CXCR4 Mutations Research Articles

Silencing Mutated Cxcr4 in Mouse Hematopoietic Stem Cells as a Genetic Cure Strategy Obviating Bone Marrow Conditioning in WHIM Syndrome

Abstract Genetic cure of diseases caused by autosomal dominant (AD) gain-of-function (GoF) mutations could require full correction of the disease allele; however, selective inactivation of the disease allele might suffice or even be superior. To test the relative efficacy of these two strategies in WHIM syndrome, a rare combined primary immunodeficiency disease caused by AD GoF mutations in chemokine receptor CXCR4 leading to impaired release of mature leukocytes from bone marrow, we have performed competitive bone marrow transplantation experiments with Cxcr4+/+, Cxcr4+/o and Cxcr4+/S338X (WHIM model) mice. Here we show a whole bone marrow donor genotype rank order of Cxcr4+/o >Cxcr4+/+> Cxcr4+/S338X for repopulating the blood of lethally irradiated recipient mice. Importantly, the strong competitive advantage of transplanted Cxcr4 haploinsufficient bone marrow cells is durable, intrinsic to hematopoietic stem cells (HSCs) and evident in the blood at the level of cell frequency and total cell numbers in both the myeloid and lymphoid lineages in both irradiated and unconditioned congenic recipients. Mechanisms included enhanced engraftment of hyperproliferative Cxcr4+/o HSCs in irradiated recipients and enhanced release of mature Cxcr4+/o leukocytes to the blood in unconditioned recipients. These results provide proof of principle that inactivation of the CXCR4 disease allele in HSCs may be a safe, effective and superior genetic cure strategy able to obviate toxic bone marrow conditioning in patients with WHIM syndrome.

Preference of Genetic Diagnosis of CXCR4 Mutation Compared with Clinical Diagnosis of WHIM Syndrome

Preference of Genetic Diagnosis of CXCR4 Mutation Compared with Clinical Diagnosis of WHIM Syndrome

Detection of MYD88 L265P and WHIM-like CXCR4 mutation in patients with IgM monoclonal gammopathy related disease.

A broad spectrum of diseases are associated with IgM monoclonal gammopathy, including Waldenstrom macroglobulinemia (WM), various types of B cell non-Hodgkin's lymphoma (NHL), multiple myeloma (MM), primary amyloidosis (AL), and monoclonal gammopathy of undetermined significance (MGUS); these are called IgM monoclonal gammopathy related diseases (IgM-RD). We investigated MYD88 L265P and WHIM-like CXCR4 mutations in various IgM-RD. Patients with serum immunofixation electrophoresis confirmed IgM monoclonal gammopathy who had enough material for DNA extraction and presented between January 2008 and October 2016 at Peking Union Medical College Hospital were enrolled in this cohort. We performed real-time allele-specific-polymerase chain reaction and Sanger sequencing to explore the presence of MYD88 L265P and WHIM-like CXCR4 mutations. One hundred and twelve patients (64 male and 48 female patients) were included in this retrospective study. The median age at diagnosis was 62years (range, 30-84years). In total, 64 patients (57.1%) carried the MYD88 L265P mutation and 14 patients (12.5%) carried the CXCR4 WHIM-like mutation. We identified the MYD88 L265P somatic variant in cases with WM (39/42), MGUS (8/18), NHL (14/41, including 4/13 diffuse large B cell lymphoma (DLBCL), 1/8 mucosa-associated lymphoid tissue, 3/6 splenic marginal zone lymphoma (SMZL), 1/4 chronic lymphocytic leukemia, 2/3 nodal marginal zone lymphoma (NMZL), 1/2 mantle cell lymphoma, 1 Burkitt lymphoma, and 1 B cell NHL that could not be classified), primary AL (2/2), and IgM-PN (1/1). The mutation was absent in five patients with Cryoglobulinemia, two with primary cold agglutinin disease and one with MM. The CXCR4 WHIM-like mutation was present in 10/42 patients with WM, 3/41 with NHL (1 DLBCL, 1 SMZL, and 1 NMZL), and 1/18 patients with IgM MGUS. Among the patients with NHL, those with the mutated MYD88 L265P genotype were younger and had lower level of IgG and IgA than the patients with the wild-type genotype. Patients with the mutated MYD88 L265P genotype with WM and MZL were compared. More male patients, higher levels of IgM and lower levels of LDH were found in the WM group. There was no significant difference in overall survival between the two groups. We present a study of the prevalence of the MYD88 L265P mutation and CXCR4 WHIM-like mutation in IgM RD. The MYD88 L265P mutation may play a key role in the pathogenesis of IgM monoclonal gammopathies. It would be interesting in the future to use MYD88 mutation status to differentiate among diseases.

Genomics, Signaling, and Treatment of Waldenström Macroglobulinemia.

Next-generation sequencing has revealed recurring somatic mutations in Waldenström macroglobulinemia (WM). Commonly recurring mutations include MYD88 (95% to 97%), CXCR4 (30% to 40%), ARID1A (17%), and CD79B (8% to 15%). Diagnostic discrimination of WM from overlapping B-cell malignancies is aided by MYD88 mutation status. Transcription is affected by MYD88 and CXCR4 mutations and includes overexpression of genes involved in VDJ recombination, CXCR4 pathway signaling, and BCL2 family members. Among patients with MYD88 mutations, those with CXCR4 mutations show transcriptional silencing of tumor suppressors associated with acquisition of mutated MYD88. Deletions involving chromosome 6q are common and include genes that modulate nuclear factor-κB, BCL2, BTK, apoptosis, differentiation, and ARID1B. Non-chromosome 6q genes are also frequently deleted and include LYN, a regulator of B-cell receptor signaling. MYD88 and CXCR4 mutations affect WM disease presentation and treatment outcome. Patients with wild-type MYD88 show lower bone marrow disease burden and serum immunoglobulin M levels but show an increased risk of death. Patients with CXCR4 mutations have higher bone marrow disease burden, and those with nonsense CXCR4 mutations have higher serum immunoglobulin M levels and incidence of symptomatic hyperviscosity. Mutated MYD88 triggers BTK, IRAK1/IRAK4, and HCK growth and survival signaling, whereas CXCR4 mutations promote AKT and extracellular regulated kinase-1/2 signaling and drug resistance in the presence of its ligand CXCL12. Ibrutinib is active in patients with WM and is affected by MYD88 and CXCR4 mutation status. Patients with mutated MYD88 and wild-type CXCR4 mutation status exhibit best responses to ibrutinib. Lower response rates and delayed responses to ibrutinib are associated with mutated CXCR4 in patients with WM. MYD88 and CXCR4 mutation status may be helpful in treatment selection for symptomatic patients. Novel therapeutic approaches under investigation include therapeutics targeting MYD88, CXCR4, and BCL2 signaling.

