Fritz Offner

Waldenström Macroglobulinemia (WM) is an indolent lymphoplasmacytic lymphoma, characterized by the production of excess immunoglobulin M monoclonal protein. WM belongs to the spectrum of IgM gammopathies, ranging from asymptomatic IgM monoclonal gammopathy of undetermined significance (IgM-MGUS), through IgM-related disorders and asymptomatic WM to symptomatic WM. In recent years, its complex genomic and transcriptomic landscape has been extensively explored, hereby elucidating the biological mechanisms underlying disease onset, progression and therapy response. An increasing number of mutations, cytogenetic abnormalities, and molecular signatures have been described that have diagnostic, phenotype defining or prognostic implications. Moreover, cell-free nucleic acid biomarkers are increasingly being investigated, benefiting the patient in a minimally invasive way. This review aims to provide an extensive overview of molecular biomarkers in WM and IgM-MGUS, considering current shortcomings, as well as potential future applications in a precision medicine approach.

Selinexor, an oral selective inhibitor of nuclear export, was evaluated in the Phase 2b SADAL study in patients with diffuse large B-cell lymphoma (DLBCL) who previously received two to five prior systemic regimens. In post hoc analyses, we analyzed several categories of patient characteristics (age, renal function, DLBCL subtype, absolute lymphocyte count, transplant status, number of prior lines of therapy, refractory status, Ann Arbor disease stage, and lactate dehydrogenase) at baseline, i.e., during screening procedures, to determine their potential contributions to the efficacy (overall response rate [ORR], duration of response [DOR], overall survival [OS]) and tolerability of selinexor. Across most categories of characteristics, no significant difference was observed in ORR or DOR. OS was significantly longer for patients < 65 vs. ≥ 65 years, and for those with lymphocyte counts ≥ 1000/µL vs. < 1000/µL or lactate dehydrogenase ≤ ULN vs. > ULN. The most common adverse events (AEs) across the characteristics were thrombocytopenia and nausea, and similar rates of grade 3 AEs and serious AEs were observed. With its oral administration, novel mechanism of action, and consistency in responses in heavily pretreated patients, selinexor may help to address an important unmet clinical need in the treatment of DLBCL.

Multiple myeloma (MM) is a hematological malignancy characterized by plasma cells’ uncontrolled growth. The major barrier in treating MM is the occurrence of primary and acquired therapy resistance to anticancer drugs. Often, this therapy resistance is associated with constitutive hyperactivation of tyrosine kinase signaling. Novel covalent kinase inhibitors, such as the clinically approved BTK inhibitor ibrutinib (IBR) and the preclinical phytochemical withaferin A (WA), have, therefore, gained pharmaceutical interest. Remarkably, WA is more effective than IBR in killing BTK-overexpressing glucocorticoid (GC)-resistant MM1R cells. To further characterize the kinase inhibitor profiles of WA and IBR in GC-resistant MM cells, we applied phosphopeptidome- and transcriptome-specific tyrosine kinome profiling. In contrast to IBR, WA was found to reverse BTK overexpression in GC-resistant MM1R cells. Furthermore, WA-induced cell death involves covalent cysteine targeting of Hinge-6 domain type tyrosine kinases of the kinase cysteinome classification, including inhibition of the hyperactivated BTK. Covalent interaction between WA and BTK could further be confirmed by biotin-based affinity purification and confocal microscopy. Similarly, molecular modeling suggests WA preferably targets conserved cysteines in the Hinge-6 region of the kinase cysteinome classification, favoring inhibition of multiple B-cell receptors (BCR) family kinases. Altogether, we show that WA’s promiscuous inhibition of multiple BTK family tyrosine kinases represents a highly effective strategy to overcome GC-therapy resistance in MM.

The Complement 1 trial investigated the efficacy and safety of ofatumumab + chlorambucil with chlorambucil monotherapy in patients with previously untreated chronic lymphocytic leukaemia (CLL). On long‐term follow‐up in the chemoimmunotherapy arm vs. the chemotherapy arm there was an estimated 12% (not significant) and 39% risk reduction in overall survival and progression‐free survival, respectively. A high rate (61%) of treatment with next‐line therapies in both the treatment arms may dilute any potential OS difference and confound the interpretation of the OS results. Addition of ofatumumab to chlorambucil demonstrated clinical benefit and tolerability as a frontline treatment option in patients unfit for fludarabine‐containing therapy, with no new safety concerns.

Temsirolimus, a selective inhibitor of the mammalian target of rapamycin, has demonstrated clinical benefit versus investigator’s choice (INV) of therapy in patients with relapsed/refractory mantle cell lymphoma (MCL). This post hoc study retrospectively assigned simplified Mantle Cell Lymphoma International Prognostic Index (MIPI) scores (ie, secondary MIPI) based on parameters at the time of randomization in patients with MCL (N = 162) who received temsirolimus 175 mg once weekly for 3 weeks followed by once-weekly 75 mg or 25 mg or the INV of active therapy. Outcomes were analyzed according to the low-, intermediate- or high-risk category. Patient distribution by MIPI risk category was 31%, 39%, and 30% in the low-, intermediate-, and high-risk groups, respectively. Among patients in all categories, objective response rate (complete response + partial response) was higher in patients in the temsirolimus 175/75-mg group versus the INV group, respectively: 42% versus 0% (low-risk); 33% versus 5% (intermediate-risk); 10% versus 0% (high-risk). Median progression-free survival was significantly longer with temsirolimus 175/75 mg versus INV, respectively, in patients with intermediate (4.3 vs 1.9 months; P = 0.035) or high (4.5 vs 1.6 months; P = 0.0025) risk, and a trend toward improvement was observed in patients with low risk (5.3 vs 2.6 months; P = 0.091). Improvement in median overall survival was observed with temsirolimus 175/75 mg versus INV in low-risk patients (18.0 vs 10.5 months, respectively; P = 0.069). This analysis suggests that, compared with INV, temsirolimus demonstrated benefit in all MIPI risk categories in patients with MCL. In all treatment groups, patients with high secondary MIPI scores at baseline faced a dismal prognosis. ClinicalTrials.gov NCT00117598 .

