BackgroundMultiple Myeloma (MM) is an incurable plasma cell malignancy residing within the bone marrow (BM). We aim to develop allogeneic Natural Killer (NK) cell immunotherapy for MM. As the BM contains hypoxic regions and the tumor environment can be immunosuppressive, we hypothesized that hypoxia inhibits NK cell anti-MM responses.MethodsNK cells were isolated from healthy donors by negative selection and NK cell function and phenotype were examined at oxygen levels representative of hypoxic BM using flowcytometry. Additionally, NK cells were activated with IL-2 to enhance NK cell cytotoxicity under hypoxia.ResultsHypoxia reduced NK cell killing of MM cell lines in an oxygen dependent manner. Under hypoxia, NK cells maintained their ability to degranulate in response to target cells, though, the percentage of degranulating NK cells was slightly reduced. Adaptation of NK- or MM cells to hypoxia was not required, hence, the oxygen level during the killing process was critical. Hypoxia did not alter surface expression of NK cell ligands (HLA-ABC, -E, MICA/B and ULBP1-2) and receptors (KIR, NKG2A/C, DNAM-1, NCRs and 2B4). It did, however, decrease expression of the activating NKG2D receptor and of intracellular perforin and granzyme B. Pre-activation of NK cells by IL-2 abrogated the detrimental effects of hypoxia and increased NKG2D expression. This emphasized that activated NK cells can mediate anti-MM effects, even under hypoxic conditions.ConclusionsHypoxia abolishes the killing potential of NK cells against multiple myeloma, which can be restored by IL-2 activation. Our study shows that for the design of NK cell-based immunotherapy it is necessary to study biological interactions between NK- and tumor cells also under hypoxic conditions.

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

RAS pathway mutations have been linked to relapse and chemotherapy resistance in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, comprehensive data on the frequency and prognostic value of subclonal mutations in well-defined subgroups using highly sensitive and quantitative methods are lacking. Targeted deep sequencing of 13 RAS pathway genes was performed in 461 pediatric BCP-ALL cases at initial diagnosis and in 19 diagnosis-relapse pairs. Mutations were present in 44.2% of patients, with 24.1% carrying a clonal mutation. Mutation frequencies were highest in high hyperdiploid, infant t(4;11)-rearranged, BCR-ABL1-like and B-other cases (50–70%), whereas mutations were less frequent in ETV6-RUNX1-rearranged, and rare in TCF3-PBX1- and BCR-ABL1-rearranged cases (27–4%). RAS pathway-mutated cells were more resistant to prednisolone and vincristine ex vivo. Clonal, but not subclonal, mutations were linked to unfavorable outcome in standard- and high-risk-treated patients. At relapse, most RAS pathway mutations were clonal (9 of 10). RAS mutant cells were sensitive to the MEK inhibitor trametinib ex vivo, and trametinib sensitized resistant cells to prednisolone. We conclude that RAS pathway mutations are frequent, and that clonal, but not subclonal, mutations are associated with unfavorable risk parameters in newly diagnosed pediatric BCP-ALL. These mutations may designate patients eligible for MEK inhibitor treatment.

Pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is associated with a high frequency of copy number alterations (CNAs) in IKZF1, EBF1, PAX5, CDKN2A/B, RB1, BTG1, ETV6, and/or the PAR1 region (henceforth: B-cell development genes). We aimed to gain insight in the association between CNAs in these genes, clinical outcome parameters, and cellular drug resistance. 71% of newly diagnosed pediatric BCP-ALL cases harbored one or more CNAs in these B-cell development genes. The distribution and clinical relevance of these CNAs was highly subtype-dependent. In the DCOG-ALL10 cohort, only loss of IKZF1 associated as single marker with unfavorable outcome parameters and cellular drug resistance. Prednisolone resistance was observed in IKZF1-deleted primary high hyperdiploid cells (~1500-fold), while thiopurine resistance was detected in IKZF1-deleted primary BCR-ABL1-like and non-BCR-ABL1-like B-other cells (~2.7-fold). The previously described risk stratification classifiers, i.e. IKZF1plus and integrated cytogenetic and CNA classification, both predicted unfavorable outcome in the DCOG-ALL10 cohort, and associated with ex vivo drug cellular resistance to thiopurines, or L-asparaginase and thiopurines, respectively. This resistance could be attributed to overrepresentation of BCR-ABL1-like cases in these risk groups. Taken together, our data indicate that the prognostic value of CNAs in B-cell development genes is linked to subtype-related drug responses.