Ibrutinib in Waldenström's macroglobulinemia: revamping the landscape

In The Lancet Oncology, Meletios Dimopoulos and colleagues 1 Dimopoulos MA Trotman J Tedeschi A et al. Ibrutinib monotherapy for patients with rituximab-refractory Waldenström's macroglobulinaemia (iNNOVATE): an open-label substudy of an international, multicentre, phase 3 trial. Lancet Oncol. 2016; (published online Dec 9.)http://dx.doi.org/10.1016/S1470-2045(16)30632-5 Google Scholar report that the activity of the BTK inhibitor, ibrutinib, in patients with rituximab-refractory Waldenström's macroglobulinemia is not only remarkable, but eclipses the other available treatment options. When attempting to place these results into perspective, a couple of adages regarding clinical trials come to mind. First, “the better the therapy, the less relevant the prognostic factors and the more important the predictive factors.” In almost all B-cell malignancies, including Waldenström's macroglobulinemia, an anti-CD20 antibody, generally rituximab, has become a standard component of most first-line treatment strategies because this drug improves progression-free survival and, often overall survival. Nonetheless, initial chemoimmunotherapy is associated with three potential outcomes: cure for the curable subtypes, such as diffuse large B-cell lymphoma, and for the remaining subtypes, either response with eventual relapse, in which case retreatment with a CD20-based regimen is reasonable, or the absence of a response where the role of additional rituximab is questionable. As such, rituximab refractoriness is considered highly unfavourable, and is traditionally associated with a poor outcome. Few drugs have provided exceptions to this rule. However, in the GADOLIN study, 2 Sehn LH Chua N Mayer J et al. Obinutuzumab plus bendamustine versus bendamustine monotherapy in patients with rituximab-refractory indolent non-Hodgkin's lymphoma (GADOLIN): a randomised, controlled, open-label, multicentre, phase 3 trial. Lancet Oncol. 2016; 17: 1081-1093 Summary Full Text Full Text PDF PubMed Scopus (232) Google Scholar , 3 Cheson BD Trneny M Bouabdallah K et al. Obinutuzumab plus bendamustine followed by obinutuzumab maintenance prolongs overall survival compared with bendamustine alone in patients with rituximab-refractory indolent non-Hodgkin lymphoma: updated results of the GADOLIN study. Blood. 2016; 128 (abstr).: 623 Google Scholar the use of the next generation anti-CD20 obinutuzumab prolonged not only progression-free survival but also overall survival in rituximab-refractory follicular and low-grade lymphoma. In the present study of patients with rituximab-refractory disease, the proportions of patients with a response were similar to those in another cohort 4 Treon SP Tripsas CK Meid K et al. Ibrutinib in previously treated Waldenström's macroglobulinemia. New Engl J Med. 2016; 372: 1430-1440 Crossref Scopus (655) Google Scholar who were not refractory to this drug, thus overcoming that adverse feature. Progression-free survival and overall survival results were also very promising. Traditional prognostic factors used in the International Waldenström Scoring System include age, serum IgM level, β-2 microglobulin, and presence of anaemia and thrombocytopenia. 5 Morel P Duhamel A Gobbi P et al. International prognostic scoring system for Waldenström macroglobulinemia. Blood. 2009; 113: 4163-4170 Crossref PubMed Scopus (280) Google Scholar Nevertheless, the activity of ibrutinib in the present study does not seem to be affected by any of those features. More importantly are the findings that suggest that mutations in MYD88 and CXCR4 are predictive of outcome. Although the present substudy enrolled a fairly small sample size (n=31) as part of a larger randomized trial of ibrutinib in this population, the quality of response seemed to be affected by MYD88 and CXCR4 mutational status; although these data must be interpreted with caution due to the small numbers. 4 Treon SP Tripsas CK Meid K et al. Ibrutinib in previously treated Waldenström's macroglobulinemia. New Engl J Med. 2016; 372: 1430-1440 Crossref Scopus (655) Google Scholar More data will be forthcoming on correlations between genotype and outcome after analysis of the main portion of the current study. Ibrutinib for patients with rituximab-refractory Waldenström's macroglobulinaemia (iNNOVATE): an open-label substudy of an international, multicentre, phase 3 trialThe sustained responses and median progression-free survival time, combined with a manageable toxicity profile observed with single-agent ibrutinib indicate that this chemotherapy-free approach is a potential new treatment choice for patients who had heavily pretreated, rituximab-refractory Waldenström's macroglobulinaemia. Full-Text PDF

Mutated MYD88 Zygosity and CXCR4 Mutation Status Are Important Determinants of Ibrutinib Response and Progression Free Survival in Waldenstrom's Macroglobulinemia

