Waldenstrom’s Macroglobulinemia (WM) is a lymphoplasmacytic lymphoma characterized by bone marrow (BM) infiltration of immunoglobulin M (IgM)-secreting lymphoplasmacytic cells.[1][1] Activating mutations in MYD88 are present in 93-97% of WM and 50-70% of IgM monoclonal gammopathy of undetermined

Somatic mutations affecting MYD88 are present in >90% of Waldenstrom's Macroglobulinemia (WM) patients, with nearly all of them being heterozygous [mutant allele frequency (MAF) <50%) and resulting from a change of leucine to proline at amino acid position 265. Previous studies in small study sets have observed that increased MYD88 homozygosity, i.e. MAFs >50% were present in 10-12% of untreated WM patients, a phenomenon that was attributed to acquired uniparental disomy (aUPD) and copy number alterations (Treon et al, NEJM 2012; Poulain et al, Blood 2013) at chromosome 3p. CXCR4 WHIM-like mutations are the second most common mutations in WM, affecting 30-40% of patients and almost always are associated with mutated MYD88 (Hunter et al, Blood 2014). Further to these studies, we evaluated the MAF of MYD88 in 236 WM patients (130 untreated and 106 previously treated), and observed MAFs >50% in 42/236 WM patients (17.8%), that was particularly striking in previously treated (26/106, 24.5%) versus untreated (16/130, 12.3%) patients (p = 0.017). To better understand the mechanism for increased MYD88 MAFs in WM, Sanger sequencing, copy number (CNA), and aUPD analysis were performed using CD19-selected BM mononuclear cells. Sanger sequencing of MYD88 L265P was used to establish the ratio of mutant versus wild-type allele expression. TaqMan real-time PCR assay was used to determine the MYD88 copy number. In total, 42 patients were screened for MYD88 copy number, with 6 patients (14.3%) being copy number altered (3 amplifications, 3 deletions).

We propose a novel self-homodyne optical-electrical-optical clock recovery technique for binary phase-shift keying (BPSK) signals using commercial optical and electrical components. We present the principle of operation as well as a proof-of-concept experiment for a 10.7 Gb/s BPSK signal clock recovery transmitted over a dispersion-compensated link of 20 km of single-mode fiber. Suppression of pattern-related frequency noise at the output of the recovered clock is shown. The timing jitter of the recovered clock at 10.7 GHz was measured to be ∼450  fs (integration range: 100 Hz-10 MHz).

Summary MYD 88 mutations are present in 95% of Waldenstrom Macroglobulinaemia ( WM ) patients, and support diagnostic discrimination from other IgM‐secreting B‐cell malignancies. Diagnostic discrimination can be difficult among suspected wild‐type MYD 88 ( MYD 88 WT ) WM cases. We systematically reviewed the clinical, pathological and laboratory studies for 64 suspected MYD 88 WT WM patients. World Health Organization and WM consensus guidelines were used to establish clinicopathological diagnosis. Up to 30% of suspected MYD 88 WT WM cases had an alternative clinicopathological diagnosis, including IgM multiple myeloma. The estimated 10‐year survival was 73% (95% confidence interval [ CI ] 52–86%) for MYD 88 WT versus 90% (95% CI 82–95%) for mutated ( MYD 88 MUT ) WM patients (Log‐rank P < 0·001). Multivariate analysis only showed MYD 88 mutation status ( P < 0·001) as a significant determinant for overall survival. Diffuse large B‐cell lymphoma ( DLBCL ) was diagnosed in 7 (15·2%) and 2 (0·76%) of MYD 88 WT and MYD 88 MUT patients, respectively (Odds ratio 23·3; 95% CI 4·2–233·8; P < 0·001). Overall survival was shorter among MYD 88 WT patients with an associated DLBCL event (Log‐rank P = 0·08). The findings show that among suspected MYD 88 WT WM cases, an alternative clinicopathological diagnosis is common and can impact clinical care. WM patients with MYD 88 WT disease have a high incidence of associated DLBCL events and significantly shorter survival versus those with MYD 88 MUT disease.

