BUSCADOR RECOLECTA

SUPPLEMENTAL MATERIAL 1.- Description of genetic analyses performed in the IMCL-2015 study 2.- Treatment discontinuation events and relationship with IR combination 3.- Supplemental Table S1 . Histological review according to the tissue sample 4.- Supplemental Table S2. Clinical and biological features according to TP53 mutational status 5.- Supplemental Table S3. Clinical and biological features according to nnMCL or cMCL molecular subtypes 6.- Supplemental Table S4. Responses after 12 cycles of IR combination according to genomic complexity 7.- Supplemental Table S5. Molecular response in peripheral blood and bone marrow at 6,12, 18 and 24 months 8.- Supplemental Figure S1. PFS from the time of ibrutinib stop in patients in response and sustained undetectable MRD 9.- Supplemental Figure S2. PFS according to MRD status at PB (Cycle 12) 10.- Supplemental Figure S3. A) OS by MIPI and B) OS by TP53 mutational status 2 Supplemental Figure Legends Supplemental Figure S1. PFS from the time of ibrutinib stop in patients in response and sustained undetectable MRD PFS, progression-free survival; MRD, minimal residual disease Supplemental Figure S2. PFS according to MRD status at PB (Cycle 12) (P=.063) PFS, progression-free survival ;MRD, minimal residual disease; PB, peripheral blood Supplemental Figure S3. A) OS by MIPI (P= .007) and B) OS by TP53 mutational status (P= .0002) OS, overall survival; MIPI, Mantle cell lymphoma International Prognostic Index; wt, wild-type; mut, mutated, Peer reviewed

Figure S1. CONSORT diagram. aCrossed over to receive single-agent ibrutinib after PD (n = 35). bOf the 29 patients in the placebo-rituximab arm who discontinued treatment due to investigator decision, 24 patients discontinued due to study unblinding per Data Monitoring Committee recommendation. cDiscontinued rituximab before the completion of 8 infusions. Figure S2. Progression-free survival benefit across prespecified subgroups by prior treatment status. CI, confidence interval; IgG, immunoglobulin G; Hgb, hemoglobin; HR, hazard ratio; RTX, rituximab; IPSSWM, International Prognostic Scoring System for Waldenström’s Macroglobulinemia. Figure S3. Serum immunoglobulin M (IgM) improvement over time. Figure S4. Overall survival. Not censored at crossover. aCounterfactual survival times in crossover patients (n = 35) were calculated using the two-stage Accelerated Failure Time model (Latimer NR, et al: Med Decis Mak. 2014;34(3):387-402). CI, confidence interval; HR, hazard ratio. TABLE S1. Baseline Demographics and Clinical Characteristics by Genotype TABLE S2. Reasons for Ibrutinib Discontinuation. TABLE S3. Key Concomitant Medications of Interest TABLE S4. Adverse events leading to ibrutinib dose reductions., Peer reviewed

Figure A.1: Selection of Comparator Arms for ITC Analyses Figure A.2: Results of sensitivity analyses with OIs removed for OS at all (A) and first (B) index dates Figure A.3: Results of sensitivity analyses with LocoMMotion removed for OS at all (A) and first (B) index dates, and PF at first index dates (C) Table A.1: Characteristics of Data Sources for PCT arms in ITCs Table A.2: Published ITC Results and Augmented Results Included in Meta-analyses (All Index Dates) Table A.3: Published ITC Results and Augmented Results Included in Meta-analyses (First Index Dates) Table A.4: Baseline Covariates After Adjustment (mITT Populations; All Index Dates) Table A.5: Baseline Covariates After Adjustment (mITT Populations; First Index Dates) Table A.6: Outcome Definitions in ITC Analyses, [Objective]: In the absence of head-to-head trials, indirect treatment comparisons (ITCs) between ciltacabtagene autoleucel (cilta-cel; in CARTITUDE-1) and treatments used in real-world clinical practice (physician’s choice of treatment [PCT]), were previously conducted. We conducted multiple meta-analyses using available ITC data to consolidate the effectiveness of cilta-cel versus PCT for patients with triple-class exposed relapsed or refractory multiple myeloma (RRMM). [Methods]: Five ITCs were assessed for similarity to ensure robust comparisons using