Activating somatic mutations in MYD88 and CXCR4 are present in 90-95% and 30-40% of untreated WM patients, respectively. Nearly all MYD88 somatic mutations involve a single nucleotide mutation that results in a change from leucine to proline at amino acid position 265, and most are heterozygous. In approximately 10-12% of untreated WM patients, MYD88 L265P is homozygous due to an acquired uniparenteral disomy (aUPD) or copy number alterations (Treon et al, NEJM 2012; Poulain et al, Blood 2013). Over 30 different types of nonsense and frameshift mutations in CXCR4 have been described in WM, and almost always are associated with mutated MYD88 (Hunter et al, Blood 2014). Mutated MYD88 activates BTK, and the BTK inhibitor ibrutinib is highly active in WM (Yang et al, Blood 2013; Treon et al NEJM 2015; Dimopoulos et al, ASH 2015). MYD88 mutations predict for response, while CXCR4 mutations are associated with slower response kinetics and lower response rates among mutated MYD88 WM patients on ibrutinib (Treon et al, NEJM 2015). In this study, we sought to clarify the impact of MYD88 homozygosity on ibrutinib activity in WM. We evaluated 33 previously treated, symptomatic WM patients who received ibrutinib on a clinical trial (NCT01614821), and for whom baseline and serial bone marrow (BM) CD19-selected cells were available for sequencing, copy number (CNA) and aUPD analysis. Mutated MYD88 homozygosity was determined by establishing the ratio of mutant (Mut) versus wild-type (WT) allele expression by Sanger sequencing. CNA status was determined using TaqMan real-time PCR assays. For patients with unaltered copy number, the presence of aUPD was determined by analyzing the tumor/germline allele balance using established TaqMan genotyping assays. At baseline, 9/33 (27.3%) of patients were homozygous for mutated MYD88. Mutated MYD88 homozygosity was confirmed to be due to deletion of the wild-type MYD88 allele by CNA in one patient, and amplification of the mutant MYD88 allele in another patient. TaqMan genotyping assays confirmed the presence of an aUPD in 6 patients that were confined to Chr. 3p. CXCR4 mutations were highly prevalent (8/9; 88.8%) among mutated MYD88 homozygous versus heterozygous (7/24; 29.2%) patients (p=0.014). We next assessed the impact of mutated MYD88 zygosity and CXCR4 mutation status on ibrutinib treatment outcome. The baseline characteristics for patients based on mutated MYD88 zygosity and CXCR4 mutation status are shown in Table 1. Following ibrutinib treatment, serum IgM levels declined by -0.89%, -0.54%, and -0.61% among MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Hemoglobin levels improved by 40.5%, 16.2% and 21.6% among MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Major responses (PR or better) were observed in 100%, 50%, and 89% of MYD88Mut/WTCXCR4WT, MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. VGPR occurred in 50% of MYD88Mut/WTCXCR4WT patients, and 16.7% and 22.2% of MYD88Mut/WTCXCR4Mut, and MYD88Mut/Mut patients, respectively. Progression-free survival was also impacted by mutated MYD88 zygosity and CXCR4 mutation status (Figure 1). Median PFS was not reached for patients with MYD88Mut/WTCXCR4WT and MYD88Mut/Mut, and was 25.5 months for those with a MYD88Mut/WTCXCR4Mut mutation status (Log-rank Chi-square: 14.74, p=0.0006).Conclusions: MYD88 homozygosity is strongly associated with activating mutations in CXCR4, and appears to confer a better outcome for WM patients with CXCR4 mutations on ibrutinib therapy. [Display omitted] [Display omitted] DisclosuresTreon:Pharmacyclics: Consultancy, Research Funding; Janssen: Consultancy. Castillo:Pharmacyclics: Honoraria; Abbvie: Research Funding; Otsuka: Consultancy; Biogen: Consultancy; Millennium: Research Funding; Janssen: Honoraria. Palomba:Pharmacyclics: Consultancy.

Targeted Next Generation Sequencing Identifies Novel Genetic Mutations in Patients with Waldenstrom's Macroglobulinemia/Lymphoplasmacytic Lymphoma or IgM-Monoclonal Gammopathies of Undetermined Significance

Background. TheMYD88 (L265P) somatic mutation is present in more than 90% of patients (pts) with Waldenström’s Macroglobulinemia (WM)/lymphoplasmacitic lymphoma (LPL). The second most common mutations are nonsense (NS) or frameshift (FS) mutations in the CXCR4 gene, detectable in approximately 25-30% of WM pts by Sanger sequencing. Limited data are available about other genetic mutations in WM/LPL and its precursor condition IgM-monoclonal gammopathy of undetermined significance (IgM-MGUS).Pts and methods. Using targeted next generation sequencing (NGS), we evaluated the prevalence of somatic mutations of 11 genes selected on the basis of evidences available from the literature (MYD88, CXCR4, ARID1A, KMT2D, TP53, NOTCH2, PRDM1, CD79b, TRAF3,TNFAIP3, MYDBBP1A) in 119 pts, classified as WM/LPL (n=63) or IgM-MGUS (n=56) according to International Consensus Criteria. Median age of pts (67 males, 52 females) was 65 years (range: 38-82). Samples were collected at diagnosis (n=101), after diagnosis but before any treatment (n=9) or at progression after therapy (n=9). Paired tumor and germline DNA extracted respectively from CD19-selected and CD19-depleted bone marrow (BM) mononuclear cells was available in all pts. Mean resequencing depth across gene panel was 1009x. Only mutations tagged as oncogenic or possibly oncogenic based on information derived from the literature and on in silico prediction effect were considered in the analysis. For MYD88 (L265P) and CXCR4 mutations, results obtained with NGS were compared with those obtained respectively with allele-specific PCR (AS-PCR) and Sanger sequencing.Results. Overall, we found 151 mutations in 88 pts (74%). The median number of mutations was significantly higher in WM/LPL as compared with IgM-MGUS and in pts previously treated as compared with untreated ones (median 2 versus 1, P < 0.001 for both comparisons). MYD88 mutations were found in 80/119 pts (67%), with a median allele burden of 34.2% (range: 2.5-93.3%). The prevalence of MYD88 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (86% versus 46%, P <0.001). MYD88 mutations other than classical L265P (n=76) were found in 4 pts and were represented by V217F (n=2), S219C (n=1), M232T (n=1). Fifteen pts who were MYD88 (L265P) wild-type by NGS were found to be mutated by AS-PCR (K coefficient of concordance between NGS and AS-PCR: 70%, P < 0.001). CXCR4 mutations were found in 19/119 pts (16%), with a median allele burden of 34% (range: 4.2-84%). The prevalence of CXCR4 mutations was significantly higher in WM/LPL as compared with IgM-MGUS (24% versus 7%, P < 0.02). The K coefficient of concordance between NGS and Sanger was 83% (P < 0.001), with 2 pts mutated only by NGS and 2 pts mutated only by Sanger. Somatic mutations were also found in KMT2D (formerly known as MLL2) (16% of pts), TP53 (8%), NOTCH2 (7%), PRDM1 (4%), ARID1A (3%), CD79b (2%), and TRAF3 (1%). No mutations were found in MYBBP1A and TNFAI3. Overall, the prevalence of these mutations was significantly lower in pts wild-type either for MYD88 or CXCR4 as compared with those with MYD88 and/or CXCR4 mutations (15% versus 41%, P = 0.04). The prevalence of KMT2D mutations was significantly higher in WM/LPL as compared with MGUS (25% versus 5%), while for the other genes the distribution was not statistically different according to diagnosis. With a median follow-up of 20 months (range: 0-264), we did not find a statistically significant correlation between genetic mutations and pts’ outcome in terms of overall survival or time to first treatment.Conclusions. In this cohort of pts with WM/LPL and IgM-MGUS studied with NGS we could demonstrate that: i) NGS identifies MYD88 mutations other than L265P in a small proportion of pts; ii) the prevalence of CXCR4 mutations by Sanger is confirmed by NGS, despite the higher sensitivity of the latter method; iii) the subgroup of pts wild type either for MYD88 or CXCR4 shows a low incidence of other genetic mutations; iv) 25% of pts with WM/LPL were found to carry KMT2D mutations, a prevalence similar to that reported in marginal zone lymphoma; v) genetic mutations are more common in WM/LPL than in IgM-MGUS in agreement with the hypothesis that multiple genetic hits are required for progression from a pre-benign condition to a neoplastic disease; vi) due to the indolent nature of these disorders, longer follow-up is probably needed to see the prognostic impact of these mutations, if any. DisclosuresNo relevant conflicts of interest to declare.

