Miguel Quintela-Fandino

Differential requirement of MALT1 for BAFF-induced outcomes in B cell subsets

B cell activation factor of the TNF family (BAFF) activates noncanonical nuclear factor κB (NF-κB) heterodimers that promote B cell survival. We show that although MALT1 is largely dispensable for canonical NF-κB signaling downstream of the B cell receptor, the absence of MALT1 results in impaired BAFF-induced phosphorylation of NF-κB2 (p100), p100 degradation, and RelB nuclear translocation in B220+ B cells. This corresponds with impaired survival of MALT1−/− marginal zone (MZ) but not follicular B cells in response to BAFF stimulation in vitro. MALT1−/− MZ B cells also express higher amounts of TRAF3, a known negative regulator of BAFF receptor–mediated signaling, and TRAF3 was found to interact with MALT1. Furthermore, phenotypes associated with overexpression of BAFF, including increased MZ B cell numbers, elevated serum immunoglobulin titers, and spontaneous germinal center formation, were found to be dependent on B cell–intrinsic MALT1 expression. Our results demonstrate a novel role for MALT1 in biological outcomes induced by BAFF-mediated signal transduction.

Phase I Combination of Sorafenib and Erlotinib Therapy in Solid Tumors: Safety, Pharmacokinetic, and Pharmacodynamic Evaluation from an Expansion Cohort

The aims of this study were to further define the safety of sorafenib and erlotinib, given at their full approved monotherapy doses, and to correlate pharmacokinetic and pharmacodynamic markers with clinical outcome. In addition, a novel pharmacodynamic marker based on the real-time measurement of RAF signal transduction capacity (STC) is described. Sorafenib was administered alone for a 1-week run-in period, and then both drugs were given together continuously. RAF STC was assessed in peripheral blood monocytes prior to erlotinib initiation. Epidermal growth factor receptor (EGFR) expression and K-RAS mutations were measured in archival tumor samples. Changes in pERK and CD31 were determined in fresh tumor biopsies obtained pretreatment, prior to erlotinib dosing, and during the administration of both drugs. In addition, positron emission tomography–computed tomography scans and pharmacokinetic assessments were done. Eleven patients received a total of 57 cycles (median, 5; range, 1–10). Only four patients received full doses of both drugs for the entire study course, with elevation of liver enzymes being the main reason for dose reductions and delays. Among 10 patients evaluable for response, 8 experienced tumor stabilization of ≥4 cycles. Pharmacokinetic analysis revealed no significant interaction of erlotinib with sorafenib. Sorafenib-induced decrease in RAF-STC showed statistically significant correlation with time-to-progression in seven patients. Other pharmacodynamic markers did not correlate with clinical outcome. This drug combination resulted in promising clinical activity in solid tumor patients although significant toxicity warrants close monitoring. RAF-STC deserves further study as a predictive marker for sorafenib. Mol Cancer Ther; 9(3); 751–60

Analysis of Paired Primary-Metastatic Hormone-Receptor Positive Breast Tumors (HRPBC) Uncovers Potential Novel Drivers of Hormonal Resistance

We sought to identify genetic variants associated with disease relapse and failure to hormonal treatment in hormone-receptor positive breast cancer (HRPBC). We analyzed a series of HRPBC with distant relapse, by sequencing pairs (n = 11) of tumors (primary and metastases) at >800X. Comparative genomic hybridization was performed as well. Top hits, based on the frequency of alteration and severity of the changes, were tested in the TCGA series. Genes determining the most parsimonious prognostic signature were studied for their functional role in vitro, by performing cell growth assays in hormonal-deprivation conditions, a setting that mimics treatment with aromatase inhibitors. Severe alterations were recurrently found in 18 genes in the pairs. However, only MYC, DNAH5, CSFR1, EPHA7, ARID1B, and KMT2C preserved an independent prognosis impact and/or showed a significantly different incidence of alterations between relapsed and non-relapsed cases in the TCGA series. The signature composed of MYC, KMT2C, and EPHA7 best discriminated the clinical course, (overall survival 90,7 vs. 144,5 months; p = 0.0001). Having an alteration in any of the genes of the signature implied a hazard ratio of death of 3.25 (p<0.0001), and early relapse during the adjuvant hormonal treatment. The presence of the D348N mutation in KMT2C and/or the T666I mutation in the kinase domain of EPHA7 conferred hormonal resistance in vitro. Novel inactivating mutations in KMT2C and EPHA7, which confer hormonal resistance, are linked to adverse clinical course in HRPBC.