Proteome studies on hematological malignancies contribute to the understanding of the disease mechanism and to the identification of new biomarker candidates. With the isobaric tag for relative and absolute quantitation (iTRAQ) method we analyzed the protein expression between B-cells of healthy people and chronic lymphocytic leukemia (CLL) B-cells. CLL is the most common lymphoid cancer of the blood and is characterized by a variable clinical course. By comparing samples of patients with an aggressive vs. indolent disease, we identified a limited list of differentially regulated proteins. The enhanced sensitivity attributed to the iTRAQ labels led to the discovery of a previously reported but still not clarified proteolytic product of histone H2A (cH2A) which we further investigated in light of the suggested functional properties of this modification. In the exploratory proteome study the Histone H2A peptide was up-regulated in CLL samples but a more specific and sensitive screening of a larger patient cohort indicated that cH2A is of myeloid origin. Our subsequent quantitative analysis led to a more profound characterization of the clipping in acute monocytic leukemia THP-1 cells subjected to induced differentiation.

We studied the relation of clonal evolution (CE) in Chronic B‐lymphocytic leukemia (CLL) with prognostic factors and the correlation between CE and disease progression and overall survival. With interphase fluorescence in situ hybridization (FISH) analysis, we looked for 11q22 deletion, 17p13 deletion, and trisomy 12. A second FISH was performed approximately 3 yr after the first one or earlier in case of disease progression. High‐risk CE, defined as the acquisition of a new 11q or 17p deletion, was observed in 11.5% (11/95) of patients with CLL. The relative risk of CE was not influenced by CD38 and ZAP‐70 expression, mutational status of the immunoglobulin heavy chain gene (IgVH), lymphocyte doubling time, and genomic aberrations observed with the first FISH or by treatment given between the sequential genetic analyses. Patients with high‐risk CE had a significant shorter survival time (59 months vs. not reached, P = 0.0367). Multivariate analysis identified CE as the strongest independent prognostic marker regarding survival [hazard ratio (HR) 4.1, P = 0.01]. Clonal fluctuation, defined as disappearance of the 11q or 17p deletion, was seen in 11.5% (11/95) of patients. Most patients lost the high‐risk clone after treatment despite persistence of a malignant clone. The disappearance of these genomic aberrations did not ameliorate outcome. A few patients have lost spontaneously a small 17p clone. This study confirms that CE and clonal fluctuation are common phenomena in CLL. CE was not limited to patients with pre‐existing adverse prognostic factors. Acquiring high‐risk CE was identified as the strongest independent prognostic factor for impaired survival.

We explored the mechanism of cell death of the polymethoxyflavone tangeretin (TAN) in K562 breakpoint cluster region‐abelson murine leukemia (Bcr‐Abl+) cells. Flow cytometric analysis showed that TAN arrested the cells in the G₂/M phase and stimulated an accumulation of the cells in the sub‐G₀ phase. TAN‐induced cell death was evidenced by poly(ADP)‐ribose polymerase cleavage, DNA laddering fragmentation, activation of the caspase cascade and downregulation of the antiapoptotic proteins Mcl‐1 and Bcl‐x_L. Pretreatment with the pancaspase inhibitor Z‐VAD‐FMK_blocked caspase activation and cell cycle arrest but did not inhibit apoptosis which suggest that other cell killing mechanisms like endoplasmic reticulum (ER)‐associated cell death pathways could be involved. We demonstrated that TAN‐induced apoptosis was preceded by a rapid activation of the proapoptotic arm of the unfolded protein response, namely PKR‐like ER kinase. This was accompanied by enhanced levels of glucose‐regulated protein of 78 kDa and of spliced X‐box binding protein 1. Furthermore, TAN sensitized K562 cells to the cell killing effects of imatinib via an apoptotic mechanism. In conclusion, our results suggest that TAN is able to induce apoptosis in Bcr‐Abl+ cells via cell cycle arrest and the induction of the unfolded protein response, and has synergistic cytotoxicity with imatinib.

Chronic lymphocytic leukemia (CLL) is an incurable disease with a natural history of increasing resistance to chemotherapy. A novel approach to overcome chemotherapy resistance may be targeting the endoplasmic reticulum (ER). The involvement of the unfolded protein response (UPR) in the cell killing effect of xanthohumol (X) was examined in 18 patient samples. X-induced apoptosis of CLL cells was accompanied by the induction of glucose-regulated protein of 78 kDa (GRP78) and heat-shock protein of 70 kDa (Hsp70) protein levels and by sustained phosphorylation of the eukaryotic translation initiation factor 2 (eIF2alpha), suggesting the involvement of the ER stress transducer, the double-stranded RNA-activated protein kinase (PKR)-like ER kinase (PERK). The X-box-binding protein 1 (XBP1) mRNA was spliced but no clear activation of activating transcription factor 6 (ATF6) was observed. The proapoptotic outcome was further demonstrated by the up-regulation of CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), down-regulation of myeloid cell leukemia 1 (Mcl-1) and B-cell lymphoma 2 (Bcl-2), cleavage of poly-(ADP)-ribose polymerase (PARP) and processing of caspase-3, -4 and -9. Furthermore, X showed proteasome inhibitory activity. X stimulates the proapoptotic arm of the UPR in ex vivo CLL cells, suggesting that ER stress may play an important role during X-induced apoptosis.