Introduction: MYD88 L265P mutations have been described in more than 90% of Waldenstrom9s Macroglobulinemia (WM) patients. MYD88 wild-type (wt) WM represent less than 10% of WM, and associate with shorter overall survival versus MYD88 mutated (mut) WM, as well as with lack of major responses and inferior progression-free survival to ibrutinib. Mutated MYD88 triggers WM cells growth and survival through NF-kB activation via IRAK1/4, BTK and HCK transactivation. CXCR4 mutations promote pro-survival AKT and ERK1/2 signaling and are associated with delayed response to ibrutinib. Deletions involving the long arm of chromosome 6 (Del6q) are highly recurrent in various B-cell malignancies, particularly those that are driven by mutated MYD88, and have been observed in up to half of WM patients. Chr6q genomic loss in WM affect important modulators of NFkB (TNFAIP3, HIVEP2), BCL2 protein family (BCLAF1), apoptosis and IGF1/PI3K/AKT signaling (FOXO3), and BTK (IBTK). The frequency and impact of Del6q on the different patient genotype remains to be clarified. Patients and Methods: Based on location and known regulation of key pathways in WM, copy number alterations (CNA) and gene expression level were assessed for IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 genes, along with CXCR4 transcriptional levels. DNA and RNA from CD19+ sorted BM lymphoplasmacytic cells from 32 untreated WM and one IgM and IgG-secreting lymphoplasmacytic lymphoma patients were analyzed. Paired CD19-depleted peripheral-blood mononuclear cells (PBMCs) were used as germline controls. Paired CD19+ and CD19- PBMCs from 6 healthy donors were analyzed to rule out possible B-cell specific findings. CNA were measured in quadruplicate and gene expression in triplicate using TaqMan real-time polymerase chain reaction (RT-PCR) assays. Deletions affecting less than 20% of WM cells were considered to be beneath the RT-PCR detection threshold. The cohort included 33 patients (21 males, 12 females), with a median age of 62 (range 35-91) years, BM involvement of 60% (range 2.5-90%), serum IgM levels of 3010 (range 257-6910) mg/dl, and hemoglobin of 10.9 (range 8.4-14.4) g/dl. MYD88 L265P mutations were detectable in 25 (76%) patients, 11 of whom (44%) also carried CXCR4WHIM mutations. Eight patients (24%) were MYD88 and CXCR4 wt. Results: Somatic Del6q were observed in 4/8 (50%) and in 20/25 (80%) patients in the MYD88 wt and mut population, respectively (p=NS). In contrast, no CNA were present in the HDs CD19+/CD19- compartments. IBTK remained intact in all 8 MYD88 wt WM, whereas resulted deleted in 13/25 (52%) MYD88 mut patients (p=0.01). BCLAF1 deletion frequency was significantly different among the two subgroups, with only 2/8 deleted MYD88wt (25%) and 19/25 (76%) deleted MYD88 mut patients (p=0.02; Figure 1). Eight MYD88 mut WM (32%) showed contiguous fully clonal Del6q, that spanned all studied genes in 7/8 (87.5%) cases; in contrast, contiguous subclonal deletions occurred only in 3/12 (25%) cases (p=0.02). All 8 fully clonal Del6q patients were CXCR4 wt; in contrast, CXCR4 mut were detected in 9/12 (75%) patients with subclonal Del6q (p=0.001; Figure 2). Unlike the MYD88 mut population, all 4 MYD88 wt patients presented with non-contiguous subclonal deletions (p=NS). Fully clonal deletions of IBTK, BCLAF1 and HIVEP2 significantly reduced the respective gene transcriptional levels (p= 0.03, 0.01 and 0.01, respectively), while CNA in TNFAIP3 and FOXO3 did not affect their expression. The MYD88 wt patients showed significant decreases in the transcriptional levels of BCLAF1 related to gene subclonal deletions, and reduced CXCR4 levels compared to MYD88 mut WM (p=0.02 and p=0.02, respectively). Conclusions: Our findings demonstrate that gene loss of IBTK, FOXO3, BCLAF1, TNFAIP3 and HIVEP2 occurs in most WM patients, but at a lower frequency in MYD88 wt. Unlike the MYD88 mut patients, those MYD88 wt showed no contiguous fully clonal deletions. The latter correlate with CXCR4 wt and reduce only IBTK, BCLAF1 and HIVEP2 levels, suggesting that regulatory mechanisms may compensate for FOXO3 and TNFAIP3 gene loss. Significant variation in IBTK and BCLAF1 deletion rate suggest that BCR and BCL2 signaling may play a different role in MYD88 mut and wt WM. The findings provide valuable insights into MYD88 wt WM pathogenesis, and may be relevant to understanding therapeutic outcome with agents that target MYD88, CXCR4, BTK and BCL2. Disclosures Castillo: Millennium: Research Funding; Abbvie: Research Funding; Janssen: Consultancy, Research Funding; Pharmacyclics: Consultancy, Research Funding. Arcaini: Pfizer, Celgene, Bayer, Roche: Membership on an entity9s Board of Directors or advisory committees; Celgene, Roche, Sandoz: Consultancy; Gilead: Research Funding. Varettoni: Jannsen: Consultancy, Other: travel expenses. Treon: Pharmacyclics: Consultancy, Research Funding.