Personalising and targeting antiangiogenic resistance: a complex and multifactorial approach

Pathological angiogenesis involves complex and dynamic interactions between tumour cells and other lineages existing in the microenvironment of the tumour. Preclinical and clinical data suggest that tumours can show dual, different adaptive responses against antiangiogenic agents: one successful adaptation is vascular normalisation, whereas the second adaptation is elicited through vascular trimming and increased hypoxia. These phenomena depend on the type of tumour and the type of agent. The classical approach for investigating acquired resistance against antiangiogenic agents is to identify compensatory signalling pathways emerging in response to VEGF blockade, which has led to the development of highly effective drugs; however, ultimately these drugs fail. Here we review how the dual stromal adaptive patterns determine the mechanisms of escape that go beyond the reprogramming of signal transduction pathways, which obliges us to investigate the tumour as an ecosystem and to develop uni- and multicompartmental models that explain drug resistance involving metabolic and immune reprogramming. We also propose a method for facilitating personalised therapeutic decisions, which uses 18F-fluoromisonidazole-positron emission tomography to monitor the dual stromal response in tumours of individual patients.

In vivo RAF signal transduction as a potential biomarker for sorafenib efficacy in patients with neuroendocrine tumours.

Background:Targeted therapies elicit anticancer activity by exerting pharmacodynamic effects on specific molecular targets. Currently, there is limited use of pharmacodynamic assessment to guide drug administration in the routine oncology setting.Methods:We developed a phosphoshift (pShift) flow cytometry-based test that measures RAF signal transduction capacity in peripheral blood cells, and evaluated it in a phase II clinical trial of oral sorafenib plus low-dose cyclophosphamide in patients with advanced neuroendocrine tumours (NETs), in order to predict clinical course and/or guide individual dose-titration.Results:Twenty-two patients were enrolled. Median progression-free survival (PFS) was 3 months (95% CI 2–10.7), and one patient had a partial response. PFS was longer among five patients who demonstrated an increase in pShift after 7 days of sorafenib compared with those who did not (14.9 months vs 2.8 months; P=0.047). However, pShift did not add value to toxicity-based dose-titration.Conclusion:The pharmacodynamic assessment of RAF transduction may identify selected patients with advanced NETs most likely to benefit from the combination of sorafenib plus cyclophosphamide. Further investigation of this test as a potential biomarker is warranted.

18F-fluoromisonidazole PET and Activity of Neoadjuvant Nintedanib in Early HER2-Negative Breast Cancer: A Window-of-Opportunity Randomized Trial

Purpose: We previously detected promising efficacy of neoadjuvant nintedanib (a multityrosine kinase inhibitor, TKI) in early HER2-negative breast cancer. In a preclinical study, we monitored stromal hypoxia with 18F-fluoromisonidazole-positron emission tomography (18F-FMISO-PET); we found that reoxygenation of tumors (or lack of it) during a window-of-opportunity (WoO) treatment with TKIs correlated with the benefit (or lack of it) from TKI-plus-chemotherapy combinations. We studied the predictive role of 18F-FMISO-PET for the TKI nintedanib in the neoadjuvant setting in a phase II WoO randomized trial. Experimental Design: Patients were randomized to a 14-day WoO of nintedanib preceded and followed by an 18F-FMISO-PET, followed by nintedanib plus weekly paclitaxel (Arm A) or an 18F-FMISO-PET followed by weekly paclitaxel (Arm B) before surgery. The endpoint was residual cancer burden (RCB). The objective was to detect the patients with no response (RCB-III) on the basis of the baseline or evolutive 18F-FMISO-PET values/changes. Results: One-hundred and thirty HER2-negative patients were randomized. Seventeen (27.9%), 34 (55.7%), and 8 (13.1%) patients had an RCB of III, II, and I/0, respectively, in Arm A. In this arm, baseline hypoxic tumors had a 4.4-fold higher chance of experiencing RCB = 3 (P = 0.036) compared with baseline normoxic tumors. Nintedanib WoO induced tumor reoxygenation in 24.5% of the patients; those not reoxygenating showed a trend toward higher chance of experiencing RCB-III (6.4-fold; P = 0.09). In Arm B, 18F-FMISO-PET lacked predictive/prognostic value. Conclusions: Baseline hypoxic tumors (measured with 18F-FMISO-PET) do not benefit from neoadjuvant nintedanib. Clin Cancer Res; 23(6); 1432–41. ©2016 AACR.

