Microfluidic-based dynamic BH3 profiling predicts anticancer treatment efficacy

Cancer therapy; Predictive markers; Translational researchTerapia del cáncer; Marcadores predictivos; Investigación traslacionalTeràpia del càncer; Marcadors predictius; Recerca translacionalPrecision medicine is starting to incorporate functional assays to evaluate anticancer agents on patient-isolated tissues or cells to select for the most effective. Among these new technologies, dynamic BH3 profiling (DBP) has emerged and extensively been used to predict treatment efficacy in different types of cancer. DBP uses synthetic BH3 peptides to measure early apoptotic events (‘priming’) and anticipate therapy-induced cell death leading to tumor elimination. This predictive functional assay presents multiple advantages but a critical limitation: the cell number requirement, that limits drug screening on patient samples, especially in solid tumors. To solve this problem, we developed an innovative microfluidic-based DBP (µDBP) device that overcomes tissue limitations on primary samples. We used microfluidic chips to generate a gradient of BIM BH3 peptide, compared it with the standard flow cytometry based DBP, and tested different anticancer treatments. We first examined this new technology’s predictive capacity using gastrointestinal stromal tumor (GIST) cell lines, by comparing imatinib sensitive and resistant cells, and we could detect differences in apoptotic priming and anticipate cytotoxicity. We then validated µDBP on a refractory GIST patient sample and identified that the combination of dactolisib and venetoclax increased apoptotic priming. In summary, this new technology could represent an important advance for precision medicine by providing a fast, easy-to-use and scalable microfluidic device to perform DBP in situ as a routine assay to identify the best treatment for cancer patients.Ramon y Cajal Programme, Ministerio de Economia y Competitividad grant RYC-2015–18357. (J.M.). Ministerio de Ciencia, Innovación y Universidades grant RTI2018-094533-A-I00 (J.M.). CELLEX foundation (J.M., A.M.). Beca Trienal Fundación Mari Paz Jiménez Casado (J.M.). European Research Council, grant ERC-StG-DAMOC 714317 (J.R.-A.). European Research Council, H2020 EU framework FET-open BLOC 863037 (J.R.-A.). Spanish Ministry of Economy and Competitiveness, “Severo Ochoa” Program for Centers of Excellence in R&D SEV-2020-2023 (J.R.-A.). Generalitat de Catalunya. CERCA Programme 2017-SGR-1079 (J.R.-A., J.S.). Fundación Bancaria “la Caixa”- Obra Social “la Caixa” (project IBEC-La Caixa Health Ageing) (J.R.-A.). Fero Foundation (C.S.). Networking Biomedical Research Center (CIBER). CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund (J.S.)

MCL-1 Inhibition Overcomes Anti-apoptotic Adaptation to Targeted Therapies in B-Cell Precursor Acute Lymphoblastic Leukemia

Multiple targeted therapies are currently explored for pediatric and young adult B-cell precursor acute lymphoblastic leukemia (BCP-ALL) treatment. However, this new armamentarium of therapies faces an old problem: choosing the right treatment for each patient. The lack of predictive biomarkers is particularly worrying for pediatric patients since it impairs the implementation of new treatments in the clinic. In this study, we used the functional assay dynamic BH3 profiling (DBP) to evaluate two new treatments for BCP-ALL that could improve clinical outcome, especially for relapsed patients. We found that the MEK inhibitor trametinib and the multi-target tyrosine kinase inhibitor sunitinib exquisitely increased apoptotic priming in an NRAS-mutant and in a KMT2A-rearranged cell line presenting a high expression of FLT3, respectively. Following these observations, we sought to study potential adaptations to these treatments. Indeed, we identified with DBP anti-apoptotic changes in the BCL-2 family after treatment, particularly involving MCL-1 – a pro-survival strategy previously observed in adult cancers. To overcome this adaptation, we employed the BH3 mimetic S63845, a specific MCL-1 inhibitor, and evaluated its sequential addition to both kinase inhibitors to overcome resistance. We observed that the metronomic combination of both drugs with S63845 was synergistic and showed an increased efficacy compared to single agents. Similar observations were made in BCP-ALL KMT2A-rearranged PDX cells in response to sunitinib, showing an analogous DBP profile to the SEM cell line. These findings demonstrate that rational sequences of targeted agents with BH3 mimetics, now extensively explored in clinical trials, may improve treatment effectiveness by overcoming anti-apoptotic adaptations in BCP-ALL.JM acknowledges the Ramon y Cajal Program, Ministerio de Economia y Competitividad (RYC-2015-18357) and the Spanish National Plan “Retos Investigacion” I + D + i (RTI2018-094533-A-I00) from Ministerio de Ciencia, Innovación y Universidades. This work was supported by the CELLEX foundation and the Networking Biomedical Research Center (CIBER), Spain. CIBER is an initiative funded by the VI National R&D&i Plan 2008–2011, Iniciativa Ingenio 2010, Consolider Program, CIBER Actions, and the Instituto de Salud Carlos III (RD16/0006/0012), with the support of the European Regional Development Fund (ERDF). This work was also partially funded by the CERCA Program and by the Commission for Universities and Research of the Department of Innovation, Universities, and Enterprise of the Generalitat de Catalunya (2017 SGR 1079). FS: Medical Faculty of Ulm University (Clinician Scientist Programme). K-MD and LM: German Research Foundation (DFG, SFB 1074)