Activating mutations in MYD88 and CXCR4 are present in 93-95% and 30-40% of patients with Waldenstrom's Macroglobulinemia (WM), respectively. Mutations in MYD88 trigger NF-KB dependent growth and survival of WM cells through BTK/IRAK, and AKT/ERK through HCK (Yang et al, Blood 2013; 2016). Patients with MYD88 mutations show high levels of response activity to ibrutinib, that targets BTK and HCK, while responses are poor in MYD88WT patients denoting biological differences between these two subgroups (Treon et al, NEJM 2015). The mutational landscape of MYD88 wild-type (WT) patients is under investigation, though is likely to involve alternative signaling pathways based on transcriptome studies (Hunter et al, Blood 2016). In a previous study, we observed that patients with MYD88WT were at higher risk of death, while CXCR4 mutation status had no impact on overall survival (Treon et al, Blood 2014). Given the uncommon nature of MYD88WT WM disease, we sought in this study to investigate the impact of MYD88WT status in a larger population of WM patients. WHO and WM Consensus guidelines were utilized to establish WM diagnosis, and thorough pathological and laboratory testing was undertaken to exclude non-WM IgM secreting B-cell entities. We utilized sorted CD19+ lymphoplasmacytic cells (LPC) obtained from the bone marrow of WM patients, and determined MYD88 mutation status by highly sensitive and specific AS-PCR for MYD88L265P as before (Xu et al, Blood 2013). Patients with wild-type MYD88 by AS-PCR underwent Sanger sequencing to exclude non-L265P MYD88 mutations as before (Treon et al, NEJM 2015). CXCR4 mutation status was determined in sorted LPC by AS-PCR and Sanger sequencing as before (Xu et al, BJH 2016). We identified 46 MYD88WT WM patients by these efforts, and compared their findings at time of diagnosis, and survival outcome to 262 patients with MYD88 mutated (MYD88MUT) disease who were diagnosed over the same time-period. The median follow-up for all patients was 74.7 (0.5-324.9 months), and was similar for MYD88WT and MYD88MUT WM patients (64.1 vs. 73.7 months, respectively; p=0.71). Patient characteristics are shown in Table 1.

