ALBERT

Description: Multiple myeloma (MM) pathogenesis has been explained for many years by the cancer biology dogma introduced by Peter Nowell: first, a single plasma cell would be immortalized by an error in the immunoglobulin genes rearrangement process; then, a progressive stepwise acquisition of somatic cell mutations would induce a sequential selection and domination by the fittest clone. In line with this idea of “myeloma stability”, SNP arrays studies in diagnostic-relapse paired samples have revealed the presence of common clonal characteristics. Biologically, the M-protein remains usually constant across MM evolution and further, the variable domain of the rearranged immunoglobulin heavy chain genes (or CDR3 region) has been used as a patient-specific myeloma fingerprint in minimal residual disease (MRD) studies. However, massive genome studies with Next Generation Sequencing (NGS) have challenged this concept, showing a significant intraclonal heterogeneity at diagnosis with the possible presence of several clonal progenitors or tumor-initiating cells. In this study, we have characterized and compared the CDR3 region in 52-paired samples from 26 MM patients aiming: 1) to assess mono-clonality in MM evolution through the analysis of the CDR3 sequence and, 2) to validate ASO RQ-PCR approaches for MRD in MM, based on the constancy and specificity of the CDR3 region. Samples were obtained at diagnosis and progression (19 pairs) or at 2 different timepoints of progressive disease (7 pairs). Median time between sampling was 2 years. M-protein subtype remained stable in all pairs but 1, associated with a light-chain escape phenomenon. All samples proceeded from bone marrow (BM) except for 2 pairs, composed by BM and extramedullary disease (spleen and testes). Two major cytogenetic changes were identified: increased 13q14 deletion (from 7 to 54%) in 1 pair and increased 17p (p53) deletion (from 5 to 87%) in a further one. Treatments administered between sampling included most of the current approaches used in MM (data not shown). Genomic DNA isolation, PCR amplification and sequencing were performed following conventional methods. Germline VH, DH and JH segments were identified by comparison with public databases. CDR3 region was first identified in all samples and then compared between the two samples in the 26 pairs: the sequence of nucleotides was constantly identical in each pair, including those associated with major cytogenetic changes, a light-chain escape, extramedullar vs. BM infiltration and relapsed (and therefore, treatment selected) vs. refractory disease. Therefore, we can first conclude that the main tumor clone in MM retains a specific signature across all stages of disease evolution that allows the identification of samples as evolutionary related. This major clone signature is not modified by clinical or biological changes in the disease nor under different treatment pressures and would thus identify disease relapse and progression. Our results have also a clear impact on the validity of molecular MRD techniques. The high rate of complete responses (up to 50-60%) currently achieved in MM has prompted the use of new techniques for disease assessment. Today, ASO RQ-PCR, based on the use of specific primers and probes complementary of the VDJH rearrangement, continues to be the most sensitive approach. One pitfall of this technique would be the potential instability of PCR targets over time, which would induce false negative results. In B-cell precursor ALL, this is estimated to happen in 30-40% of cases but has not been deeply evaluated in MM yet. With the present study, we can also conclude that the junction region of the VDJH rearrangement in MM constantly identifies the myeloma cells responsible for relapse and therefore can be used as a reliable target for MRD assessment by ASO RQ-PCR and more recently, by NGS methods. If the CDR3 region remains stable, the novel concept of clonal tiding in MM should not be interpreted as a poly- or oligoclonal but subclonal. In MM, tides can be subclonal, but the ocean remains monoclonal. Disclosures No relevant conflicts of interest to declare.