Sequential combinations of chemotherapeutic agents with BH3 mimetics to treat rhabdomyosarcoma and avoid resistance

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood and adolescence. Refractory/relapsed RMS patients present a bad prognosis that combined with the lack of specific biomarkers impairs the development of new therapies. Here, we utilize dynamic BH3 profiling (DBP), a functional predictive biomarker that measures net changes in mitochondrial apoptotic signaling, to identify anti-apoptotic adaptations upon treatment. We employ this information to guide the use of BH3 mimetics to specifically inhibit BCL-2 pro-survival proteins, defeat resistance and avoid relapse. Indeed, we found that BH3 mimetics that selectively target anti-apoptotic BCL-xL and MCL-1, synergistically enhance the effect of clinically used chemotherapeutic agents vincristine and doxorubicin in RMS cells. We validated this strategy in vivo using a RMS patient-derived xenograft model and observed a reduction in tumor growth with a tendency to stabilization with the sequential combination of vincristine and the MCL-1 inhibitor S63845. We identified the molecular mechanism by which RMS cells acquire resistance to vincristine: an enhanced binding of BID and BAK to MCL-1 after drug exposure, which is suppressed by subsequently adding S63845. Our findings validate the use of DBP as a functional assay to predict treatment effectiveness in RMS and provide a rationale for combining BH3 mimetics with chemotherapeutic agents to avoid tumor resistance, improve treatment efficiency, and decrease undesired secondary effects

Comparative external validation of the PRECISE-DAPT and PARIS risk scores in 4424 acute coronary syndrome patients treated with prasugrel or ticagrelor

Background: The PRECISE-DAPT and PARIS risk scores (RSs) were recently developed to help clinicians at individualizing the optimal dual antiplatelet therapy duration (DAPT) after percutaneous coronary intervention (PCI). Nevertheless, external validation of these RSs it has not yet been performed in ACS (acute coronary syndrome) patients treated with prasugrel or ticagrelor in a real- world scenario. Methods: 4424 ACS patients who underwent PCI and survived to hospital discharge, from January 2012 to December 2016 at 12 European centers, were included. PRECISE-DAPT and PARIS bleeding RS, as well as PARIS ischemic RS, were computed, and their performance at predicting major bleeding (MB; BARC type 3 or 5) and ischemic events (MI and stent thrombosis) during follow up was compared. Results: After a median follow-up of 14 (interquartile range 12–20.9) months, 83 (1.88%) patients developed MB and 133 (3.0%) suffered an ischemic episode. PRECISE-DAPT performed better than PARIS bleeding RS (c-statistic = 0.653 vs. 0.593; p =.01 for comparison) in predicting MB. The RSs performance for MB prediction remained consistent in STEMI patients (c-statistic = 0.632 vs 0.575) or in those treated with prasugrel (c-statistic = 0.623 vs 0.586). PARIS ischemic RS exhibited superior discrimination in predicting ischemic complications compared to PRECISE-DAPT (c-statistic = 0.604 vs 0.568 p =.05 for comparison). Conclusion: Our data provide support to the use of PRECISE-DAPT in MB risk stratification for patients receiving DAPT in form of aspirin and prasugrel or ticagrelor whereas the PARIS ischemic RS has potential to complement the risk prediction with respect to ischemic events