meta-analysis. Effectiveness outcomes were overall survival (OS), progression-free survival (PFS), time to next treatment (TTNT), and overall response rate (ORR). A robust variance estimator was used to account for the use of CARTITUDE-1 in each pairwise ITC. Analyses were conducted in both treated and enrolled populations of CARTITUDE-1. [Results]: Four ITCs were combined for evaluation of OS. Results were statistically significantly in favor of cilta-cel versus PCT in treated patients (hazard ratio [HR]: 0.24, 95% confidence interval [CI]: 0.22–0.26). Three ITCs were combined for evaluation of PFS and TTNT. Cilta-cel reduced the risk of progression and receiving a subsequent treatment by 80% (HR: 0.20 [95% CI: 0.06, 0.70]) and 83% (HR: 0.17 [95% CI: 0.12, 0.26]), respectively. Three ITCs were combined for evaluation of ORR. Cilta-cel increased the odds of achieving an overall response by 86-times versus PCT in treated patients. Findings were consistent in the enrolled populations and across sensitivity analyses. [Conclusions]: Evaluating multiple indirect comparisons, cilta-cel demonstrated a significantly superior advantage over PCT, highlighting its effectiveness as a therapy in patients with triple-class exposed RRMM., Peer reviewed

Table S1. Genetically modified animals. Table S2. PCR primers. Table S3. Antibodies used for flow cytometry, confocal immunofluorescence microscopy, immunohistochemistry, immunoprecipitation and western blot. Table S4. Deletion of C3G did not modify platelet counts and its parameters. Platelet number and parameters in C3G-KO mice and their C3G-wt siblings, male and female. The counts were made using an Advia 120 Hematology Analyzer (Bayer). Values are the mean of five, 10-week, mice of each genotype. There were no significant differences between genotypes or genders. Figure S1. Platelet C3G regulates ischemia-induced angiogenesis. (A) 3LL cells were injected in C3G-KO mice and their controls and tumors removed after 15 days. Histograms represent the number of vessels per area (upper) and the vessels surface per area (lower) (mean ± SEM) in tumor sections. (B) Blood collected at the indicated days 5 after implantation of 3LL cells in tgC3G, C3G-KO mice and their controls was incubated with anti-CXCR4-PE and anti-VEGFR-APC to determine the percentage of hemangiocytes. Histograms represent the mean ± SEM of the percentage of CXCR4+ (upper) or VEGFR+ (lower) cells in peripheral blood from the indicated genotypes. (C) Histograms represent the mean ± SEM of the percentage of CXCR4/VEGFR-double positive cells in blood from tgC3G mice and their controls (left) and the percentage of VEGFR-positive cells in blood from C3G-KO mice and their controls (right), collected at the indicated times post-ischemia. (D) Histogram represents the quantification of SDF-1 levels in thrombin-induced secretome from tgC3G, C3G-KO platelets and their controls, using a Mouse Angiogenesis Array Kit (n=2, each per duplicated). Representative images of the arrays are depicted in the left panels. SDF-1 spots are marked with red boxes. a. u, arbitrary units. Reference spots, which are not suitable for quantification, are indicated. (E) Western blot analysis of TSP-1 and VEGF levels in cytosolic (left) or membrane (right) fractions from resting, thrombin (TH)- or ADP- stimulated C3G-KO or C3G-wt platelets. β-actin was used as loading control. Values were normalized to those of resting C3G-wt platelets. Figure S2. C3G ablation or overexpression does not modify physiological MK or platelet production, nor does MK production after TPO injection or 5-FU-induced BM depletion. (A) Box plots showing the median of the number of platelets in C3G-KO mice and their control siblings. (B) Expression of CD41 and CD61 markers in BM cells from the indicated genotypes was determined by flow cytometry using CD41-FITC and CD61-PE. Histograms represent the mean ± SD of the percentage of CD41+, CD61+ or double-positive cells (megakaryocytes). (C) Expression of CD42 (left), CD41 and CD61 markers (middle) and ploidy status (right) was analyzed by flow cytometry in BM from C3G-KO and control mice, 