in patients with breast cancer showing different HER2-pathway dependence and immune phenotypes. Herein, we report a NeoALTTO exploratory analysis evaluating the clinical value of 22 types of tumor-infiltrating immune cells by CIBERSORT and 5 immune-related metagenes in the overall patient population, and in subgroups defined by the TRAR classifier as HER2-addicted (TRAR-low) or not (TRAR-high). Patients and Methods: Association of baseline TRAR, immune-related metagenes, and CIBERSORT data with pathologic complete response (pCR) and event-free survival (EFS) were assessed using logistic and Cox regression models. Corrections for multiple testing were performed by the Bonferroni method. Results: A total of 226 patients were analyzed: 80 (35%) achieved a pCR, and 64 (28%) experienced a relapse with a median follow-up of 6.7 (interquartile range 6.1–6.8) years; 108 cases were classified as TRAR-low, and 118 TRAR-high. Overall, γδ T-cell fraction [OR = 2.69; 95% confidence interval (CI), 1.40–5.18], and no immune-related metagenes were predictive of pCR. Notably, lymphocyte-specific kinase (LCK) predicted pCR to combination (OR = 2.53; 95% CI, 1.12–5.69), but not to single-agent trastuzumab or lapatinib [OR = 0.74; 95% CI, 0.45–1.22 (Pinteraction = 0.01)]. Integrating LCK with γδ T cells in a multivariate model added to the discriminatory capability of clinical and molecular variables with a shift in AUC from 0.80 (95% CI, 0.74–0.86) to 0.83 (95% CI, 0.78–0.89). In TRAR-low cases, activated mast cells, IFN and MHCII were reduced, and STAT1, HCK1, and γδ T cells were associated with pCR. STAT1 was broadly associated with improved EFS regardless of pCR, and nodal status in overall (HR = 0.68; 95% CI, 0.49–0.94) and in TRAR-low cases (HR = 0.50; 95% CI, 0.30–0.86). Conclusions: Immuno-phenotyping holds the promise to complement current predictive models in HER2-positive breast cancer and to assist in new therapeutic development.

Germline pathogenic variants (PVs) in the BRCA1 or BRCA2 genes cause high breast cancer risk. Recurrent or founder PVs have been described worldwide including some in the Bergamo province in Northern Italy. The aim of this study was to compare the BRCA1/2 PV spectra of the Bergamo and of the general Italian populations. We retrospectively identified at five Italian centers 1019 BRCA1/2 PVs carrier individuals affected with breast cancer and representative of the heterogeneous national population. Each individual was assigned to the Bergamo or non-Bergamo cohort based on self-reported birthplace. Our data indicate that the Bergamo BRCA1/2 PV spectrum shows less heterogeneity with fewer different variants and an average higher frequency compared to that of the rest of Italy. Consistently, four PVs explained about 60% of all carriers. The majority of the Bergamo PVs originated locally with only two PVs clearly imported. The Bergamo BRCA1/2 PV spectrum appears to be private. Hence, the Bergamo population would be ideal to study the disease risk associated with local PVs in breast cancer and other disease-causing genes. Finally, our data suggest that the Bergamo population is a genetic isolate and further analyses are warranted to prove this notion.