MYD88 mutations are present in 95% of patients with Waldenstrom's Macroglobulinemia (WM), and permit diagnostic discrimination from other overlapping IgM-secreting B-cell malignancies. However, such diagnostic discrimination can often be difficult among suspected WM patients with wild-type MYD88 (MYD88WT), and can result in either diagnostic misclassification or mistreatment. We therefore examined a relatively large cohort of patients with suspected MYD88WT WM, and performed a systematic review of their clinical, pathologic and laboratory studies. Sixty-four patients with suspected MYD88 wild-type WM were identified. All had a monoclonal IgM protein, and bone marrow (BM) disease involvement. AS-PCR testing for MYD88 L265P mutation, and exclusion of non-L265P mutations by Sanger sequencing was performed to confirm MYD88WT status utilizing CD19-selected and unselected BM mononuclear cells (Xu et al, Blood 2013). WHO and WM consensus guidelines were used to establish clinicopathological diagnosis.

Whole-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.

The incidence and prognostic impact of nephropathy related to Waldenström macroglobulinaemia (WM) is currently unknown. We performed a retrospective study to assess biopsy-confirmed WM-related nephropathy in a cohort of 1391 WM patients seen at a single academic institution. A total of 44 cases were identified, the estimated cumulative incidence was 5·1% at 15 years. There was a wide variation in kidney pathology, some directly related to the WM: amyloidosis (n = 11, 25%), monoclonal-IgM deposition disease/cryoglobulinaemia (n = 10, 23%), lymphoplasmacytic lymphoma infiltration (n = 8, 18%), light-chain deposition disease (n = 4, 9%) and light-chain cast nephropathy (n = 4, 9%), and some probably related to the WM: thrombotic microangiopathy (TMA) (n = 3, 7%), minimal change disease (n = 2, 5%), membranous nephropathy (n = 1, 2%) and crystal-storing tubulopathy (n = 1, 2%). The median overall survival in patients with biopsy-confirmed WM-related nephropathy was 11·5 years, shorter than for the rest of the cohort (16 years, P = 0·03). Survival was better in patients with stable or improved renal function after treatment (P = 0·05). Based on these findings, monitoring for renal disease in WM patients should be considered and a kidney biopsy pursued in those presenting with otherwise unexplained renal failure and/or nephrotic syndrome.

Using photovoltage (PV) spectroscopy we analyse the electronic structure of a series of GaBixAs1-x/(Al) GaAs dilute bismide quantum well (QW) laser structures. The use of polarisation-resolved PV measurements allows us to separately identify transitions involving bound light-and heavy-hole states in the QWs, as well as bound-to-continuum transitions from the QWs to the barriers. Analysis of these transitions enables us to probe the GaBixAs1-x/(Al) GaAs conduction and valence band offsets, thereby quantifying the band offsets. Using a 12-band k . p Hamiltonian, we extract the band offsets in the QWs explicitly by constraining the Bi-related parameters of the model against the experimentally measured transition energies. The PV measurements and k . p calculations we present provide the first explicit confirmation of a type-I band offset at the GaBixAs1-x/GaAs heterointerface near x = 2%. This result, combined with the theory we present for calculating the band offsets at GaBixAs1-x/(Al) GaAs heterointerfaces, can be used to determine the band offsets at arbitrary Bi composition x.

