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Introduction Patients with Multiple Myeloma (MM) often have a significant delay between onset of symptoms and diagnosis of disease. As a result, a significant number of patients present via emergency routes with severe co-morbidities which affect survival rates. Timely diagnosis relies on the early recognition of symptoms and blood test results which may indicate disease. Methods We examined the medical records of 142 newly diagnosed MM patients (121 intact immunoglobulin and 21 light chains) across 2 UK Hospitals. Patients included had not previously been diagnosed with a plasma cell dyscrasia, including Monoclonal Gammopathy of Undetermined Significance (MGUS). Clinical symptoms and blood test results were examined from the time of initial presentation to the healthcare system with symptoms indicative of MM, to the point of diagnosis in order to highlight patterns of symptoms and blood tests results which may give an early indication of disease. Blood tests results recorded included globulin, calcium, creatinine, erythroid sedimentation rate and haemoglobin. Time to diagnosis from presentation with symptoms indicative of MM was also measured as well as the patient pathway from the point of presentation to the point of diagnosis. Results The median time to diagnosis from initial presentation was 77 days (range 0 - 12,986). Initial presentation was most commonly via primary care (58.1%). Urgent secondary care presentation was documented in 28.5% which included acute medical unit (15.6%), the emergency department (7.1%), and other secondary care specialities (5.7%) respectively. Multiple GP visits were common prior to haematology referral with a median of 3 visits (range 1 - 40). Initial presenting symptoms varied, but of those with data recorded (n=107) back/bone pain was the most common (58.2%) followed by anaemia (18.7%), fracture (7.5%), recurrent infection (7.5%) and renal impairment (3.7%) respectively. Interestingly, analysis of evaluable blood test results revealed a raised globulin was most often evident prior to diagnosis with 58% of patients recording an abnormal globulin a median of 140 days (range 3 - 4297) prior to diagnosis of disease. Conclusions Multiple GP visits prior to establishing a diagnosis of myeloma is very common. Inclusion of abnormal globulin to reflex electrophoresis request and serum free light chain assay may serve as a useful trigger for investigation when interpreted alongside presenting symptoms and other blood test results. Increased awareness of myeloma warning signs in primary care may reduce diagnostic delay and avoid presentation with severe co-morbidities in emergency settings. Disclosures No relevant conflicts of interest to declare.

Introduction: Chronic Lymphocytic Leukemia (CLL) is an incurable B-cell malignancy which disproportionately affects the elderly. Although first-line chemoimmunotherapy (CIT) improved CLL clinical outcomes, recent randomized trials revealed superior outcomes with novel chemotherapy-free combinations (CFC) incorporating anti-CD20 monoclonal antibodies and inhibitors of BTK or Bcl-2. So far, these CFC have not been compared head-to-head. We conducted this network meta-analysis to evaluate their relative efficacy to each other. Methods: A review of the medical literature was conducted using online databases. Inclusion criteria consisted of English language; diagnosis of CLL; trials that explored the efficacy of first-line CFC with Obinutuzumab (O), Rituximab (R), Ibrutinib (IB), Acalabrutinib (ACAL), Venetoclax (VEN) compared to standard CIT that included Chlorambucil (CHLOR) with either R or O, Bendamustine+Rituximab, or Fludarabine+ Cyclophosphamide+R; and phase 3 randomized studies reporting responses, progression, death, and adverse (AE) events. A frequentists network meta-analysis was conducted using netmeta package and random-effects model. Results: Five studies comprising a total of 2,272 participants were included. When O-based CFC data was analyzed, only ACAL-O had a significant lower relative risk (RR) of progression and death (P&D). There were no significant differences with respect to overall response rates (ORR), complete remission (CR), minimal residual disease (MRD), or grade >3 adverse events (Grd3+) among O-based CFC. When R-based CFC data was analyzed, IB and IB-R were not different with respect to RR of P&D, ORR, CR, MRD, or Grd3+. When the data was analyzed as CFC versus combined CIT, only ACAL-O was found to be significantly superior to other O- and R-based CFC with respect to RR of P&D. ORR and Grad3+ rates of O- and R-based CFC were not significantly different. While ACAL-O, IB-O, and VEN-O had superior CR and MRD rates compared to other CFC, there were no significant differences among each other. Conclusions: This network meta-analysis is the first to compare and rank first-line CFC therapies in CLL. It indicates that ACAL-O has a superior profile having the lowest RR of P&D without significant difference in Grd3+ among CFC. Disclosures No relevant conflicts of interest to declare.