Simple Summary The Italian population is characterized by a high genetic heterogeneity mostly due to its long history of migration and colonization and to its geographical conformation. Consistently, several BRCA1/2 pathogenic variants (PVs) have been reported to be recurrent or even founder in defined geographical areas including the Bergamo province in Northern Italy. In this study, we retrospectively analyzed the data from 1019 women affected with breast cancer with BRCA1/2 PVs. We compared the BRCA1/2 PVs spectrum found in the carrier individuals from the Bergamo province (BGP) with that of the general Italian population. We found that the majority of the BGP PVs had a local origin and remained confined to the BGP or to the surrounding Lombardy region. We also observed that the BGP BRCA1/2 PV spectrum is private and conserved comprising a smaller number of variants with an average higher frequency with respect to that of carrier individuals from the rest of Italy. Abstract Germline pathogenic variants (PVs) in the BRCA1 or BRCA2 genes cause high breast cancer risk. Recurrent or founder PVs have been described worldwide including some in the Bergamo province in Northern Italy. The aim of this study was to compare the BRCA1/2 PV spectra of the Bergamo and of the general Italian populations. We retrospectively identified at five Italian centers 1019 BRCA1/2 PVs carrier individuals affected with breast cancer and representative of the heterogeneous national population. Each individual was assigned to the Bergamo or non-Bergamo cohort based on self-reported birthplace. Our data indicate that the Bergamo BRCA1/2 PV spectrum shows less heterogeneity with fewer different variants and an average higher frequency compared to that of the rest of Italy. Consistently, four PVs explained about 60% of all carriers. The majority of the Bergamo PVs originated locally with only two PVs clearly imported. The Bergamo BRCA1/2 PV spectrum appears to be private. Hence, the Bergamo population would be ideal to study the disease risk associated with local PVs in breast cancer and other disease-causing genes. Finally, our data suggest that the Bergamo population is a genetic isolate and further analyses are warranted to prove this notion.

Circulating microRNA (ct-miRNAs) are able to identify patients with differential response to HER2-targeted therapy. However, their dynamics are largely unknown. We assessed 752 miRNAs from 52 NeoALTTO patients with plasma pairs prior and two weeks after trastuzumab. Increased levels of ct-miR-148a-3p and ct-miR-374a-5p were significantly associated with pathological complete response (pCR) (p = 0.008 and 0.048, respectively). At a threshold &ge, the upper limit of the 95%CI of the mean difference, pCR resulted 45% (95%CI 24%&ndash, 68%), and 44% (95%CI 22%&ndash, 69%) for ct-miR-148a-3p and ct-miR-374a-5p, respectively. Notably, ct-miR-148a-3p retained its predictive value (OR 3.42, 95%CI 1.23&ndash, 9.46, p = 0.018) in bivariate analysis along with estrogen receptor status. Combined information from ct-miR-148a-3p and ct-miR140-5p, which we previously reported to identify trastuzumab-responsive patients, resulted in greater predictive capability over each other, with pCR of 54% (95%CI 25%&ndash, 81%) and 0% (95%CI 0%&ndash, 31%) in ct-miR-148a/ct-miR-140-5p high/present and low/absent, respectively. GO and KEGG analyses showed common enriched terms between the targets of these ct-miRNAs, including cell metabolism regulation, AMPK and MAPK signaling, and HCC progression. In conclusion, early modulated ct-miR-148-3p may inform on the functional processes underlying treatment response, integrate the information from already available predictive biomarkers, and identify patients likely to respond to single agent trastuzumab-based neoadjuvant therapy.

Motor deficits are common outcomes of neurological conditions such as stroke. In order to design personalised motor rehabilitation programmes such as robot-assisted therapy, it would be advantageous to predict how a patient might respond to such treatment. Spontaneous neural activity has been observed to predict differences in the ability to learn a new motor behaviour in both healthy and stroke populations. This study investigated whether spontaneous resting-state functional connectivity could predict the degree of motor adaptation of right (dominant) upper limb reaching in response to a robot-mediated force field. Spontaneous neural activity was measured using resting-state electroencephalography (EEG) in healthy adults before a single session of motor adaptation. The degree of beta frequency (β; 15–25 Hz) resting-state functional connectivity between contralateral electrodes overlying the left primary motor cortex (M1) and the anterior prefrontal cortex (aPFC) could predict the subsequent degree of motor adaptation. This result provides novel evidence for the functional significance of resting-state synchronization dynamics in predicting the degree of motor adaptation in a healthy sample. This study constitutes a promising first step towards the identification of patients who will likely gain most from using robot-mediated upper limb rehabilitation training based on simple measures of spontaneous neural activity.

