Immunomodulation of NK, NKT and B/T cell subtypes in relapsed/refractory multiple myeloma patients treated with pomalidomide along with velcade and dexamethasone and its association with improved progression-free survival.

Background: Multiple Myeloma (MM) patients exhibit dysregulated immune system, which is further weakened by chemotherapeutic agents. While cereblon-modulating agents, such as pomalidomide and lenalidomide, have been found to improve the immune profile, the efficacy of their impact in combination with other treatments is yet unknown. Methods: We conducted an immune-profiling of a longitudinal cohort of 366 peripheral blood samples from the CC4047-MM-007 (OPTIMISMM, NCT01734928) study. This study followed relapsed/refractory Multiple Myeloma (RRMM) patients who were treated with Velcade + dexamethasone (Vd), or Vd with pomalidomide (PVd). 366 blood samples from 186 patients were evaluated using multi-color flow cytometry at 3 timepoints: screening, day 8 of cycle 1, and cycle 3. Results: Among NK and NKT cell populations, adding pomalidomide showed no inhibition in the frequency of NK cells. When expression of double positivity for activation markers like, p46/NKG2D, on NK cells was higher than the median, PVd treated patients showed significantly better (p=0.05) progression-free survival (PFS) (additional 15 months) than patients with lower than the median expression of p46/NKG2D on NK cells. PVd treated patients who expressed CD158a/b below the median at cycle 1 demonstrated a significantly better PFS (more than 18months). Among B cell subtypes, PVd treatment significantly increased the abundance of B1b cells (p<0.05) and decreased Bregs (p<0.05) at day 8 of both cycle 1 and cycle 3 when compared to screening samples. Of all the B cell-markers evaluated among paired samples, a higher expression of MZB cells at day 8 of cycle 1 has resulted in enhanced PFS in PVd treated patients. Within T cells, pomalidomide treatment did not decrease the frequency of CD8 T cells when compared with screening samples. The higher the surface expression of OX-40 on CD8 T cells and the lower the expression of PD-1 and CD25 on CD4 T cells by PVd treatment resulted in improved PFS. Conclusion: The prognostic significance for the number of immune markers is only seen in the PVd arm and none of these immune markers exhibit prognostic values in the Vd arm. This study demonstrates the importance of the immunomodulatory effects and the therapeutic benefit of adding pomalidomide to Vd treatment.

Genomic analyses of flow-sorted Hodgkin Reed-Sternberg cells reveal complementary mechanisms of immune evasion.

Classical Hodgkin lymphoma (cHL) is composed of rare malignant Hodgkin Reed-Sternberg (HRS) cells within an extensive, but ineffective, inflammatory/immune cell infiltrate. HRS cells exhibit near-universal somatic copy gains of chromosome 9p/9p24.1, which increase expression of the programmed cell death protein 1 (PD-1) ligands. To define genetic mechanisms of response and resistance to PD-1 blockade and identify complementary treatment targets, we performed whole-exome sequencing of flow cytometry-sorted HRS cells from 23 excisional biopsies of newly diagnosed cHLs, including 8 Epstein-Barr virus-positive (EBV+) tumors. We identified significantly mutated cancer candidate genes (CCGs) as well as somatic copy number alterations and structural variations and characterized their contribution to disease-defining immune evasion mechanisms and nuclear factor κB (NF-κB), JAK/STAT, and PI3K signaling pathways. EBV- cHLs had a higher prevalence of genetic alterations in the NF-κB and major histocompatibility complex class I antigen presentation pathways. In this young cHL cohort (median age, 26 years), we identified a predominant mutational signature of spontaneous deamination of cytosine- phosphate-guanines ("Aging"), in addition to apolipoprotein B mRNA editing catalytic polypeptide-like, activation-induced cytidine deaminase, and microsatellite instability (MSI)-associated hypermutation. In particular, the mutational burden in EBV- cHLs was among the highest reported, similar to that of carcinogen-induced tumors. Together, the overall high mutational burden, MSI-associated hypermutation, and newly identified genetic alterations represent additional potential bases for the efficacy of PD-1 blockade in cHL. Of note, recurrent cHL alterations, including B2M, TNFAIP3, STAT6, GNA13, and XPO1 mutations and 2p/2p15, 6p21.32, 6q23.3, and 9p/9p24.1 copy number alterations, were also identified in >20% of primary mediastinal B-cell lymphomas, highlighting shared pathogenetic mechanisms in these diseases.

Characterization of midostaurin as a dual inhibitor of FLT3 and SYK and potentiation of FLT3 inhibition against FLT3-ITD-driven leukemia harboring activated SYK kinase.

Oncogenic FLT3 kinase is a clinically validated target in acute myeloid leukemia (AML), and both multi-targeted and selective FLT3 inhibitors have been developed. Spleen tyrosine kinase (SYK) has been shown to be activated and increased in FLT3-ITD-positive AML patients, and has further been shown to be critical for transformation and maintenance of the leukemic clone in these patients. Further, over-expression of constitutively activated SYK causes resistance to highly selective FLT3 tyrosine kinase inhibitors (TKI). Up to now, the activity of the multi-targeted FLT3 inhibitor, midostaurin, against cells expressing activated SYK has not been explored in the context of leukemia, although SYK has been identified as a target of midostaurin in systemic mastocytosis. We compared the ability of midostaurin to inhibit activated SYK in mutant FLT3-positive AML cells with that of inhibitors displaying dual SYK/FLT3 inhibition, targeted SYK inhibition, and targeted FLT3 inhibition. Our findings suggest that dual FLT3/SYK inhibitors and FLT3-targeted drugs potently kill oncogenic FLT3-transformed cells, while SYK-targeted small molecule inhibition displays minimal activity. However, midostaurin and other dual FLT3/SYK inhibitors display superior anti-proliferative activity when compared to targeted FLT3 inhibitors, such as crenolanib and quizartinib, against cells co-expressing FLT3-ITD and constitutively activated SYK-TEL. Interestingly, additional SYK suppression potentiated the effects of dual FLT3/SYK inhibitors and targeted FLT3 inhibitors against FLT3-ITD-driven leukemia, both in the absence and presence of activated SYK. Taken together, our findings have important implications for the design of drug combination studies in mutant FLT3-positive patients and for the design of future generations of FLT3 inhibitors.