Using whole genome sequencing, we identified highly recurrent activating mutations in MYD88 and CXCR4 in patients with Waldenström's Macroglobulinemia (WM). Using highly sensitive AS-PCR assays, we and others confirmed the presence of MYD88L265Pmutations in 95% of untreated WM patients which occur from a C>T transversion at position 38182641 at 3p22.2. Recent studies by others and us have shown that additional activating MYD88 mutations including S243N, M232T, L265RPP can also occur in WM patients. In contrast to diffuse large B-cell lymphoma where non-L265P mutations make up a quarter of all MYD88 mutations, non-L265P mutations are rare in WM. By in vitro modeling, we have shown that MYD88 mutations can trigger BTK, IRAK4/IRAK1 and PI3K dependent survival signaling in WM cells, which can be abrogated by exogenous administration or endogenous lentiviral transduced expression of peptides which block MYD88 homodimerization. Mutations in the C-terminal domain of CXCR4 are present in 40% of untreated WM patients, and are almost always present in MYD88 mutated patients. CXCR4 mutations are subclonal in most WM patients supporting their acquisition subsequent to the MYD88 mutations in WM oncogenesis. Multiple mutations resulting in compound heterozygous and homozygous CXCR4 mutation may occur within individual patients suggestive of ongoing tumor evolution and genomic instability with respect to CXCR4 in WM patients. Unsupervised clustering and principal component analysis of RNASeq data from WM patients demonstrates that MYD88 and CXCR4 mutation status are primary determinants of gene expression in WM. MYD88 and CXCR4 mutation status also impact bone marrow disease burden, serum IgM levels, and extra-medullary disease presentation. Patients with mutated MYD88 exhibit longer overall survival (>10 years) versus those with wild-type MYD88 (4.7 years), and also demonstrate higher overall and major responses to ibrutinib. The findings highlight the importance of MYD88 and CXCR4 mutations in the pathogenesis and clinical outcome of patients with WM.

We report a unidirectional frequency dissemination scheme for high-fidelity optical carriers deployable over telecommunication networks. For the first time, a 10 Gb/s Binary Phase Shift Keying (BPSK) signal from an ultra-narrow linewidth laser was transmitted through a field-installed optical fibre with round-trip length of 124 km between Cork City and town of Clonakilty, without inline optical amplification. At the receiver, using coherent communication techniques and optical injection-locking the carrier was recovered with noise suppression. The beat signal between the original carrier at the transmitter and recovered carrier at the receiver shows a linewidth of 2.8 kHz. Long term stability measurements revealed fractional instabilities (True Allan deviation) of 3.3 × 10(-14) for 1 s averaging time, prior to phase noise cancellation.

In this paper we will discuss the implications of using all-optical OFDM in the network, the need for synchronisation and clock recovery, and demonstrate a simple all-optical clock recovery system for such superchannels. The recovered analogue clock was used to generate a second comb synchronised to the original from the transmitter.

We report for the first time an ultra-stable optical-carrier dissemination technique for transmission over a 20 km unidirectional fibre link. The optical-linewidth of the recovered carrier matches closely that of the original carrier.

In this paper we will discuss a range of applications, pertinent to phase-modulated OFDM superchannels, for which optical signal processing can be used. We demonstrate a novel technique for clock recovery of a 53.5 Gbit/s BPSK superchannel, which uses the well-known four-wave mixing process in a SOA.

The genetic basis for Waldenström macroglobulinemia (WM) remains to be clarified. Although 6q losses are commonly present, recurring gene losses in this region remain to be defined. We therefore performed whole genome sequencing (WGS) in 30 WM patients, which included germline/tumor sequencing for 10 patients. Validated somatic mutations occurring in >10% of patients included MYD88, CXCR4, and ARID1A that were present in 90%, 27%, and 17% of patients, respectively, and included the activating mutation L265P in MYD88 and warts, hypogammaglobulinemia, infection, and myelokathexis-syndrome-like mutations in CXCR4 that previously have only been described in the germline. WGS also delineated copy number alterations (CNAs) and structural variants in the 10 paired patients. The CXCR4 and CNA findings were validated in independent expansion cohorts of 147 and 30 WM patients, respectively. Validated gene losses due to CNAs involved PRDM2 (93%), BTG1 (87%), HIVEP2 (77%), MKLN1 (77%), PLEKHG1 (70%), LYN (60%), ARID1B (50%), and FOXP1 (37%). Losses in PLEKHG1, HIVEP2, ARID1B, and BCLAF1 constituted the most common deletions within chromosome 6. Although no recurrent translocations were observed, in 2 patients deletions in 6q corresponded with translocation events. These studies evidence highly recurring somatic events, and provide a genomic basis for understanding the pathogenesis of WM.