Driver Mutation in Waldenstrom's Macroglobullinemia and Their Clonal Heterogeneity during Progression and Relapse

Background: Whole genome sequencing has recently shown the presence of recurrent mutations, such as MYD88, CXCR4 and ARID1A, in patients with Waldenstrom's Macroglobulinemia (WM) at diagnosis. Nevertheless, the contribution of each genomic aberration within the clonal evolution of the tumor during WM progression has not been reported. We therefore aimed to investigate whether sequential genomic events sustain WM disease progression by using whole exome sequencing (WES) and targeted sequencing of serial samples. In addition, we investigated if specific genomic alterations can change in response to treatment with samples before and after proteasome or BTK inhibitor treatments.Methods: We have sequenced 74 samples from 32 patients using WES or targeted sequencing technology. DNA was collected from bone marrow CD19-selected cells that were isolated from 6 patients with WM at different stages of disease (2-3 serial samples per patient, total 15 samples), and was subjected to library construction, followed by Agilent Sure-Select Human All Exon v2.0-based hybrid selection. Germline DNA was isolated from matched CD19-depleted peripheral blood samples. All libraries were sequenced with Illumina Hiseq 2500 instrument (New York Genome Center, Rockefeller University, New York, NY). Reads were aligned to GRCh37, and quality control, mutation calling, insertion and deletion identification, copy number variation detection, coverage calculations were accomplished via Firehose at Broad. Somatic Single Nucleotide Variations (SSNVs) were identified and annotated using MuTect and Oncotator, respectively. Insertions and deletions were detected using Strelka. In addition, the deep targeted sequencing was done with a customized bait set in 59 independent serial samples obtained from 26 patients who were treated with either proteasome inhibitor (Pi)- or BTK inhibitor (BTKi)-based therapies in 12 and 14 cases, respectively.Results: Whole exome sequencing, performed at a total average depth of 83X for germline and 85X for tumor samples, led to the identification of average of 14 non-silent mutations per sample (range 2-60). Base conversion signature showed dominant A>C/G transitions. Copy number analysis showed 6q deletion being the most prevalent one. MYD88 was the most recurrent somatic variants in WM patients (>93%), followed by several genes including IRS4, VCAN, CXCR4 and ALDH2, being detected in ~20-30% of the samples. Specifically, changes in the Cancer Cell Fraction (CCF) of these mutations, such as MYD88 and CXCR4, occurred in the serial samples depending on progression and response to therapy.The mutated genes mentioned above, including MYD88, CXCR4, LRG1 and VCAN, were also observed in targeted sequencing of 59 independent serial samples (mean target coverage 434X). CXCR4 was linked to disease progression exposed to Pi or BTK; but not detected in patients responding to therapies. MYD88 was present in patients with either progressive disease or PR/VGPRs. Specifically, different frequencies of MYD88 mutations were identified in patients with progression or PR/VGPR to BTKi-based therapies compared to Pi-based therapies. LRG1 was detected in a patient in response to Pi, while VCAN was observed in both patients showing progression or response to Pi regimens only.Conclusion: These findings reveal the occurrence of clonal variations in patients with WM during disease progression and response to therapies. MYD88 was confirmed to be the most prevalent somatic aberration, and was present in post-treatment samples of progressors and responders to Pi- or BTKi-based regimens. In contrast, mutations in CXCR4 were enriched in patients with WM progressing to either Pi or BTKi therapies. This study demonstrates that WES and targeted sequencing of serial samples of WM patients can detect clonal variations during disease progression. DisclosuresGhobrial:Celgene: Honoraria, Research Funding; Noxxon: Honoraria; Amgen: Honoraria; BMS: Honoraria, Research Funding; Novartis: Honoraria; Takeda: Honoraria. Roccaro:Takeda Pharmaceutical Company Limited: Honoraria.