BACKGROUND: As a neoadjuvant regimen for HER2-positive early breast cancer (BC), the use of two HER2-directed agents is more effective in producing pathological complete (pCR) responses than trastuzumab alone. Nevertheless, highly effective dual anti-HER2 combination may be unnecessary in patients who already benefit from a single agent. We previously reported that our 41-gene TRAR score is an accurate predictor of response to trastuzumab, with low scores being predictive of response to trastuzumab and favorable prognosis (Triulzi T. et al., 2015). PATIENTS AND METHODS: Tissue specimens from HER2-positive BC patients of Neo-ALTTO trial who received neoadjuvant trastuzumab and/or lapatinib plus paclitaxel were included in this study. Analysing RNA from fresh tissue using the 41-gene signature test, the area under the ROC curve (AUC) and the corresponding 95% confidence interval was computed to evaluate the predictive ability of TRAR score with respect to pCR, the primary endpoint of Neo-ALTTO. The prognostic role of TRAR score was investigated using a Cox regression model in univariate fashion. The patterns of Event Free Survival (EFS) according to the dichotomized TRAR score were estimated using the Kaplan–Meier method. RESULTS: The TRAR score was assessed for 226 of the 455 (49.7%) patients enrolled in the Neo-ALTTO study: 136 (60%) presented with T2 tumors, 188 (83%) with N0/1 and 128 (56.6%) with estrogen receptor negative BC. In details, basal TRAR score was available for 69, 79 and 78 patients assigned to neoadjuvant trastuzumab, lapatinib, and their combination, respectively. Overall, patients achieving a pCR showed significantly lower levels of TRAR score than those with residual disease (p CONCLUSION: Overall, we show that our 41-gene signature is accurate in predicting patient response to neoadjuvant HER2 targeted therapy in terms of pCR. In particular, low levels of TRAR score can identify a HER2-positive breast cancer subgroup highly responsive to trastuzumab as monotherapy for whom combination with other HER2-targeted drugs does not appear justified and may be one tool used for exploring de-escalating strategies without sacrificing outcomes. Citation Format: Di Cosimo S, Triulzi T, De Cecco L, Pizzamiglio S, de Azambuja E, Fumagalli D, Pusztai L, Harbeck N, Izquierdo M, de la Pena L, Huober J, Baselga J, Piccart M, Verderio P, Tagliabue E. Identifying clinically relevant subgroups of women with HER2-positive breast cancer: An analysis of Neo-ALTTO using the 41-gene TRAR score [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P2-09-03.

Central nervous system (CNS) tumors are the most common solid tumors in childhood. Since the sensitivity of combined cerebrospinal fluid (CSF) cytology and radiological neuroimaging in detecting meningeal metastases remains relatively low, we sought to characterize the CSF proteome of patients with CSF tumors to identify biomarkers predictive of metastatic spread. CSF samples from 27 children with brain tumors and 13 controls (extra-CNS non-Hodgkin lymphoma) were processed using core-shell hydrogel nanoparticles, and analyzed with reverse-phase liquid chromatography/electrospray tandem mass spectrometry (LC-MS/MS). Candidate proteins were identified with Fisher's exact test and/or a univariate logistic regression model. Reverse phase protein array (RPPA), Western blot (WB), and ELISA were used in the training set and in an independent set of CFS samples (60 cases, 14 controls) to validate our discovery findings. Among the 558 non-redundant proteins identified by LC-MS/MS, 147 were missing from the CSF database at http://www.biosino.org. Fourteen of the 26 final top-candidate proteins were chosen for validation with WB, RPPA and ELISA methods. Six proteins (type 1 collagen, insulin-like growth factor binding protein 4, procollagen C-endopeptidase enhancer 1, glial cell-line derived neurotrophic factor receptor α2, inter-alpha-trypsin inhibitor heavy chain 4, neural proliferation and differentiation control protein-1) revealed the ability to discriminate metastatic cases from controls. Combining a unique dataset of CSFs from pediatric CNS tumors with a novel enabling nanotechnology led us to identify CSF proteins potentially related to metastatic status.