By whole-genome and/or Sanger sequencing, we recently identified a somatic mutation (MYD88 L265P) that stimulates nuclear factor κB activity and is present in >90% of Waldenström macroglobulinemia (WM) patients. MYD88 L265P was absent in 90% of immunoglobulin M (IgM) monoclonal gammopathy of undetermined significance (MGUS) patients. We therefore developed conventional and real-time allele-specific polymerase chain reaction (AS-PCR) assays for more sensitive detection and quantification of MYD88 L265P. Using either assay, MYD88 L265P was detected in 97 of 104 (93%) WM and 13 of 24 (54%) IgM MGUS patients and was either absent or rarely expressed in samples from splenic marginal zone lymphoma (2/20; 10%), CLL (1/26; 4%), multiple myeloma (including IgM cases, 0/14), and immunoglobulin G MGUS (0/9) patients as well as healthy donors (0/40; P < 1.5 × 10(-5) for WM vs other cohorts). Real-time AS-PCR identified IgM MGUS patients progressing to WM and showed a high rate of concordance between MYD88 L265P ΔCT and BM disease involvement (r = 0.89, P = .008) in WM patients undergoing treatment. These studies identify MYD88 L265P as a widely present mutation in WM and IgM MGUS patients using highly sensitive and specific AS-PCR assays with potential use in diagnostic discrimination and/or response assessment. The finding of this mutation in many IgM MGUS patients suggests that MYD88 L265P may be an early oncogenic event in WM pathogenesis.

Abstract Background Waldenstrom’s Macroglobulinaemia (WM) is an IgM secreting lymphoplasmocytic lymphoma characterized by involvement mainly in the bone marrow, and occasionally in the lymph nodes and spleen. Central nervous system involvement of WM, known as Bing-Neel syndrome (BNS), is very uncommon and as such the clinical characteristics and treatment outcomes remain to be clarified. Methods We evaluated the incidence, clinical characteristics, and treatment outcome of BNS among patients with WM who were diagnosed at our Institution. Results We identified 13 patients with BNS from our database of 1,523 patients diagnosed with WM from 1999 -2013. The median age at diagnosis of BNS was 60 (range 51-75 years). The median time to development of BNS after WM diagnosis was 6.3 (range 0.3-11.9 years). Patients presented with a variety of neurological signs and symptoms including seizures, hearing loss, cognitive impairment, gait instability and lower extremity weakness. The diagnosis of BNS was confirmed by MRI in 11, and by examination of the CSF in 12 patients. CSF cytopathologic analysis demonstrated definitive evidence of malignant lymphoplasmacytic cells in 6 patients, and was suspicious for malignant cells in 2 patients. CSF flow cytometry was positive in 8, and a clonal immunoglobulin heavy chain gene rearrangement was identified in 6 patients. MYD88 L265P was identified in CSF sample of two patients in whom this examination was undertaken, and who demonstrated malignant disease by flow cytometry and cytological examination. Seven patients were treated with high-dose methotrexate (HD-MTX), 2 with intrathecal liposomal cytarabine, 1 with rituximab and intrathecal methotrexate, and 1 with rituximab and bendamustine. One patient was recommended treatment with HD-MTX and was lost to follow-up, 1 has been initiated on HD-MTX. Of 11 patients, 5 had a response in CSF and/or MRI, 4 had stable disease and 1 progressed following therapy. Of the responders, 2 patients had received HD-MTX, 2 intrathecal liposomal cytarabine and 1 rituximab-bendamustine. Four patients with stable disease had received HD-MTX. One patient who received intrathecal MTX developed treatment related “chemical meningitis”, and was subsequently treated with HD-MTX to which she had a CSF response. The median overall survival was not reached. Eleven patients are alive, 1 died and 1 was lost to follow-up. Conclusions BNS is an uncommon late complication of WM. MYD88 L265P may help in the diagnosis of BNS, and may represent a novel approach for targeted therapy of WM. HD-MTX is active in the treatment of BNS, though prospective studies are required to standardize treatment and improve outcomes. Disclosures: No relevant conflicts of interest to declare.