Critically short telomeres and toxicity of chemotherapy in early breast cancer

Cumulative toxicity from weekly paclitaxel (myalgia, peripheral neuropathy, fatigue) compromises long-term administration. Preclinical data suggest that the burden of critically short telomeres (< 3 kilobases, CSTs), but not average telomere length by itself, accounts for limited tissue renewal and turnover capacity. The impact of this parameter (which can be modified with different therapies) in chemotherapy-derived toxicity has not been studied. Blood from 115 treatment-naive patients from a clinical trial in early HER2-negative breast cancer that received weekly paclitaxel (80 mg/m2 for 12 weeks) either alone or in combination with nintedanib and from 85 healthy controls was prospectively obtained and individual CSTs and average telomere lenght were determined by HT Q-FISH (high-throughput quantitative FISH). Toxicity was graded according to NCI common toxicity criteria for adverse events (NCI CTCAE V.4.0). The variable under study was “number of toxic episodes” during the 12 weeks of therapy. The percentage of CSTs ranged from 6.5%–49.4% and was directly associated with the number of toxic events (R2 = 0.333; P < 0.001). According to a linear regression model, each 18% increase in the percentage of CSTs was associated to one additional toxic episode during the paclitaxel cycles; this effect was independent of the age or treatment arm. Patients in the upper quartile (> 21.9% CSTs) had 2-fold higher number of neuropathy (P = 0.04) or fatigue (P = 0.019) episodes and >3-fold higher number of myalgia episodes (P = 0.005). The average telomere length was unrelated to the incidence of side effects. The percentage of CSTs, but not the average telomere size, is associated with weekly paclitaxel-derived toxicity.

Abstract OT3-01-02: CNIO-BR-008 trial: Reversion of T-cell exhaustion caused by chronic treatment with hypoxia-inducing antiangiogenic treatment by durvalumab in HER2-negative breast cancer: A pilot proof-of-concept trial

Background: The role of immune checkpoints in tumor progression is less relevant in breast cancer than in other malignancies. However, in preclinical experimentation we found a niche for immune checkpoint inhibitors. In an immunocompetent model of HER2-negative breast cancer (MMTV-PyMT) we observed that antiangiogenic agents may induce either a tumor normoxic or hypoxic adaptation. Normoxic adaptation leads to a therapeutically exploitable dependence on mitochondrial metabolism (Cell Rep 2016; 15: 1-14). Hypoxic adaptation (usually caused by monoclonal antibody type antiangiogenics) induces an increase in PGE2 and PGA synthesis, followed by a switch of the tryptophan metabolism from 5HIAA to kinurenine that culminates in differentiation of naive T-cells to Tregs and expression of PD-L1 in the hypoxic areas. The exhausted T-cell response observed in this model can be restored with PD-L1 inhibitors. We sought to prove the relevance of T-cell exhaustion an its reversibility by the anti-PD-L1 antibody durvalumab in patients receiving chronic bevacizumab (Bev). Trial design Single-arm, prospective, multicentric, phase II open-label trial. Patients receiving Bev maintenance after a chemotherapy+Bev regimen administered in the first line of metastatic disease that experience disease progression (PD) during maintenance will be candidates. The treatment will start by adding durvalumab (10 mg/kg q2w) to the ongoing Bev (15 mg/kg q3 w). Patients will undergo serial tumor biopsies, tumor-cfDNA sequencing (baseline and progression), and immunophenotyping (baseline and q4w). RECIST/I-RECIST and NCI CTC AE V4.03 criteria will be used for assessing disease response and toxicity. Elegibility criteria Women >18 year old diagnosed of HER2-negative advanced breast cancer; 2) have received chemotherapy plus Bev for the first line treatment and experienced PR, CR or SD, 3) followed by maintenance with three-weekly Bev in monotherapy for at least 6 weeks before diagnosis of PD. 4) Concurrent hormonal therapy is allowed for ER+ patients, but reception of previous immunotherapy is precluded. 5) Adequate organ function defined according to standard parameters. Specific aims Primary: 1) To determine the relative percentages of innate and adaptive immune cell subpopulations and ascertain the status of T-cell function and polarization by multiparametric flow cytometry in patients with acquired resistance against Bev 2) To assess the reversibility of the abnormalities evidenced in (1) by durvalumab 3) To determine the disease control rate of the combination, and its relationship with (1) and (2) Secondary: 4) To determine potential tumor neoantigens generated by chronic tumor hypoxia secondary to the antiangiogenic treatment. Statistical methods The sample size is calculated on the basis of the expected change in the Treg percentage in peripheral blood (10%), with an alpha and beta errors of 5% and 20% respectively. The minimum number of patients necessary to observe a 10% decrease is 25. Changes in lymphocytes will be compared with intra-subject measurements and Z-scores. Accrual: 2 of 25 (target accrual) patients have been recruited. Contact info: mquintela@cnio.es. Citation Format: Quintela-Fandino M, Manso L, Mouron S, Lopez-Acosta JF, Garcia-Saenz JA, Holgado E, Pascual-Martinez T, Medicna L, Guerra J, Gonzalez-Cortijo L, Manes S. CNIO-BR-008 trial: Reversion of T-cell exhaustion caused by chronic treatment with hypoxia-inducing antiangiogenic treatment by durvalumab in HER2-negative breast cancer: A pilot proof-of-concept trial [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr OT3-01-02.