14 days after injection of TPO. (D, E) Expression of CD42 (left), CD41 and CD61 markers (middle) and ploidy status (right) was analyzed by flow cytometry in BM from C3G-KO and C3G-wt mice (D) or tgC3G and wtC3G mice (E), 21 after 5-FU injection. Figure S3. C3G regulates c-Mpl levels and its ubiquitination and interacts with c-Cbl. (A) Western blot analysis of c-Mpl protein levels in platelets (left) or MKs (right) from the indicated genotypes. Values are relative to β-actin expression and were normalized 8 against those of each wild-type. (B) C3G-KO and C3G-wt platelets were treated with thrombin (TH, 0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min) in the presence or absence or the SFK inhibitor PP2 (10 µM) and labeled with anti-c-Mpl + Alexa FluorTM-568 (red) or anti-Ubiquitin + Alexa FluorTM-647 (green). Upper panels: representative immunofluorescence images of platelets taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of c-Mpl (left) or ubiquitin (right). (C) The graph shows the Pearson’s Correlation Coefficients (mean ± SD) of c-Mpl and ubiquitin under the indicated experimental conditions. (D) tgC3G and wtC3G platelets were treated with TPO (100 ng/ml, 5 min) in the presence or absence of the SFK inhibitor PP2 (10 µM) and labeled with anti-c-Mpl + Alexa FluorTM-568 (red) or anti-Ubiquitin + Alexa FluorTM-647 (green). Left panels: representative immunofluorescence images of platelets of each genotype under each treatment condition, taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of c-Mpl (left) or ubiquitin (right). (E) The graph shows the Pearson’s Correlation Coefficients (mean ± SD) of c-Mpl and ubiquitin under the indicated experimental conditions. (F) TPO induces C3G and c-Cbl colocalization. Representative immunofluorescence images of tgC3G platelets treated with TH (0.5 U/ml, 1 min), ADP (25 µM, 5 min) or TPO (100 ng/ml, 5 min) and labeled with anti-c-Cbl + Alexa FluorTM-568 (red) and anti-C3G + Alexa FluorTM-647 (green). Histograms show the Pearson’s Correlation Coefficients (mean ± SD) of C3G and c-Cbl under the indicated experimental conditions. Figure S4. C3G promotes c-Cbl phosphorylation by Src. (A) Representative immunofluorescence images of tgC3G, C3G-KO and control platelets treated with thrombin (TH, 0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min) and labeled with antiphospho-c-Cbl + Alexa FluorTM-647 (red) and Phalloidin (green). All images were taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of phospho-c-Cbl (p-c-Cbl). (B) Representative immunofluorescence images of C3G-KO platelets and their controls treated with TH (0.5 U/ml, 1 min) or TPO (100 ng/ml, 5 min), in the presence or absence of PP2, and labeled with anti-phospho-c-Cbl + Alexa FluorTM-647 (green) and anti-phospho-Src + Alexa FluorTM-568 (red). All images were taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SD of the fluorescence intensities of phospho-Src (pSrc) (upper) and phospho-c-Cbl (lower) under the indicated treatments. (C) Graph showing the Pearson’s Correlation Coefficients (mean ± SD) of phospho-c-Cbl and phospho-Src under the indicated experimental conditions. (D) Representative immunofluorescence images of tgC3G platelets and their controls treated with TPO (100 ng/ml, 5 min), in the presence or absence of PP2, and labeled with anti-phospho-c-Cbl + Alexa Fluor TM-647 (green) and anti-phospho-Src + Alexa FluorTM-568 (red). All images were taken at the same exposure time. Bar: 2.5 µm. Histograms represent the mean ± SEM of the fluorescence intensities of phospho-Src (p-Src) (left) and phospho-c-Cbl (right) under the indicated treatments., C3G is a Rap1 guanine nucleotide exchange factor that controls platelet activation, aggregation, and the release of α-granule content. Transgenic expression of C3G in platelets produces a net proangiogenic secretome through the retention of thrombospondin-1. In a physiological context, C3G also promotes megakaryocyte maturation and proplatelet formation, but without affecting mature platelet production. The aim of this work is to investigate whether C3G is involved in pathological