Description: Background and objectives. PIM kinases (PIM1, PIM2, PIM3) are proteins known to be overexpressed in several hematological malignancies. In particular, in chronic lymphocytic leukemia (CLL) they are involved in cell survival, resistance to apoptosis (especially PIM2 and PIM3) and interactions with the microenvironment (PIM1). The aim of this study was dual: I) to evaluate the preclinical efficacy of PIM447, a pan PIM kinase inhibitor, in CLL and to study potential synergies with other drugs; and II) to evaluate the expression of PIM-kinases in different stages of the disease and correlate it with the prognosis and the sensitivity to the drug. Methods. Peripheral blood samples from untreated patients with different stages of the disease (monoclonal B lymphocytosis (MBL), stable CLL not requiring treatment (sCLL), and active CLL requiring treatment (aCLL)) were collected after informed consent. The ex vivo efficacy of PIM447 was analyzed by flow cytometry with annexin V in these samples. Moreover, PIM447 efficacy was also analyzed in two cell lines (MEC-1 and JVM-2) by MTT assay. Synergy with other drugs effective in CLL (bendamustine and fludarabine) was evluated with the calcusyn software. Protein levels of PIM Kinase proteins were evaluated by capillary electrophoresis immunoassay (WESTM ProteinSimple) in monoclonal B cells purified by CD19 selection with anti-CD19 magnetic microbeads and the autoMacs Cell separator (both from Miltenyi Biotec) from a subset of patients. Results. The pan PIM inhibitor, PIM447 was active in both cell lines tested, MEC-1 (IC50 5μM) and JVM2 (IC50 7μM), and also in monoclonal B cells from freshly isolated patients samples (sCLL=11; aCLL=5), with no difference in sensitivity between the different stages of the disease (IC50 of 4,8 μM and 4,7 μM for sCLL and aCLL respectively). There was a clear therapeutic window as treatment with PIM447 at doses toxic for monoclonal B cells, preserved T lymphocytes (figure 1) (median % of apoptosis for B cells and T lymphocytes respectively of 23 vs 20 at 5μM and 87 vs 35 at 10 μM). Moreover, PIM447 demonstrated to potentiate the activity of both bendamustine and fludarabine, being especially synergistic with this last one (combination index 0.1-0.6). A second objective was to analyze PIM2 protein expression by western blot in monoclonal B cells from these samples and correlate it with clinical and biological features. Up to now, it has been evaluated in 18 samples (MBL=4; sCLL=8; aCLL=6,). All of them expressed PIM-2. Expression levels of this protein were significantly higher in active CLL as compared with indolent stages of the disease (p=0,012). Patients with an unmutated IGHV status also displayed higher levels of PIM2 (p=0,01). Finally, samples with high PIM2 levels were slightly more resistant to PIM447 as compared with samples with lower protein levels (IC50 of 7,7 μM vs 5 μM, respectively). We are currently completing the analysis of the PIM2 levels of remaining samples and we are also measuring the levels of PIM1 protein, what will be available at the meeting. Conclusions: PIM-Kinase inhibition with PIM447 is effective in vitro in CLL cell lines and ex vivo in samples from patients. It synergizes with other agents especially fludarabine. PIM2 protein levels correlated with the clinical activity of CLL and with the mutational state of IGHV. Although all patients appear sensitive ex vivo to PIM447, further work is required to define PIM2 expression as a marker of sensitivity. Figure 1. Figure 1. Disclosures Ocio: Array BioPharma: Consultancy, Research Funding; Celgene: Honoraria, Research Funding; Amgen/Onyx: Consultancy, Honoraria, Research Funding; Bristol Myers Squibb: Consultancy; Mundipharma: Consultancy, Research Funding; Novartis: Consultancy, Research Funding; MSD: Research Funding; Pharmamar: Consultancy, Research Funding; Jassen: Honoraria.

Description: Obinutuzumab (GA101; G) is an anti-CD20 monoclonal antibody with activity in relapsed/refractory follicular lymphoma (FL) as a single agent and in combination with chemotherapy. G-chemotherapy induction followed by G-maintenance has not been evaluated for untreated FL. The open-label, randomized, phase Ib GAUDI study (NCT00825149) investigated safety and efficacy of G + CHOP (G-CHOP) or bendamustine (G-B) as first-line treatment for FL. We report data from patients (pts) who received G-maintenance after responding to G-chemotherapy induction. Pts with treatment-naïve CD20+ B-cell FL and ≥1 bi-dimensionally measurable lesion (CT scan; largest dimension 〉1.5cm) were allocated on a per-center basis to receive induction G IV 1000mg + standard CHOP (3-weekly, 6–8 cycles) or B IV 90mg/m2(4-weekly, 4–6 cycles). Induction responders received G-maintenance every 3 months for 2 years or until disease progression (PD). Pts completing maintenance were followed for a further 2 years, until PD or start of new anti-lymphoma therapy. Anti-infective prophylaxis was used at investigator discretion. The primary objective was safety. Secondary objectives included progression-free survival (PFS) and response rates. The overall safety population comprised 81 pts (G-B: n=41; G-CHOP: n=40). Baseline characteristics (age, sex, FLIPI status, bone marrow involvement, bulky disease [lesion ≥7cm], time from diagnosis) were balanced between arms. Median observation time from study start was 31 months (G-B) and 33 months (G-CHOP). The maintenance safety population comprised 72 pts (n=36 from each induction therapy arm). Most pts completed maintenance (G-B: 81%; G-CHOP: 72%). There were 17 discontinuations (24%; G-B: n=7; G-CHOP: n=10), due to an adverse event (AE)/intercurrent illness (n=9), insufficient therapeutic response (n=5), administrative/other (n=2) and death (n=1). During 2 years’ maintenance most pts had AEs: G-B, 100% (44% grade ≥3); G-CHOP, 78% (31% grade ≥3). The most common AE (all grades) was cough (G-B: 17%; G-CHOP: 11%). The grade ≥3 AEs mainly reflected infections and cytopenia. Six G-B pts (17%) experienced 7 grade ≥3 infections; 4 were considered treatment related (genital infection, oral herpes, pneumonia klebsiella, neutropenic infection). Five G-CHOP pts (14%) had one grade ≥3 infection each; 4 were considered treatment related (viral meningitis, respiratory tract infection [RTI], bacterial pneumonia [2 events]). Six G-B pts (17%) experienced 10 grade ≥3 cytopenia AEs; 7 were considered treatment related (anemia, febrile neutropenia, pancytopenia, neutropenia [2 events], thrombocytopenia [2 events]). No G-CHOP pt experienced a grade ≥3 cytopenia AE. AEs led to dose delays in 17% (G-B) and 6% (G-CHOP) of pts. Three pts (G-B: n=1; G-CHOP: n=2) had treatment-related AEs during, or within 24 hours of, an infusion (all grade 1–2). Two deaths (both G-CHOP) occurred during maintenance or maintenance follow-up; 1 due to PD and 1 due to a G-related AE (RTI leading to fatal lactic acidosis). At the end of maintenance, all pts with data available (G-B: n=41; G-CHOP: n=39) had experienced B-cell depletion (0.07x109cells/L). Median IgG levels remained within normal range during maintenance. In the overall safety population, complete response (CR) rate (based on CT scan rather than PET) as best overall response increased from end of induction (G-B: 37%; G-CHOP: 35%) to end of maintenance (G-B: 61%; G-CHOP: 70%). PFS rate at 32 months after first study drug was 92% (G-B) and 84% (G-CHOP). Median PFS was not reached; 10 pts (G-B: n=4; G-CHOP: n=6) had PD, including one transformation to diffuse large B-cell lymphoma. G-maintenance after G-chemotherapy induction was associated with a high CR rate in pts with previously untreated FL. Opportunistic infections occurred infrequently. Clinically relevant neutropenia was experienced by 14% of pts who received G-B induction but was not observed in G-CHOP pts. A phase III trial (GALLIUM) is investigating G versus rituximab in chemoimmunotherapy induction followed by immunotherapy maintenance in pts with untreated indolent non-Hodgkin lymphoma. Disclosures Off Label Use: Obinutuzumab is a type II CD20 monoclonal antibody which is licensed for use in combination with chlorambucil in untreated patients with CLL but is not currently approved for use in follicular lymphoma.. Grigg:Roche: Consultancy. Dreyling:Roche: Honoraria, Research Funding. Rule:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees. Lei:Roche Products Ltd.: Employment. Wassner-Fritsch:Roche: Employment. Fingerle-Rowson:F. Hoffmann–La Roche: Employment. Marlton:Roche: Honoraria, Membership on an entity's Board of Directors or advisory committees.