Background Despite significant improvements in the outcome of children with B-cell precursor acute lymphoblastic leukemia (BCP-ALL), therapeutic strategies for high risk and relapsed patients are limited and cause severe side effects. Reliable risk assessment and new therapeutic targets with high specificity are therefore warranted. The RAS pathway is the most frequently mutated pathway in cancer, and the RAF-MEK-ERK kinase axis is crucial for mediating the oncogenic effects of RAS. We and others have previously shown that in pediatric BCP-ALL, RAS pathway mutations can be retrospectively linked to relapse and chemotherapy resistance. However, data on the frequency of (sub-)clonal mutations at diagnosis and hence information about the prognostic relevance at initial diagnosis is lacking. Aim Guide therapy adaptation in pediatric BCP-ALL by evaluating the prognostic relevance of RAS pathway mutations and investigating the sensitivity to MEK inhibition. Methods We performed targeted next-generation sequencing of mutational hotspots in 13 RAS pathway genes to determine the frequency and clonality of RAS pathway mutations in a large, clinically and biologically characterized cohort of BCP-ALL patients. Initial diagnosis samples of 461 patients and 19 matched diagnosis-relapse sets were included. Mutations were considered clonal at ≥25% variant allele frequency, and high coverage allowed detection of subclones with down to 1% variant allele frequency. Clinical outcome was evaluated in 244 patients treated according to a contemporary, minimal residual disease (MRD)-based protocol (DCOG ALL10). The evolution of RAS pathway mutations was studied in 19 matched sets from diagnosis and relapse. Ex vivo sensitivity of RAS pathway mutated cells towards chemotherapeutic agents and trametinib was evaluated in an MTT-based cytotoxicity assay. Results Variants in RAS pathway genes were observed in 44% of initial diagnosis pediatric BCP-ALL cases, mostly affecting NRAS, KRAS, PTPN11, and FLT3. Clonal and subclonal mutations were found in 24% and 20% of patients, respectively. The mutation frequency was highest in high hyperdiploid, infant t(4;11)-positive, BCR-ABL1-like, and B-other cases (50-70%), whereas mutations were rare in ETV6-RUNX1-positive (27%), TCF3-PBX1-positive (8%) and BCR-ABL1-positive cases (4%). In matched diagnosis-relapse sets, clonal mutations at diagnosis were preserved at relapse, whereas the kinetics of subclones was variable. Interestingly, most RAS pathway mutations at relapse were clonal and exclusive. Cells carrying RAS pathway mutations, especially KRAS G13 mutations, were more often ex vivo resistant to prednisolone and vincristine. No association was found with ex vivo response to daunorubicine, L-asparaginase, 6-mercaptopurine, and 6-thioguanine. Mutant primary leukemic cells were ex vivo sensitive to the MEK-inhibitor trametinib. In addition, trametinib could enhance the cytotoxic effect of prednisolone ex vivo. In DCOG-ALL10 and COALL-97/-03 patients with clonal but not subclonal mutations, MRD levels tended to be more often high compared to wildtype cases (31% vs. 19%, p=0.057), while other risk factors (age, gender, white blood cell count, CNS, prednisone response) where not different. Event-free survival was lower in the standard risk and high risk arms of the DCOG ALL10 protocol (69% vs. 96%, p=0.027 and 56% vs. 100%, p=0.015, respectively). Conclusions Collectively, analysis of 461 diagnostic BCP-ALL patient samples identified RAS pathway mutations in 44% of patients, and one out of four carried a clonal mutation. MRD was the only risk factor associated with clonal RAS pathway mutations. MRD is essential to treatment stratification in many contemporary protocols, such as the DCOG ALL10 protocol, where only patients with negative MRD after induction courses are treated with a reduced regimen (standard risk arm). Given their unfavorable event-free survival, therapy should be adapted for mutated patients in future protocols. Since treatment intensification is not feasible for high risk or relapsed cases, addition of MEK inhibitors may be of benefit especially because they enhance the cytotoxicity of prednisolone. RAS pathway mutation status may therefore serve as biomarker to select patients for MEK-inhibitor treatment in new treatment protocols for children with BCP-ALL. Disclosures No relevant conflicts of interest to declare.

JAK2 abnormalities may serve as target for precision medicines in pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL). In the current study we performed a screening for JAK2 mutations and translocations, analyzed the clinical outcome and studied the efficacy of two JAK inhibitors in primary BCP-ALL cells. Importantly, we identify a number of limitations of JAK inhibitor therapy.JAK2 mutations mainly occurred in the poor prognostic subtypes BCR-ABL1-like and non- BCR-ABL1-like B-other (negative for sentinel cytogenetic lesions). JAK2 translocations were restricted to BCR-ABL1-like cases. Momelotinib and ruxolitinib were cytotoxic in both JAK2 translocated and JAK2 mutated cells, although efficacy in JAK2 mutated cells highly depended on cytokine receptor activation by TSLP. However, our data also suggest that the effect of JAK inhibition may be compromised by mutations in alternative survival pathways and microenvironment-induced resistance. Furthermore, inhibitors induced accumulation of phosphorylated JAK2Y1007, which resulted in a profound re-activation of JAK2 signaling upon release of the inhibitors. This preclinical evidence implies that further optimization and evaluation of JAK inhibitor treatment is necessary prior to its clinical integration in pediatric BCP-ALL.

Background: Sensitive and reliable molecular diagnostics is needed to guide therapeutic decisions for cancer patients. Although less material becomes available for testing, genetic markers are rapidly expanding. Simultaneous detection of predictive markers, including mutations, gene amplifications and MSI, will save valuable material, time and costs. Methods: Using a single-molecule molecular inversion probe (smMIP)-based targeted next-generation sequencing (NGS) approach, we developed an NGS panel allowing detection of predictive mutations in 33 genes, gene amplifications of 13 genes and microsatellite instability (MSI) by the evaluation of 55 microsatellite markers. The panel was designed to target all clinically relevant single and multiple nucleotide mutations in routinely available lung cancer, colorectal cancer, melanoma, and gastro-intestinal stromal tumor samples, but is useful for a broader set of tumor types. Results: The smMIP-based NGS panel was successfully validated and cutoff values were established for reliable gene amplification analysis (i.e. relative coverage ≥3) and MSI detection (≥30% unstable loci). After validation, 728 routine diagnostic tumor samples including a broad range of tumor types were sequenced with sufficient sensitivity (2.4% dropout), including samples with low DNA input (< 10 ng; 88% successful), low tumor purity (5-10%; 77% successful), and cytological material (90% successful). 75% of these tumor samples showed ≥1 (likely) pathogenic mutation, including targetable mutations (e.g. EGFR, BRAF, MET, ERBB2, KIT, PDGFRA). Amplifications were observed in 5.5% of the samples, comprising clinically relevant amplifications (e.g. MET, ERBB2, FGFR1). 1.5% of the tumor samples were classified as MSIhigh, including both MSI-prone and non-MSI-prone tumors.

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