First-line panitumumab plus FOLFOX4 or FOLFIRI in colorectal cancer with multiple or unresectable liver metastases: A randomised, phase II trial (PLANET-TTD)

Abstract Background: In first-line wild-type (WT)-Kirsten rat sarcoma viral oncogene homologue (KRAS) metastatic colorectal cancer (mCRC), panitumumab (Pmab) improves outcomes when added to FOLFOX [folinic acid, 5-fluorouracil, and oxaliplatin] or FOLFIRI [folinic acid, 5-fluorouracil, and irinotecan]. However no trial has directly compared these combinations. Methods: Multicentre, open-label study in untreated patients 18 years with (WT)-KRAS mCRC and multiple or unresectable liver-limited disease (LLD) randomised to either Pmab-FOLFOX4 or Pmab-FOLFIRI. The primary end-point was objective response rate (ORR). Secondary end-points included liver metastases resection rate (R0 þ R1), progression-free survival (PFS), overall survival (OS), adverse events and perioperative safety. Exploratory end-points were: response by RAS status, early tumour shrinkage (ETS) and depth of response (DpR) in WT-RAS patients. Results: Data on 77 patients were analysed (38 Pmab-FOLFOX4; 39 Pmab-FOLFIRI; WTRAS: 27/26, respectively). ORR was 74% with Pmab-FOLFOX4 and 67% with Pmab- FOLFIRI (WT-RAS: 78%/73%). Out of the above, 45% and 59% underwent surgical resection, respectively (WT-RAS: 37%/69%). The R0-R1 resection rate was 34%/46% (WTRAS: 26%/54%). Median PFS was 13/14 months (hazard ratio [HR] Pmab-FOLFIRI versus Pmab-FOLFOX4: 0.9; 95% confidence interval: [0.6e1.5]; WT-RAS:13/15; HR: 0.7 [0.4 e1.3]). Median OS was 37/41 months (HR:1.0 [0.6e1.8]; WT-RAS: 39/49; HR:0.9 [0.4 e1.9]). In WT-RAS patients with confirmed response, median DpR was 71%/66%, and 65%/77% of patients showed ETS 30%/ 20% at week 8, without significant differences between arms; these patients had longer median PFS and OS and higher resectability rates. Surgery was associated with longer survival. Perioperative and overall safety were similar, except for higher grade 3/4 neutropenia (40%/10%; p Z 0.003) and neuropathy (13%/0%; p Z 0.025) in the Pmab-FOLFOX4 arm. Conclusions: In patients with WT-KRAS mCRC and LLD, both first-line Pmab-FOLFOX4 and Pmab-FOLFIRI resulted in high ORR and ETS, allowing potentially curative resection. No significant differences in efficacy were observed between the two regimens. (clinicaltrials.gov:NCT00885885

Average daily ischemic versus bleeding risk in patients with ACS undergoing PCI: Insights from the BleeMACS and RENAMI registries