Description: Background Idelalisib is a PI3-kinase inhibitor specific for the delta isoform, approved (in combination with rituximab or ofatumumab) for the treatment of adult patients with Relapsed/Refractory Chronic Lymphocytic Leukemia (R/R CLL). Although the efficacy of idelalisib was reported in clinical trials, it is unclear how this translates into Real World. Methods A non-interventional retrospective study was conducted in Spain to describe the clinical characteristics and outcomes of patients diagnosed with R/R CLL that started treatment with idelalisib between 1 February 2015 and 31 December 2017. The Time from start of idelalisib treatment to either the start of a new anti CLL therapy or death (Time to Next Treatment or Death - TNTD) was defined as primary endpoint. Secondary endpoints included overall survival (OS), time to discontinuation (TTD) and safety, especially Adverse Events of Special Interest (AESI): ≥ grade 3 transaminase elevations, diarrhea /colitis, pneumonitis, neutropenia,Cytomegalovirus, bacterial, fungal and respiratory virus infections (RVI),Pneumocystis jirovecii pneumonia (PjP). Results Investigators from 21 centers included 77 patients in the study. The median age was 72.1 years (48-86) and 67.5% (n=52) were male. The main baseline characteristics were: Binet stage B-C in 32.5% (n=25), 17p deletion or TP53 mutation in 35.1% (n=27) and the median number previous lines of therapy was 4 (1-16). With a median follow up time of 14.4 months (1.2-44.4), 27 patients (35.1%) had started a new treatment and 19 (24.7%) had died. The median TNTD was 17.1 months (95% CI 14.0-22.3) in the overall population and 13.8 months (95% CI 7.9-15.2) in patients that discontinued idelalisib. The median OS has not been reached, with a cumulative probability of surviving at 1 and 2 years of 0.84 (95% CI 0.73-0.91) and 0.67 (95% CI 0.52-0.78). During follow-up 54 patients (70.1%) discontinued idelalisib: 39 (72.3%) due to toxicity and 10 (18.5%) due to disease progression. The median TTD was 13.0 months (95% CI 7.7-15.8); 5.3 months (95%CI 3.8-8.0) in patients who discontinued treatment due to serious adverse events (SAE) or AESI and 10.5 months (95% CI 0.5-16.0) in those who did so due to progressive disease. A total of 355 AEs were reported, of which 29.0% (n=103) were SAE. 181 AESIs were recorded: the most frequent were neutropenia (n=67; 22 grade 〉3), diarrhea/colitis (n=37; 14 grade 〉3) and bacterial infections (n=24; 14 grade 〉3). Other AESI were CMV infections (n=11), grade ≥ 3 pneumonitis (n=7), RVI (n=6), fungal infections (n=5), grade ≥ 3 transaminase elevations (n=3) and PjP (n=1). The median time from Idelalisib start to first AESI was 9.5 months (95%CI 5.5-15.3). During follow-up, 19 deaths were registered, 10 of which were caused by idelalisib-related SAEs (4 respiratory infections, 2 pneumonitis, 1 diarrhea/colitis). The time from SAE related to idelalisib that led to death, to death was 0.8 months (95% CI 0.7-1.7). Conclusions This real world study shows results of effectiveness of idelalisib consistent with the efficacy findings of the 312-0116 clinical trial. Toxicity was the most common reason for idelalisib discontinuation. Findings of adverse events were consistent with the known safety profile of idelalisib and no new drug-related adverse events were identified. This is a Gilead Sciences sponsored study. Disclosures Perez Encinas: GILEAD SCIENCES: Research Funding; CELGENE: Consultancy; JANSSEN: Consultancy. Lopez Jimenez:GILEAD SCIENCES: Honoraria, Other: Education funding. Ortiz:GILEAD SCIENCES: Research Funding. Cordoba:Pfizer: Consultancy; Celgene: Consultancy, Honoraria, Speakers Bureau; FUNDACION JIMENEZ DIAZ UNIVERSITY HOSPITAL: Employment; Roche: Honoraria, Speakers Bureau; Kyowa-Kirin: Consultancy, Honoraria, Speakers Bureau; Gilead: Consultancy, Research Funding, Speakers Bureau; Servier: Consultancy, Honoraria, Speakers Bureau; Janssen: Consultancy, Honoraria, Speakers Bureau. Ramirez Payer:GILEAD SCIENCES: Research Funding. González-Barca:Roche: Consultancy, Honoraria; Janssen: Consultancy, Honoraria; Celtrion: Consultancy; AbbVie: Consultancy, Honoraria; Kiowa: Consultancy; Takeda: Honoraria; Celgene: Consultancy. Martín Sánchez:GILEAD SCIENCES: Research Funding. Sanchez:Gilead Sciences: Research Funding. Baltasar Tello:JANSSEN: Consultancy, Honoraria; ABBVIE: Honoraria; ROCHE: Honoraria; GILEAD: Honoraria. Amutio:NOVARTIS: Consultancy; JANSSEN: Consultancy, Honoraria; CELGENE: Consultancy, Honoraria; ROCHE: Honoraria; GILEAD SCIENCES: Consultancy, Honoraria; TAKEDA: Consultancy; GSK: Honoraria; BMS: Honoraria; JAZZ PHARMACEUTICALS: Honoraria; MUNDIPHARMA: Consultancy. Vidal Maceñido:GILEAD SCIENCES: Research Funding. Fernandez:GILEAD SCIENCES: Research Funding. Loscertales:Gilead: Honoraria; AbbVie: Honoraria; AstraZeneca: Honoraria; Janssen: Honoraria; Roche: Honoraria. Rodríguez:GILEAD SCIENCES: Research Funding. Alaez:ROCHE: Consultancy. Ramroth:Gilead Sciences: Employment. Palla:Gilead Sciences: Employment.

Description: Key Points Mutations in the TLR/MYD88 pathway occur in 4% of patients with CLL, and they are the most frequent in young patients. TLR/MYD88 mutations in CLL patients confer a good outcome, which is similar to that of the age- and gender-matched healthy population.