megakaryopoiesis, as well as its specific role in platelet mediated angiogenesis and tumor metastasis. Using megakaryocyte-specific C3G knockout and transgenic mouse models, we found that both C3G overexpression and deletion promoted platelet-mediated angiogenesis, induced by tumor cell implantation or hindlimb ischemia, through differential release of proangiogenic and antiangiogenic factors. However, only C3G deletion resulted in a higher recruitment of hemangiocytes from the bone marrow. In addition, C3G null expression enhanced thrombopoietin (TPO)-induced platelet production, associated with reduced TPO plasma levels. Moreover, after 5-fluorouracil-induced platelet depletion and rebound, C3G knockout mice showed a defective return to homeostatic platelet levels, indicating impaired platelet turnover. Mechanistically, C3G promotes c-Mpl ubiquitination by inducing Src-mediated c-Cbl phosphorylation and participates in c-Mpl degradation via the proteasome and lysosome systems, affecting TPO internalization. We also unveiled a positive role of platelet C3G in tumor cell-induced platelet aggregation, which facilitated metastatic cell homing and adhesion. Overall, these findings revealed that C3G plays a crucial role in platelet-mediated angiogenesis and metastasis, as well as in platelet level modulation in response to pathogenic stimuli., Peer reviewed

Figure S1: Distribution of major IGH gene clusters involved in the rearrangements identified in 5 different studies [37,38,39,40]; Figure S2: Visual representation of CNV data in chromosomes 1 and 17 from two different patients; Table S1: Sequencing statistics; Table S2: Raw sequencing statistics for each sample. (A) Panel data. (B) WES data; Table S3: SNVs and indels identified in MM patients; Table S4: Comparative table between FISH and NGS results, related to FISH probe locations; Table S5: IGH arrangement summary of PPCs; Table S6: IG rearrangements identified by using both MiXCR and Vidjil; Table S7: IG rearrangement identification comparative study; Table S8: Comparative mutational results from exome and panel data; Table S9: CNV events identified by panel and exome analysis across chromosomes 1 and 17; Table S10: Estimation of PPV, NPV, sensitivity, specificity and global accuracy., Peer reviewed

Additional file 1: Figure S1. a, Relative mRNA expression of RRAS2 in different types of leukemia. Data comes from (Haferlach et al., 2010) and has been retrieved from www.oncomine.org . b, Schematic representation of the overexpression cassette inserted into the Rosa26 locus. c, Relative expression of RRAS2 measured by RT-qPCR in different organs of Rosa26-RRAS2fl/flxSox2-Cre (Sox2-Cre+) mice compared to that of WT C57BL/6 J Control mice using 18S as the reference gene. All expression numbers were normalized to those of liver from WT Control mice (mean = 1). Data show relative expression of RRAS2 in the indicated organs in n = 3–4 8 month-old independent mice. d, Quantification of spleen weight from control and 6 month-old Sox2-Cre + mice. Data shown correspond to four control mice and eleven Sox2-Cre mice. Two-tailed unpaired t-test with Welch’s correction. e, Two-parameter flow cytometry of the expression of CD5 and IgM in B cells in the spleen of 6 month-old control and Sox2-Cre + mice. f, Quantification of the number of CD5 + IgM+ B cells in the spleens and bone marrow of 6 month-old control and Sox2-Cre + mice. Data correspond to triplicate measurements of one control and three Sox2-Cre mice. Unpaired t-test with Welch’s correction. g, Quantification of the serum IgM concentration in the blood of 35–40 wk-old control (n = 3) and mb1-Cre (n = 8) mice by ELISA. Unpaired t-test with Welch’s correction. h, Representative images from Giemsa stainings of blood smears of 36 wk-old control and mb1-Cre mice. i, Two-parameter flow cytometry of the forward scatter and CD5 expression in CD19+ cells in the blood of 16 wk-old mb1-Cre mice. The gated population represents large cells. j, Two-parameter flow cytometry of CD5 expression and BrdU incorporation in CD19+ cells in the blood of 16 wk-old mb1-Cre mice. k, Quantification of