TP53 Mutation in Waldenstrom Macroglobulinemia

Background. Waldenstrom macroglobulinemia (WM) is a B-cell malignancy characterized by bone marrow (BM) infiltration of clonal lymphoplasmacytic cells, which produce a monoclonal immunoglobulin M. MYD88L265P mutation may be considered as a founder event because of it high frequency in WM. WM cells may acquire additional genetic hits that may potentially promote disease progression: CXCR4 or CD79B mutations, copy number variation,…TP53 is a tumor suppressor gene that functions as regulator influencing cellular responses to DNA damage. Little is known regarding TP53 alteration in WM. Our aim was to screen TP53 mutation in a large cohort of WM at diagnosis to analyze the genomic landscape of WM using targeted next generation sequencing (NGS) and genome wide single nucleotide polymorphism array (SNPa) and to identify clinical and biological characteristics.Method. BM samples of 125 WM (mean age: 67 years) were analyzed at diagnosis. Tumoral DNA was extracted following CD19 B cell selection. TP53 mutations were analyzed by targeted NGS to scan the coding exons of TP53. MYD88L265P, CD79A, CD79B, and CXCR4mutations were analyzed by sanger sequencing and/or NGS. Genome-Wide Human SNP Array 6.0 (Affymetrix chips) was performed in 62 cases. CN-LOH (copy neutral- loss of heterozygosity) and CNA (copy number aberration) were mapped using console 3.02 software (Affymetrix). Flow cytometry was performed to assess P53 and p21 expression after nutlin3a exposition to characterize functional mutant of TP53. Viability and cell growth of treated cells were determined using the MTS assay.Results. We have identified TP53 mutations using NGS in 7.3 % of WM (6 non-sense, 3 frameshift mutations located in the DNA binding domain) (TP53mut WM). The mutation load of TP53 varied from 13% to 98.9% (mean: 62.0%) using the variant allele frequency in NGS. We next examined the effects of nutlin-3a which is an mdm2 inhibitor on WM patients CD19+ cells genotyped for TP53 mutation. Nutlin-3a increased the expression of p53 and p21 in TP53Wild WM patients using flow cytometry (n=6). In contrast, in TP53MutWM cells, no significant variation of p53, p 21 and viability using MTS assay was observed suggesting the presence of functional mutation of TP53. The minimal deleted region of 17p in 17p deleted (TP53Del) samples was mapped using SNP array and contained 79 genes, among which was systematically comprised the loss of TP53. A high correlation between TP53 mutation and deletion 17p (p<106) was observed. One case of CN-LOH was observed at TP53 locus (1,6% of cases). Overall, we have identified alteration of TP53 locus including mutation, deletion and copy neutral loss of heterozigosity in 11, 2% of WM. Using SNP array, we found a relationship between deletion 17p, alteration of TP53 locus including mutation, UPD or del17p (TP53Alt) and TP53Mutand a greater frequency of genomic aberrations in WM compared toTP53wild (p=0.01, p=0.024 and p=0.06 respectively). A higher frequency of WM patients with more than 3 CNA identified by SNPa was observed in TP35Mut group (p=0.03) and del17p group (p<0.00001). No association was observed between TP53Mut and CXCR4 and MYD88mutations.We thoughtto identify clinical and biological characteristics of WM according to TP53Mutand/or Del17pfeatures. With a median follow-up of 5 years, 33 (26%) patients had died. 69% of cases were treated. Front line therapy included rituximab-based regimens in 76%, alkylating agent in 78%, fludarabine in 6%. The WM with TP53alteration, irrespective of TP53Mut or del17p, displayed features of adverse prognosis in regards to higher serum levels of b2m (89% versus 40%, p=0.012), and also greater IPSSWM score 2 and 3 (50% versus 30%, and 43% versus 30%, p=0.041, respectively). Importantly, the presence of TP53alteration, irrespective of TP5Mut or del17p, was associated to poor outcome in overall survival in our series, TP53alteration (p=0.003), del17p (p=0.002), and TP53Mut (p=0.015). TP53 alteration prognostic value was independent of CXCR4 or MYD88L265Pmutations.Conclusion: A low frequency of TP53 mutation was observed in WM at diagnosis. We identified a genomic signature associated to their presence. In addition, a pejorative prognostic value of TP53 mutation was observed in WM highlighted the need of new therapeutic in this sub group of WM. DisclosuresLeleu:TEVA: Membership on an entity's Board of Directors or advisory committees; Novartis: Honoraria; LeoPharma: Honoraria; Pierre Fabre: Honoraria; Amgen: Honoraria; Bristol-Myers Squibb: Honoraria; Takeda: Honoraria; Celgene: Honoraria; Janssen: Honoraria.

Ixazomib, Dexamethasone and Rituximab in Previously Untreated Patients with Waldenström Macroglobulinemia

▪Introduction: Waldenström’s macroglobulinemia (WM) is an incurable B-cell lymphoma characterized by the accumulation of IgM-secreting lymphoplasmacytic cells in the bone marrow and other organs. Bortezomib in combination with rituximab and dexamethasone (BDR) is highly active as primary therapy in WM, though treatment-related neuropathy is common with BDR in WM, and often leads to premature treatment discontinuation. Ixazomib is an orally administered proteasome inhibitor with limited neuropathy that is active in myeloma, but has not been previously evaluated in WMMethods: Symptomatic, previously untreated patients with a clinicopathological diagnosis of WM were included in this prospective, single-arm phase II study evaluating ixazomib 4 mg PO on days 1, 8 and 15 + dexamethasone 20 mg PO/IV on days 1, 8 and 15 + rituximab 375 mg/m2 IV on day 1 (IDR) were administered for six 4-week cycles (induction) followed by six 8-week cycles (maintenance). Rituximab was held for the first two cycles of therapy to minimize risk of IgM flare. Zoster prophylaxis and proton pump inhibitors were administered throughout IDR therapy. The study was approved by the institutional review board at the Dana-Farber Cancer Institute, and registered under Clinicaltrials.gov ID NCT02400437.Results: Twenty-six WM patients were enrolled and were exposed to IDR therapy. The median age at WM diagnosis was 63 years (range 46-81 years) and the median age at initiation of therapy was 65 years (range 46-82 years). Baseline median hemoglobin was 10.2 g/dl (range 6.9-13.2 g/dL), median serum IgM level was 5,068 mg/dl (range 653-7,650 mg/dL), 46% of patients had lymphadenopathy and 12% had splenomegaly. The median bone marrow involvement was 55% (range 5-95%). The MYD88 L265P gene mutation was identified in all cases. CXCR4 gene mutations were identified in 15 patients (58%), of whom 10 (67%) had nonsense, and 5 (33%) frameshift mutations. Sixteen patients have completed the induction phase of therapy at this time. Following induction therapy, the median serum IgM level decreased to 2,316 mg/dl (range 287-5,820 mg/dL), median hemoglobin increased to 13.1 mg/dl (range 10.4-14.6 g/dL), and median bone marrow involvement decreased to 23% (range 0-76%). P-value <0.001 for all comparisons against baseline. Using consensus response criteria, the overall response rate was 88% (VGPR 6%, PR 44%, MR 38%) with a major response rate of 50%. Major responses (VGPR + PR) were observed in 47% of patients with CXCR4 mutations versus 64% in those who were wild-type CXCR4 (p=0.32). The median time to response was 8 weeks. The median time to response in CXCR4 mutant patients was 12 weeks versus 8 weeks in wild-type CXCR4 patients (log-rank p=0.03). Four patients have been taken off study; 2 for lack of response, 1 due to lack of clinical benefit with persistent failure to thrive while in PR, and 1 for progressive neuropathy while in PR although in part due to worsening of diabetic neuropathy. No other grade 3 or 4 adverse events were reported.Conclusion: These preliminary data suggest that the combination of IDR is an active and well-tolerated, neuropathy-sparing regimen in symptomatic untreated WM patients. DisclosuresCastillo:Biogen: Consultancy; Otsuka: Consultancy; Abbvie: Research Funding; Pharmacyclics: Honoraria; Millennium: Research Funding; Janssen: Honoraria.