Early age at onset of breast cancer (eoBC) is suggestive of an increased genetic risk. Although genetic testing is offered to all eoBC-affected women, in isolated cases the detection rate of pathogenic variants is 60% and loss of heterozygosity at chromosome 17q. The patient hypermethylated at RAD51C showed low methylation in the tumor sample, ruling out a role for methylation-induced silencing in tumor development. In isolated eoBC patients, BRCA1 constitutive promoter methylation may be a predisposing event. Further studies are required to define the impact of methylation changes occurring at BC-predisposing genes and their role in tumorigenesis.

Inappropriate handling of blood samples might induce or repress gene expression and/or lead to RNA degradation affecting downstream analysis. In particular, sample transport is a critical step for biobanking or multicenter studies because of uncontrolled variables (i.e., unstable temperature). We report the results of a pilot study implemented within the EC funded SPIDIA project, aimed to investigate the role of transport and storage of blood samples containing and not containing an RNA stabilizer.Blood was collected from a single donor both in EDTA and in PAXgene Blood RNA tubes. Half of the samples were sent to a second laboratory both at room temperature and at 4°C, whereas the remaining samples were stored at room temperature and at 4°C. Gene expression of selected genes (c-FOS, IL-1β, IL-8, and GAPDH) known to be induced or repressed by ex vivo blood handling and of blood-mRNA quality biomarkers identified and validated within the SPIDIA project, which allow for monitoring changes in unstabilized blood samples after collection and during transport and storage, were analyzed by RT-qPCR.If the shipment of blood in tubes not containing RNA stabilizer is not performed under a stable condition, gene profile studies can be affected by the effects of transport. Moreover, also controlled temperature shipment (4°C) can influence the expression of specific genes if blood is collected in tubes not containing a stabilizer.The use of dedicated biomarkers or time course experiments should be performed in order to verify potential bias on gene expression analysis due to sample shipment and storage conditions. Alternatively, the use of RNA stabilizer containing tubes can represent a reliable option to avoid ex vivo RNA changes.

Within the EU-SPIDIA project (www.spidia.eu), the quality parameters of blood genomic DNA were defined [SPIDIA-DNA: an External Quality Assessment for the pre-analytical phase of blood samples used for DNA-based analyses – [1]; Influence of pre-analytical procedures on genomic DNA integrity in blood samples: the SPIDIA experience – [2]; Combining qualitative and quantitative imaging evaluation for the assessment of genomic DNA integrity: the SPIDIA experience – [3]. DNA quality parameters were used to evaluate the laboratory performance within an External Quality Assessment (EQA) [Second SPIDIA-DNA External Quality Assessment (EQA): Influence of pre-analytical phase of blood samples on genomic DNA quality – [4]. These parameters included DNA purity and yield by UV spectrophotometric measurements, the presence of PCR interferences by Kineret software and genomic DNA integrity analysis by Pulsed Field Gel Electrophoresis.Here we present the specific laboratory report of the 2nd SPIDIA-DNA EQA as an example of data and performances evaluation.

Background In order to develop evidence-based quality guidelines for the pre-analytical phase of blood samples used for DNA molecular testing, two pan-European External Quality Assessments (EQAs) were implemented within the European Commission funded project SPIDIA. Here we report the results of the 2nd SPIDIA EQA that has been implemented on the basis of the 1st DNA EQA with the inclusion of some stringent conditions related to blood storage temperature and time. Methods SPIDIA facility sent to all the participants the same blood sample to be processed by their own procedure following SPIDIA suggestion for time and temperature storage. Evaluated genomic DNA (gDNA) quality parameters were: purity and yield by UV spectrophotometric analysis, PCR interferences by Kineret software and integrity by a dedicated algorithm. Results/conclusions 188 applications have been collected from 26 European countries. A high variability of gDNA integrity was observed whereas purity, yield and PCR interferences had a narrow distribution within laboratories. A dedicated analysis on pre-analytical variables and the evaluated gDNA quality parameters showed that blood storage and DNA extraction procedures influence gDNA integrity. The performances of the participants were improved in comparison with the 1st SPIDIA-DNA EQA, probably due to adopted more stringent pre-analytical conditions.