Abstract B9: A phosphoproteomic portrait of triple - negative breast cancer: functional taxonomy

BACKGROUND: Triple-negative breast cancer (TNBC) is a breast cancer subtype that is not defined by targetable molecular markers. Since all the aberrations that exist and ultimately contribute to the tumor phenotype converge, from a functional point of view, in the final status of the phosphorylation of the proteome in a given moment, we sought to interrogate the phosphoproteome with two aims: 1) establish a taxonomy of TNBC based on measurable markers that predict clinical course; 2) reduce the phosphoproteome that characterizes the bad- from good-prognosis cases to its targetable, driving kinases, in order to define rational therapeutic approaches in TNBC. We chose mass spectrometry as a discovery approach, because of its coverage, sensitivity, dynamic range and specificity. Here we report the part of phosphoproteomic analysis used for establishing a new taxonomy of TNBC. METHODS: We performed quantitative phosphoproteomics from a discovery set of 34 frozen tumor samples divided in two sets paired by classic prognosis factors: 1, with 13 patients relapsed in less than 3 years; 2, 21 patients, no relapse in 10 years follow up. Raw data were processed with Maxquant and cases clustered using a supervised hierarchical approach. Kinases (predicted with linear domain consensus analysis tools using a PHOSIDA) and phospho-sites discriminating the set 1 from 2 were validated by immunohistochemistry in 113 consecutive TNBC cases, spotted in TMAs, with 14 years follow up. RESULTS: Overall, 15000 unique phosphopeptides were identified. Supervised clustering of subset 1 vs. 2 showed that 161 and 541 peptides were significantly more phosphorylated in the subset 1 and 2, respectively (FDR Kinase validation was performed directing an antibody against the active kinase form, if available; if not, against the total levels. The staining of kinases and the candidate phosphosites was analyzed with an Ariol, scored with a continue value from 0 to 3, and divided in quartiles for each staining probe. So far, 12 probes have been quantified. The prognosis of patients with upper quartile staining vs. the remainder was analyzed with the log-rank test and cox proportionate hazards model for each probe. Five of them showed statistically significant association with relapse (pSTAT3Tyr705, 6.1 vs. 10.1 years, p = 0.024; CYCB1, 7 vs. 10.3 years, p = 0.027; CDK6, 7 vs. 10.3 years, p = 0.013; pp70S6KThr389, 7.2 vs. 10 years, p = 0.042 and PRKCEpsilon, 7.1 vs. 10.2 years, p = 0.021). A combined variable integrating upper-quartile staining from either of those 5 probes occurring in 67.4% patients, identified almost all patients that experienced relapse: 48.3% of them relapsed (median time 7.6 years) vs. the reminder 32.6% of which only 13.8% relapsed (median time 11.9 years), log rank p = 0.001 INTERPRETATION: High-throughput mass-spectrometry is a powerful tool for generating a disease taxonomy, and can be translated to routine techniques like IHC. The signature constituted by pSTAT3Tyr705, CYCB1, CDK6, pp70S6K and PRKCEpsilon constitutes the most parsimonious signature to date able to detect all the early TNBC patients that relapse in the long term. Citation Format: Ivana Zagorac, Tamara Mondejar Tevar, Jesus Sanchez Ruiz, Albert Heck, Maarten Altelaar, Renske Penning, Harm Post, Gonzalo Gomez Lopez, David Pisano, Javier Munoz Peralta, Javier Munoz Peralta, Manuel Morente, Luis Manso, Miguel Quintela-Fandino. A phosphoproteomic portrait of triple - negative breast cancer: functional taxonomy. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2015 Nov 5-9; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl 2):Abstract nr B9.