Detection of MYD88 L265P and Whim-like CXCR4 Mutation in Patients with IgM Monoclonal Gammopathy

Objectives The presence of IgM monoclonal gammopathy can occur in a broad spectrum of diseases, including Waldenstrom macroglobulinemia (WM), various B cell non-Hodgkin's lymphoma (NHL), monoclonal gammopathy of undetermined significance (MGUS), and so on. MYD88 L265P and WHIM-like CXCR4 mutation are highly prevalent in WM. However, the data is lacking in other IgM monoclonal gammopathy related diseases (IgM-RD). Therefore we investigated MYD88 L265P and WHIM-like CXCR4 mutation in various IgM-RD.Methods Patients with sIFE confirmed IgM monoclonal gammopathy and had enough material for DNA extraction between January 2008 and May 2016 at Peking Union Medical College Hospital were enrolled in this cohort. Bone marrow samples or paraffin-embedded tumor tissues were collected. We performed Real-time allele-specific-polymerase chain reaction (AS-PCR), AS-PCR and Sanger sequencing to explore the presence of MYD88 L265P and WHIM-like CXCR4 mutation.Results Ninety-three patients (51 male and 42 female patients) were included in this retrospective study. Median age at diagnosis was 62 years (range, 30-84 years). As for the type of light chain, 65 patients were IgM kappa (69.9%), 28 patients were IgM lambda (30.1%). The median level of serum IgM was 7.9g/L (range, 0.6-104.0g/L). There were 32 patients (34.4%) diagnosed with WM, 5 patients (5.4%) diagnosed with smoldering WM (sWM), 38 patients (40.9%) diagnosed with B cell NHL and 9 patients (9.7%) were diagnosed with MGUS. We outlined the main diagnosis, clinical characteristics and MYD88 L265P, WHIM-like CXCR4 mutation of the 93 patients in Table 1. MYD88 L265P mutation was detected in 30/32 (93.8%) patients with WM, 2/5 (40%) with sWM, 4/9 (44.4%) with IgM-MGUS, 6/14 (42.9%) with diffuse large B cell lymphoma (DLBCL), 2/6 (33.3%) with marginal zone lymphoma (MZL), 3/5 (60%) with chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL), 1/7 (4.3%) with mucosa-associated lymphoid tissue lymphoma (MALT), 1/2 (50%) with mantle cell lymphoma (MCL), 1/1 patients with primary amyloidosis (AL) and 1/1 patients with Castleman disease (CD). WHIM-like CXCR4 mutation was detected 7/32 (21.9%) patients with WM, 1/5 (20%) with sWM and 1/14 (7.1%) with DLBCL.Conclusion IgM monoclonal gammopathy is most frequently found in WM, but it contains a broad spectrum of diseases. MYD88 L265P mutation is highly prevalent in WM and IgM MGUS, and the prevalence of MYD88 mutation in IgM related NHL is higher than that in not specified NHL reported in the literature. WHIM-like CXCR4 mutation is common in WM, but rare in other IgM-RD. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Clone-specific MYD88 L265P and CXCR4 mutation status can provide clinical utility in suspected Waldenström macroglobulinemia/lymphoplasmacytic lymphoma

MYD88 L265P, a diagnostic marker for lymphoplasmacytic lymphoma (LPL)/Waldenström macroglobulinemia (WM) can also be detected in other hematopoietic malignancies. We demonstrate a novel approach to increase the specificity of this marker for WM/LPL diagnosis by combining flow cytometric cell sorting with molecular analysis.Clonal B-lymphocyte and co-occurring clonal plasma cell populations of low-grade B-cell lymphomas were sorted by flow cytometry and analyzed for immunoglobulin gene rearrangements (PCR), and for MYD88 and CXCR4 mutations.Identical clonal origin was confirmed by PCR for 21 LPL/WM cases and MYD88 L265P was detected in both B-cell and plasma cell fractions. 9/20 other B-cell lymphomas with identical light chain restriction on B-cells and plasma cells were genotypically identical by PCR and MYD88 L265P was detected in both cell fractions in 7/9 whereas in 11/20 specimens with different clonal origin, MYD88 L265P was absent (5/11), or only found in B-lymphocytes (4/11), or plasma cells (2/11). CXCR4 mutations were detected in 17/39 cases, but missed in 63% of these without cell sorting.Confirming MYD88L265P in both B-cells and plasma cell fractions can provide a novel and powerful discriminator to distinguish LPL/WM from phenotypically similar disorders. Furthermore, this approach significantly increases CXCR4 detection sensitivity.

Efficient Plasma Cell Differentiation and Trafficking Require Cxcr4 Desensitization

CXCR4 plays a central role in B cell immune response, notably by promoting plasma cell (PC) migration and maintenance in the bone marrow (BM). Gain-of-function mutations in CXCR4 affecting receptor desensitization have been reported in therare immunodeficiency called WHIM syndrome (WS). Despite lymphopenia, patients mount an immune response but fail to maintain it over time. Using a knockin mouse model phenocopying WS, we showed that, counter-intuitively, a gain of Cxcr4 function inhibited the maintenance of antibody titers after immunization. Although the Cxcr4 mutation intrinsically and locally promoted germinal center response and PC differentiation, antigen-specific PCs were barely detected in the BM, a defect mirrored by early accumulation of immature plasmablasts potentially occupying the survival niches for long-lived PCs. Therefore, fine-tuning of Cxcr4 desensitization is critically required for efficient PC differentiation and maintenance, and absence of such a regulatory process may account for the defective humoral immunity observed in WS patients.