Background: The risk of recurrent ischemia and bleeding after percutaneous coronary intervention (PCI) for acute coronary syndrome (ACS) may vary during the first year of follow-up according to clinical presentation, and medical and interventional strategies. Methods: BleeMACS and RENAMI are 2 multicenter registries enrolling patients with ACS treated with PCI and clopidogrel, prasugrel, or ticagrelor. The average daily ischemic and bleeding risks (ADIR and ADBR) in the first year after PCI were the primary end points. The difference between ADBR and ADIR was calculated to estimate the potential excess of bleeding/ischemic events in a given period or specific subgroup. Results: A total of 19,826 patients were included. Overall, in the first year after PCI, the ADBR was 0.008085%, whereas ADIR was 0.008017% (P =.886). In the first 2 weeks ADIR was higher than ADBR (P =.013), especially in patients with ST-segment elevation myocardial infarction or incomplete revascularization. ADIR continued to be, albeit non-significantly, greater than ADBR up to the third month, whereas ADBR became higher, although not significantly, afterward. Patients with incomplete revascularization had an excess in ischemic risk (P =.003), whereas non–ST-segment elevation ACS patients and those on ticagrelor had an excess of bleeding (P =.012 and P =.022, respectively). Conclusions: In unselected ACS patients, ADIR and ADBR occurred at similar rates within 1 year after PCI. ADIR was greater than ADBR in the first 2 weeks, especially in ST-segment elevation myocardial infarction patients and those with incomplete revascularization. In the first year, ADIR was higher than ADBR in patients with incomplete revascularization, whereas ADBR was higher in non–ST-segment elevation ACS patients and in those discharged on ticagrelor

Advancing cancer precision medicine through cell-based functional assays and microfluidics. Finding the needle in a haystack to improve pediatric leukemia treatment