Abstract B23: Real-time dynamic assessment of RAF-signal transduction capacity as a predictive biomarker in patients with advanced neuroendocrine tumor (aNET) treated with sorafenib (S) and metronomic cyclophosphamide (mC): The SORNET study.

Background: Preclinical rationale exists to combine VEGFR and PDGFR blockade with mC (J Clin Oncol 23:939–52), which led to this phase II trial of S and mC in patients (pts) with aNET. The dynamic evaluation of a signal transduction pathway in both its basal and stimulated states may more accurately reflect its functional status than the basal state alone. S enhances signal transduction through the RAF resulting in increased MEK phosphorylation (pMEK) in the presence of wild type RAS and RAF (Nature 464:427–30). Proof of putative pharmacodynamic effects early on during treatment may help select patients mostly likely to benefit. In this trial, a flow cytometry-based test was performed to measure pMEK dynamically in peripheral blood monocytes (PBMCs). Methods: Eligibility criteria: aNET; disease progression within 6 months prior to study entry; ECOG 0–2; concurrent octreotide and unlimited prior therapy were allowed. A phase II trial with intra-patient dose escalation of S and fixed dose mC of 50 mg daily was conducted. Patients started with a 7-day (d) run-in phase with S at 200 mg BID and fixed dose mC. Dose escalations of S from 200 to 400 to 600 to 800 mg BID occurred every 14 d until occurrence of either ≥ intolerable Gr 2 toxicity or a maximum S dose of 800 mg BID, with 28-d cycles then follow. PBMCs and plasma S trough levels were collected at baseline and at every S dose escalation. The MAPK pathway was stimulated in PBMCs by adding 20 ng/mL of IL3 to fresh blood aliquots. The difference between stimulated and basal pMEK (pShift) was quantified. The pShift value on d7 was compared to the pre-treatment value (d0), with pShift d7>d0=stimulation and pShift d0>d7=inhibition. Results:Demographics: 22 pts (18 carcinoid/4 islet cell cancers; 12 male) were enrolled; median age=58 (range 32–81); ECOG 0:1=7:15; prior treatment regimens 0:1:2:3+ = 8:10:2:2. Final S dose after the escalation process was 200, 400, 600 or 800 mg BID in 4:12:2:1 pts, 3 pts did not complete escalation due to toxicity. A total of 160 cycles of S and mC were administered (median=3; range=1–34). Toxicity: Gr 3 non-hematologic adverse events: diarrhea (3 pts), hand-foot reaction (3), hypophosphatemia (2), amylase/lipase (2/2), AST (1), hypertension, abdominal pain, ileal perforation, ALT, and vomiting (1 each). Efficacy: 1 pt had PR (5%), 13 SD (59%), 5 PD (23%) and 3 non-evaluable (14%). Median progression-free survival (PFS)=3 months (95% CI: 2–10.7); median overall survival (OS)=11.7 months (95% CI: 4.3-not reached (NR)). Biological Correlates: pShift change from d0 to d7 was predictive of clinical outcome, with 6 pts (27%) experiencing pShift stimulation and 16 pts (73%) inhibition. All 5 pts with PD had pShift inhibition. PFS in pts with pShift stimulation vs. inhibition: 14.9 (3-NR) vs. 2.8 months (1.8−6.7) (p=0.047); OS: 21.3 (7.9-NR) vs. 6.4 (2.8−6.4) (p=0.17). Neither S trough levels nor S dose were able to predict PFS, OS or pShift. pShift was not associated with toxicity (p=0.24). Conclusions: The combination of S and mC had modest antitumor activity in unselected pts with aNET. In this limited study, pShift from d0 to d7 seems to identify pts most likely to benefit, and thus may have predictive and/or prognostic significance warranting further validation Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr B23.