Background Waldenstrom's macroglobulinemia (WM) is an indolent non-Hodgkin's lymphoma characterized by the accumulation of IgM secreting lymphoplasmacytic cells in the bone marrow. CXCR4 is a chemokine receptor that promotes the survival, migration, and adhesion to the bone marrow stroma of WM lymphoplasmacytic cells (LPC) through interactions with its ligand CXCL12. Through whole genome sequencing, we identified somatic mutations in CXCR4 that affected 1/3 of WM patients. These mutations were identical or functionally similar to those associated with Warts, Hypogammaglobulinemia, Infection, and Myelokathexis (WHIM) syndrome (Hunter et al, ASCO 2012), a rare autosomal dominant genetic disorder that is caused by frame shift or nonsense mutations in the carboxyl-terminal cytoplasmic tail of CXCR4. In WHIM syndrome, loss of the c-terminal tail of CXCR4 impairs receptor internalization, thereby prolonging G-protein and β-arrestin signaling (Lugane et al, Blood 2008). Ibrutinib induces WM cell death, and is highly active in WM (Treon et al, ICML-12, 2013). Since the target of ibrutinib (BTK) is a known downstream target of CXCR4, we sought to clarify if ibrutinib activity in WM LPCs was modulated by WHIM-like mutations in CXCR4. Methods We first sought to confirm the frequency of WHIM-like mutations in 87 untreated WM patients by Sanger sequencing. The most common CXCR4 somatic mutation identified (S338X) in these studies was then cloned by PCR from CD19+ LPCs from a WM patient with this somatic mutation. Wild type (WT) and S338X CXCR4 cDNAs were subcloned into plenti-IRES-GFP vector, and transduced using an optimized lentiviral based strategy into BCWM.1 WM cells. Five days after transduction, GFP positive cells were sorted and used for functional studies. Surface expression of CXCR4 was determined by flow cytometeric analysis using a PE-conjugated anti-CXCR4 monoclonal antibody. The expression of phosphorylated BTK, AKT, and ERK1/2 was determined by western blot analysis. Cell proliferation was measured with alamar blue. Results Sanger sequencing identified nonsense or frame shift mutations (WHIM-like) in the c-terminal tail of CXCR4 in 28 of 87 (32%) patients, the most common of which was a non-sense mutation (S338X) that was present in 12 patients. BCWM.1 cells were then transduced with control vector, CXCR4 wild type or CXCR4 S338X mutant expressing vectors. Expression was confirmed by cDNA Sanger sequencing. Stably transduced cells exposed to ibrutinib (0.5uM or 1uM) showed significantly reduced cell proliferation (p<0.005). Ibrutinib treated control vector and CXCR4 wild-type transduced cells showed suppressed tumor cell growth even in the presence of the CXCR4 ligand CXCL12 (20 nM), whereas cells transduced with CXCR4 S338X WHIM-like mutation demonstrated resistance to ibrutinib growth effect (p<0.005). In turn, this rescue could be blocked by treatment with 30uM of the CXCR4 specific inhibitor AMD3100 confirming that this effect was mediated through CXCR4 (p<0.005) (Figure 1). Phosphorylated BTK, ERK1/2 and AKT signaling increased following CXCL12 stimulation in all transduced cells, while ibrutinib inhibited their activation in control vector and CXCR4 wild-type, but not CXCR4 S338X mutant cells. CXCR4 triggered signaling by CXCL12 in these experiments was confirmed by pre-treatment with AMD3100. Conclusions By Sanger sequencing, WHIM-like CXCR4 somatic mutations are observed in 1/3 of untreated WM patients. WHIM-like CXCR4 mutations are associated with resistance to ibrutinib mediated ERK1/2 and AKT signaling, as well as growth suppression in the presence of the CXCR4 ligand, CXCL12, in WM cells. These studies may have important implications for CXCR4 modulation in the treatment of WM, as well as potential use of CXCR4 mutations in predicting outcome for patients undergoing ibrutinib therapy. Disclosures: No relevant conflicts of interest to declare.