[eng] Personalized medicine has emerged to improve cancer treatment beyond classical approaches. The identification of genetic mutations by molecular analyses dominate personalized medicine initiatives promoting clinical benefit, but they often present limited predictive capacity for treatments. An alternative to these are functional assays, which expose cancer cells to different therapeutic options to identify which will be the most effectives in eliminating the tumor. Although these techniques are less widespread than those based on genetic studies, the first clinical trials using functional assays obtained good results. Even so, there are important limitations to the application of these techniques, including the obtention of sufficient biological material to perform these assays or the deterioration of the primary samples under ex vivo conditions. Dynamic BH3 Profiling (DBP) is a functional assay that allows a rapid prediction of treatment efficacy, thus avoiding sample decline. In this thesis, we focus on finding new effective treatments for pediatric B-cell Precursor Acute Lymphoblastic Leukemia (BCP-ALL), especially in the small percentage of patients who relapse after being treated with standard-of-care therapy. Using DBP in in vitro models, we identified cytotoxic chemotherapeutics and targeted therapies after short incubations. In addition, we also detected adaptations conferring resistance to the applied drug mediated by antiapoptotic proteins of the BCL-2 family. These observations pointed to the potential use of BH3 mimetics, specific inhibitors of antiapoptotic proteins already utilized in the clinic, in combination with other anticancer treatments. In our BCP-ALL models we identified antiapoptotic adaptations after treatment using DBP, which helped us to rationally design new combinations of chemotherapeutics or targeted therapies with a specific BH3 mimetic to block the observed adaptation. These new combinations boosted treatment efficacy or allowed a reduction of its concentration while maintaining cytotoxicity. DBP also predicted the efficacy of anticancer treatments and treatment-related antiapoptotic adaptations in pediatric BCP-ALL PDX models. Finally, we performed this functional assay directly on different pediatric patient samples presenting limited therapeutic options. Following clinicians’ suggestions, we tested several compassionate drug use candidates. Some of the treatments identified by DBP were added to the patients' treatment, achieving complete remission in some cases. Another limitation of DBP is the number of cells required to perform the assay, especially in solid tumors, where biopsies provide a limited number of viable cells are usually obtained. Microfluidic platforms allow to perform typical laboratory assays by automatizing and miniaturizing the process, thus reducing cell requirement. Therefore, we developed a microfluidic chip that automatically generates a BIM peptide gradient needed to perform the DBP assay on a small number of treated cancer cells in different microwells. In addition, we also adapted the detection method to be performed in situ in the same chip by fluorescence microscopy. To validate our microfluidic prototype, we used gastrointestinal stromal tumor (GIST) models to compare the results obtained in this microfluidic platform with the standard FACS-based DBP assay, achieving similar results. Finally, we performed these assays with a GIST patient sample, where we could identify a cytotoxic treatment combination, predicted by both the FACS-based DBP and the new microfluidic version, the latter with a 10-fold reduction in the required number cells.[cat] La medicina personalitzada ha emergit com una alternativa per a millorar el tractament del càncer respecte a altres aproximacions més clàssiques. La identificació de mutacions genètiques mitjançant anàlisis moleculars ha predominat en iniciatives de medicina personalitzada però no ha demostrar un clar benefici clínic. Altres iniciatives fan servir assaigs funcionals, que exposen les cèl·lules canceroses a diferents opcions terapèutiques per identificar quines seran les més efectives en eliminar el tumor. Encara que aquestes tècniques estan menys extenses que les basades en estudis genètics, els primers estudis clínics fent servir assaigs funcionals han obtingut bons resultats. Tot i això, hi han importants limitacions per tal d’aplicar aquestes tècniques, incloent la obtenció de suficient material biològic per a realitzar l'assaig o el deteriorament de la mostra primària en condicions ex vivo. Dynamic BH3 Profiling (DBP) és un assaig funcional que permet la ràpida predicció de la eficàcia dels tractaments, per tant evitant el deteriorament de les mostres. En aquesta tesis, ens hem centrat en trobar nous tractaments efectius per a leucèmia pediàtrica del tipus limfoblàstic agut de precursors de tipus B (BCP-ALL), especialment al petit percentatge de pacients que pateixen casos refractaris o recaigudes després de ser tractats amb el tractament estàndard. Fent servir models in vitro vam poder identificar la eficàcia de diferents quimioterapèutics i teràpies dirigides després de curts tractaments. A més, també vam identificar adaptacions conferint resistència al tractament, fent servir proteïnes antiapoptòtiques de la família BCL-2. Això ens va permetre l’ús de BH3 mimetics, inhibidors específics de proteïnes antiapoptòtiques que han demostrat citotoxicitat en diferents tipus de tumors, especialment afegits en combinació amb altres tractaments contra el càncer. En els nostres models de BCP-ALL vam identificar adaptacions antiapoptòtiques després de ser tractades fent servir el DBP, el que ens va ajudar a dissenyar una nova combinació de quimioteràpia o teràpia dirigida amb un BH3 mimetic per bloquejar la posterior adaptació. Aquestes noves combinacions van millorar l’efecte de tractaments ineficaços o van permetre la reducció de concentració mantenint l’efecte citotòxic. El DBP també va predir la eficàcia de tractaments contra el càncer i les adaptacions que aquests provoquen en models PDX. Finalment, vam realitzar aquest assaig funcional directament en mostres de pacients pediàtrics amb diferents malalties hematològiques sense ninguna opció terapèutica. Seguint els suggeriments dels doctors, vam provar diferents tractaments que es podrien fer servir com a ús compassiu. Gràcies a les nostres prediccions, alguns d’aquests tractaments van ser identificats com efectius per DBP i es va decidir afegir-los al tractament del pacients, aconseguint la remissió completa en alguns d’aquests casos. Una altra limitació del DBP es el número de cèl·lules necessàries per realitzar l’assaig. Especialment en tumors sòlids, una biòpsia és necessària per obtenir una mostra del tumor de la qual normalment s’obtenen un nombre limitat de cèl·lules viables. Les plataformes microfluídiques permeten realitzar assaigs típics del laboratori automatitzant i miniaturitzant el procés. Per tal d’adaptar el DBP a una versió microfluídica, vam desenvolupar un xip que genera automàticament un gradient del pèptid BIM necessari per a realitzar l’assaig i l’exposa a cèl·lules tumorals tractades en diferents pous petits, permetent la reducció del nombre de cèl·lules necessàries per a realitzar el DBP. A més, també vam adaptar el mètode d’identificació per a ser realitzat in situ al mateix xip per microscòpia de fluorescència. Per validar el nostre prototip, vam fer servir dos models de tumor gastrointestinal (GIST) per a comparar els resultats obtinguts en la nostra plataforma microfluídica i l’assaig clàssic, sent capaç la primera d’identificar la eficàcia de tractaments contra el càncer de fora comparable. Finalment, vam repetir el nou assaig amb una mostra d’un pacient de GIST, on vam poder identificar una combinació citotòxica, predita tant per l’assaig clàssic del DBP i la nova versió microfluídica, aquesta última amb una reducció de 10 cops en el número de cèl·lules requerides i amb una automatització del procés