Transcriptome sequencing reveals a profile that corresponds to genomic variants in Waldenström macroglobulinemia

AbstractWhole-genome sequencing has identified highly prevalent somatic mutations including MYD88, CXCR4, and ARID1A in Waldenström macroglobulinemia (WM). The impact of these and other somatic mutations on transcriptional regulation in WM remains to be clarified. We performed next-generation transcriptional profiling in 57 WM patients and compared findings to healthy donor B cells. Compared with healthy donors, WM patient samples showed greatly enhanced expression of the VDJ recombination genes DNTT, RAG1, and RAG2, but not AICDA. Genes related to CXCR4 signaling were also upregulated and included CXCR4, CXCL12, and VCAM1 regardless of CXCR4 mutation status, indicating a potential role for CXCR4 signaling in all WM patients. The WM transcriptional profile was equally dissimilar to healthy memory B cells and circulating B cells likely due increased differentiation rather than cellular origin. The profile for CXCR4 mutations corresponded to diminished B-cell differentiation and suppression of tumor suppressors upregulated by MYD88 mutations in a manner associated with the suppression of TLR4 signaling relative to those mutated for MYD88 alone. Promoter methylation studies of top findings failed to explain this suppressive effect but identified aberrant methylation patterns in MYD88 wild-type patients. CXCR4 and MYD88 transcription were negatively correlated, demonstrated allele-specific transcription bias, and, along with CXCL13, were associated with bone marrow disease involvement. Distinct gene expression profiles for patients with wild-type MYD88, mutated ARID1A, familial predisposition to WM, chr6q deletions, chr3q amplifications, and trisomy 4 are also described. The findings provide novel insights into the molecular pathogenesis and opportunities for targeted therapeutic strategies for WM.

Waldenström macroglobulinemia: biology, genetics, and therapy.

Waldenström macroglobulinemia (WM) is a distinct clinicopathologic entity characterized by the presence of a lymphoplasmacytic lymphoma, a non-Hodgkin lymphoma, and IgM monoclonal gammopathy. WM is an indolent, uncommon malignancy mostly affecting the elderly. Patient outcomes have modestly improved since the introduction of rituximab to conventional cytotoxic chemotherapy more than 20 years ago. However, the pivotal discovery of the somatic MYD88 L265P mutation, harbored by most patients with WM, and the somatic CXCR4 WHIM mutations, similar to germline CXCR4 mutations seen in the warts, hypogammaglobulinemia, immunodeficiency, and myelokathexis (WHIM) syndrome, present in approximately one-third of patients with WM, has fundamentally changed our understanding of this disease and expanded the potential therapeutic targets. Within this new paradigm, ibrutinib emerged as a promising new drug. Ibrutinib targets Bruton’s tyrosine kinase, a downstream protein in the B-cell receptor pathway that is overactivated by the MYD88 L265P mutation. A seminal Phase II trial of ibrutinib in previously treated WM patients showed impressive response rates and confirmed the effects of MYD88 L265P and CXCR4 WHIM mutations in response to therapy. Ibrutinib is the first and only US Food and Drug Administration–approved drug specifically for the treatment of WM. However, before ibrutinib can be established as the standard of care for WM, long-term data regarding efficacy and safety are required. Further research to address ibrutinib resistance and cost-effectiveness is also imperative before ibrutinib can gain widespread acceptance. This review will cover the present pathophysiologic understanding of WM in light of the recent MYD88 and CXCR4 discovery, as well as current and emergent treatment regimens with focus on ibrutinib.

Clinical Validation of a CXCR4 Mutation Screening Assay for Waldenstrom Macroglobulinemia.

Waldenstrom macroglobulinemia (WM) is a B-cell lymphoma characterized by the accumulation of lymphocytes and plasmacytic cells in the bone marrow and by excess production of immunoglobulin M in serum. WM has been closely linked with the MYD88(L265P) mutation. Whole genome sequencing has identified somatic mutations in the CXCR4 gene in ∼29% of WM cases with MYD88(L265P). CXCR4 mutations may interfere with treatment response to ibrutinib. The goal of this study was to design and validate a clinical assay to detect CXCR4 mutations. Thirty-three low-grade B-cell lymphomas with plasmacytic differentiation (23 MYD88(L265P) and 10 MYD88(WT)) involving various samples types (fresh and formalin-fixed tissues) formed the study group. We designed and validated Sanger sequencing and pyrosequencing assays to detect mutations in CXCR4 in a Clinical Laboratory Improvement Amendments-approved clinical laboratory. We identified 8 cases with CXCR4 mutations, including 5 single nucleotide substitutions (3 resulting in p.S338* and 1 in p.R334*), and 3 insertion/deletions. Seven of 8 CXCR4 mutated cases were also MYD88(L265P) mutant. Among the single nucleotide substitutions, we identified a novel missense variant (p.L326P) and a previously reported variant (G335S) of uncertain clinical significance. We successfully validated a set of clinical assays to detect mutations in CXCR4 mutations in a clinical laboratory.

Neutropenic Mice Provide Insight into the Role of Skin-Infiltrating Neutrophils in the Host Protective Immunity against Filarial Infective Larvae

Our knowledge and control of the pathogenesis induced by the filariae remain limited due to experimental obstacles presented by parasitic nematode biology and the lack of selective prophylactic or curative drugs. Here we thought to investigate the role of neutrophils in the host innate immune response to the infection caused by the Litomosoides sigmodontis murine model of human filariasis using mice harboring a gain-of-function mutation of the chemokine receptor CXCR4 and characterized by a profound blood neutropenia (Cxcr4+/1013). We provided manifold evidence emphasizing the major role of neutrophils in the control of the early stages of infection occurring in the skin. Firstly, we uncovered that the filarial parasitic success was dramatically decreased in Cxcr4+/1013 mice upon subcutaneous delivery of the infective stages of filariae (infective larvae, L3). This protection was linked to a larger number of neutrophils constitutively present in the skin of the mutant mice herein characterized as compared to wild type (wt) mice. Indeed, the parasitic success in Cxcr4+/1013 mice was normalized either upon depleting neutrophils, including the pool in the skin, or bypassing the skin via the intravenous infection of L3. Second, extending these observations to wt mice we found that subcutaneous delivery of L3 elicited an increase of neutrophils in the skin. Finally, living L3 larvae were able to promote in both wt and mutant mice, an oxidative burst response and the release of neutrophil extracellular traps (NET). This response of neutrophils, which is adapted to the large size of the L3 infective stages, likely directly contributes to the anti-parasitic strategies implemented by the host. Collectively, our results are demonstrating the contribution of neutrophils in early anti-filarial host responses through their capacity to undertake different anti-filarial strategies such as oxidative burst, degranulation and NETosis.