Abstract Background Over 90% of patients with Waldenström's Macroglobulinemia (WM), and 50-80% of patients with the precursor condition, IgM MGUS, express MYD88 L265P. These findings suggest that other mutations may support progression of IgM MGUS to WM. Chromosomal aberrations including large losses in 6q are commonly present in WM patients, though the gene loss accounting for WM pathogenesis remains unclear. We therefore sought to delineate copy number alterations (CNA) and structural variants using whole genome sequencing (WGS) in order to more clearly define other important gene alterations in WM. Methods DNA from CD19+ bone marrow lymphoplasmacytic lymphoma cells (LPC) and CD19-depleted peripheral blood mononuclear cells from 10 WM patients was used for paired tumor/germline analysis by WGS. Coverage in the tumor sample was divided by the coverage in the paired germline sample for each matching position, resulting in coverage ratios for each 100Kb window. Statistically significant windows within each genome were then analyzed across the cohort by randomizing the coverage positions to assess the probability of observing the given frequency of a CNA by random chance. TaqMan quantitative polymerase chain reaction (PCR) copy number assays was used to validate findings. Translocations were validated by Sanger sequencing across the breakpoint including flanking sequences. Results Functional annotation for identified CNAs was undertaken using Ingenuity Pathway Analysis that revealed a significant enrichment for pathways dysregulated in B-cell malignancies (Table 1). Iteratively randomizing the genomic position of CNAs not related to the chromosome 6 deletions revealed a greater than 3 fold increase in the targeting of COSMIC genes than expected by chance (p< 0.001). Affected genes in the COSMIC census were BTG1 (9/10; 90%), FOXP1 (7/10; 70%), FNBP1 (7/10; 70%), CD74 (7/10; 70%), TOP1 (6/10; 60%), MYB (5/10; 50%), CBLB (5/10; 50%), ETV6 (5/10; 50%), TNFAIP3 (5/10; 50%), FBXW7 (5/10; 50%), PRDM1 (5/10; 50%), TFE3 (4/10; 40%), JAK1 (4/10; 40%), MAML2 (4/10; 40%), FAM46C (4/10; 40%), EBF1 (4/10; 40%), STL (4/10; 40%), and BIRC3 (4/10; 40%). Other affected genes of interested included PRDM2 (8/10; 80%), HIVEP2 (8/10; 80%), ARID1B (7/10; 70%) as well as LYN (7/10; 70%). There were no singular regions of statistical significance in 6q to denote a minimally deleted region though neither of the previously suspected target genes for 6q loss, PRDM1 and TNFAIP3, were included in the regions of highest statistical significance. Losses in HIVEP2 (8/10; 80%) as well as ARID1B (7/10; 70%) and BCLAF1 (7/10; 70%) constituted the most common deletions in chromosome 6, and were present in patients with and without the large-scale losses in 6q. While no recurrent translocations were noted in this study, 2 or the 5 (40%) of the 6q deletions corresponded with translocation events. In one case, this was a result of chromothripsis focused on 6q while in the other case, a t(6;X) translocation linked to the amplification of Xq was identified. Validation studies confirmed presence of somatic deletions in BTG1 (4/5; 80%) at Chr. 12q21.33, HIVEP2 (4/5; 80%) at 6q24.2, LYN (3/5 60%) at 8q12.1, PLEKHG1 (3/5; 60%) at 6q25.1, ARID1B (3/5 60%) at 6q25.1, PDRM2 (2/5; 40%) at 1p36.21, FOXP1 (2/5; 40%) at 3p13, and MKLN1 (2/5 40%) at 7q32. As some CVAs were subclonal, we validated the correlation between the PCR relative copy number and WGS coverage predictions (rho = .926; p =2.2x10-16). Conclusions Highly recurrent CNAs are present in WM LPCs that include genes with critical regulatory roles in lymphocytic growth and survival signaling. Disclosures: No relevant conflicts of interest to declare.