the percentage of CD19+ cells that are CD5+ blasts and of the CD19+ CD5+ cells that have incorporated BrdU., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 2: Figure S2. a, Flow cytometry analysis of GFP populations in 23 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse spleen. Representative two-color contour plots of GFPhigh and GFPlow populations in total B cells (CD19+), CD5+ leukemic and CD23+ follicular B cells. Bottom, representation of GFP populations in T lymphocytes (CD3+). b, Percentage of GFPhigh cells in the indicated populations determined by flow cytometry. Data show means ± SEM from n = 8 mice (23 wk-old mice). ****p < 0.0001 (one-way ANOVA test). c, Western blot analysis of R-RAS2 expression of sorted GFPlow and GFPhigh leukemic cells from the spleen of a 25 wk-old Rosa26-RRAS2fl/flxSox2-Cre mouse (β-actin as loading control). d, Dot plot representation of GFPlow CD5+ leukemic B cell evolution in mb1-Cre mice over time, showing each mouse individually (n = 14). Data points were adjusted to a linear fit. These data were retrieved from the same mice as in Fig. 2i. e, Percentage of CD5+ cells in the indicated populations comparing GFPhigh and GFPlow distribution. Data show means ± SEM from n = 4 30 wk-old mice. Two-way ANOVA test. f, Heatmap of RNAseq expression data showing the genes differentially regulated in wild-type, follicular B cells (n = 6, 12wk-old), leukemic CD19 + CD5+ B cells (n = 6, 54wk-old), CD19+ GFPhigh (n = 2, 54wk-old) and CD19+ GFPlow (n = 2, 54wk-old) populations. Only genes significantly different between GFPhigh GFPlow populations (p < 0.05) and with a difference of 2-fold or more were used. Gene expression is shown in normalized log2 fold change., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 3: Figure S3. a, Representative two-color contour plots of B cell populations in a peritoneal wash and the spleen of 12 wk-old mice according to the expression of the CD11b and CD5 markers in the CD19+ population. The blue square indicates CD11b + CD5- B1b cells in the peritoneum. Red square, the presence of CD11b + CD5+ B1a cells in control mice and leukemic cells. Quantification of CD11b + CD5+ cells is shown to the right in box and whiskers plots showing all points and median value. **p < 0.01; *** p < 0.001, two-tailed unpaired t-test with Welch’s correction. b, Representative two-color contour plots of IgM and GFP expression within the CD11b + CD5+ populations shown in a. Quantification of IgMbright cells within the CD11b + CD5+ B cell population is shown to the right in box and whiskers plots showing all points and median value. **** p < 0.0001, two-tailed unpaired t-test with Welch’s correction., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

Additional file 7: Figure S7. Ingenuity Pathway Analysis (IPA) of differentially expressed genes associated with mTOR signaling, immunological development, and G1-S checkpoint regulation in leukemic versus normal follicular B cells. Pink-filled symbols: upregulated genes. Green-filled: downregulated genes. Double circle: protein complex; horizontal ellipse: transcription regulator; vertical ellipse: transmembrane receptor, diamond: enzyme; trapezium: transporter; triangle: phosphatase; inverted triangle: kinase; vertical rectangle: G protein-coupled receptor; circle: other. Black arrows: direct interactions; grey/white arrows: indirect interactions. Relationship labels: A: activation; B: binding; C: causation; CO: correlation; E: expression; EC: enzyme catalysis; I: inhibition; L: molecular cleavage; LO: localization; M: biochemical modification; miT: microRNA Targeting; P: phosphorylation/dephosphorylation; PD: protein-DNA binding; PP: protein-protein binding; PR: protein-RNA binding, RB: regulation of binding; RE: reaction; T: transcription; TR: translocation; UB: ubiquitination., Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed

Fundación Científica Asociación Española Contra el Cáncer Ministerio de Ciencia, Innovación y Universidades H2020 European Research Council Instituto de Salud Carlos III Consejería de Educación, Junta de Castilla y León, Peer reviewed