Genomic Landscape of CXCR4 Mutations in Waldenström Macroglobulinemia.

Whole-genome sequencing has revealed MYD88 L265P and CXCR4 mutations (CXCR4(mut)) as the most prevalent somatic mutations in Waldenström macroglobulinemia. CXCR4 mutation has proved to be of critical importance in Waldenström macroglobulinemia, in part due to its role as a mechanism of resistance to several agents. We have therefore sought to unravel the different aspects of CXCR4 mutations in Waldenström macroglobulinemia. We have scanned the two coding exons of CXCR4 in Waldenström macroglobulinemia using deep next-generation sequencing and Sanger sequencing in 98 patients with Waldenström macroglobulinemia and correlated with SNP array landscape and mutational spectrum of eight candidate genes involved in TLR, RAS, and BCR pathway in an integrative study. We found all mutations to be heterozygous, somatic, and located in the C-terminal domain of CXCR4 in 25% of the Waldenström macroglobulinemia. CXCR4 mutations led to a truncated receptor protein associated with a higher expression of CXCR4. CXCR4 mutations pertain to the same clone as to MYD88 L265P mutations but were mutually exclusive to CD79A/CD79B mutations (BCR pathway). We identified a genomic signature in CXCR4(mut) Waldenström macroglobulinemia traducing a more complex genome. CXCR4 mutations were also associated with gain of chromosome 4, gain of Xq, and deletion 6q. Our study panned out new CXCR4 mutations in Waldenström macroglobulinemia and identified a specific signature associated to CXCR4(mut), characterized with complex genomic aberrations among MYD88L265P Waldenström macroglobulinemia. Our results suggest the existence of various genomic subgroups in Waldenström macroglobulinemia.

Highlights of This Issue

Legat and colleagues report a clinical trial in 16 advanced-stage melanoma patients vaccinated with five tumor peptides, Montanide and IMP321/LAG-3Ig, a non-TLR agonist with interesting adjuvant properties including dendritic cell activation. The treatment was well tolerated. Combinatorial immune monitoring revealed multiple tumor antigen-specific CD8 and CD4 T-cell responses in most patients, supporting the further use of these vaccine components for the development of combination immunotherapies.Zabor and colleagues analyzed individual patient tumor data from a phase III clinical trial of vemurafenib versus dacarbazine in melanoma patients to identify criteria for tumor measures that correlated with OS. Correlation between OS and early response was weak no matter how the response was defined; however, a strong correlation was seen between OS and progression when defined as ≥50% increase in sum of tumor diameters or appearance of new tumors. These data suggest that tumor progression is a better surrogate endpoint for OS than tumor response, such as RECIST.Zhuang and colleagues studied whether targeting IL-1a inhibits NF-κB activity and suppresses PDAC cell growth. Previously, studies show that KRASG12V induces expression of IL-1a in genetically engineered mouse models that express mutant KRAS and p53 and in cell lines expressing KRASG12V with p16 knockdown. The authors also observed a correlation between mutant KRAS and IL-1a expression in PDAC patient samples. The experimental evidence suggests a combination of gemcitabine and IL-1 receptor antagonist can reduce PDAC cell growth by inhibiting IL-1a-induced NF-κB activity, thus suggesting a new therapeutic approach for PDAC.Poulain and colleagues analyzed the genomic landscape of CXCR4 somatic mutation in Waldenström macroglobulinemia (WM) combining deep sequencing and SNP array. The study showed existence of intraclonal (variation in coexpression of MYD88 and CXCR4 mutations) and interclonal (mutually exclusive BCR and CXCR4 mutations in MYD88L265P WM) heterogeneity of the molecular spectrum of CXCR4 mutation. Furthermore, CXCR4 mutated WM displayed a specific clinicobiologic and genomic and transcriptomic signature. This dataset further suggests the genomic heterogeneity of WM beyond the current indolent versus symptomatic classification.

Waldenstrom Macroglobulinemia: Familial Predisposition and the Role of Genomics in Prognosis and Treatment Selection.

Waldenstrom macroglobulinemia (WM) is a non-Hodgkin lymphoma (NHL) characterized by the presence of a CD20 + lymphoplasmacytic bone marrow (BM) infiltrate and serum immunoglobulin M monoclonal protein. Both sporadic and familial forms exist. A remarkable improvement in outcome of nearly all age groups of WM patients may be primarily a consequence of successful integration of anti-CD20 monoclonal antibody, rituximab, to the conventional chemotherapy. However, the seminal discoveries of MYD88 (L265P) mutation, present in the vast majority (85-100 %), and CXCR4 (WHIM) mutations, identified in nearly a third of patients (who almost exclusively harbor the MYD88 (L265P) variant), have laid a solid foundation for a paradigm shift in our diagnostic and therapeutic approaches towards this rare hematologic malignancy. Given that 20-25 % of patients are asymptomatic at diagnosis and early intervention does not translate into survival benefit, we follow a risk-adapted approach and actively monitor this subset of "smoldering" patients. Those with low-grade cytopenias can generally be managed with an abbreviated course of rituximab monotherapy, while those with B symptoms, bulky lymphadenopathy, or profound disease-related cytopenias require more aggressive strategies, incorporating several courses of chemoimmunotherapy. For symptoms associated with hyperviscosity, prompt plasma exchange is warranted prior to initiation of cytoreductive therapy. Prospective data are unavailable to a support a unique approach in familial WM or initiation of rituximab maintenance post-induction. A multitude of potentially effective therapies targeting cell-survival pathways are in development and offer a more precise approach for WM patients. One such agent, ibrutinib, a Bruton's tyrosine kinase inhibitor, was recently granted approval. Off-study, we limit the use of ibrutinib to relapsed-refractory WM patients who harbor MYD88 mutations. Autologous stem-cell transplantation (ASCT) is another viable option in the relapsed setting for chemosensitive transplant-eligible patients. Unfortunately, despite substantial progress, achieving a minimal residual disease-negative state-a prerequisite for cure-is rare in WM.