Introduction: Serum M-protein levels, measured by serum immunoelectrophoresis, have been one of the major tests that determine the type of response to therapy in Multiple Myeloma. Achieving an M-protein of zero is one of the criteria determining complete remission which in turn translates into longer progression-free survival and overall survival. Triplet therapy tends to be the standard therapeutic approach in younger, fit, and transplant candidates. However, in older non-fit patients many oncologists start with doublets and only escalate to triplet combinations upon less optimal response. The predictive value of the velocity of the M-spike decline (VMD) has not been adequately studied. We conducted this study to find the optimal VMD that predicts a serum M-protein of zero. Methods: A sample of mostly non-fit older Multiple Myeloma patients was identified. All patients had regular serum protein electrophoresis with M-protein levels documented every 4-8 weeks until they achieved M-protein levels of zero, plateauing without attaining the target of zero, or rebounding with progression. Best-fit line or curve was constructed and the slope of the decline was calculated. VMD was defined as the slope of the best fit-curve/line. VMD values corresponding to the binary outcomes of achieving or failing to achieve an M-protein value of zero were collected. A ROC curve was constructed to determine the cut-off VMD value that best predicts the outcome of M-protein of zero. Results: A total of 47 patients of mostly non-fit older Multiple Myeloma patients were included in our final sample. The median age of the sample was 71 years. Thirteen percent had triplet therapy. Thirty four percent had proteasome inhibitor based combinations while 64% had IMiD based combinations. Fifty seven percent achieved the target M-protein level of zero while 43% failed. The area under the ROC curve, AUC, was 0.98 (p Conclusions: This exploratory study established the VMD value that best predicts the outcome of M-protein of zero in response to Multiple Myeloma therapy. This may be potentially used by oncologists to make early decisions regarding the need for further optimization or intensification of their therapeutic combinations as they treat their older non-fit Multiple Myeloma patients. Disclosures No relevant conflicts of interest to declare.

INTRODUCTION Thrombotic episodes represent significant morbidity and mortality in patients with hematologic malignancies and may be influenced by several risk factors. Current predictive and prognostic models do not take into account several risk factors. Furthermore, prophylactic and therapeutic management of thrombosis remains to be standardized for patients with AML. Few studies report the impact of catheters to the likelihood of thrombosis. We comprehensively report our institution's experience with thrombosis in AML patients. METHODS The Total Cancer Care (TCC) database was used to retrospectively chart review patients with histologically confirmed AML from 2000 to 2018. Several parameters were extracted including but not limited to history of thrombosis and recurrences, chemotherapy regimens, and laboratory values for significant events. All statistical analyses were performed using SPSS v24.0 and GraphPad Prism 7. RESULTS: A total of 1101 patients were diagnosed with either de novo (516, 46.87%) or secondary (584, 53.04%) AML. The median age of diagnosis was 75 years (52, 95) and men were the majority, 726 (65.94%). In terms of cytogenetics, 25 (2.27%) had good/favorable, 616 (55.95%) had intermediate, and 334 (41.75%) had poor cytogenetics. At the time of diagnosis, CBC revealed median WBC of 3 (0, 420), ANC 1 (0, 132), Plt 52 (1, 996), and hgb 9 (1, 15). Median ECOG score was 1. 159, 14.4% patients had a history of thrombosis prior to AML diagnosis; with a median of 1 episode of thrombosis (1, 4). After AML diagnosis 70 patients had one episode of thrombosis, 9 had two episodes, and 2 patients had three recurrent episodes. As far as the first-line induction therapy, 247 (22.43%) patients received 7+3, 12 (1.08%) received CLAG, 36 (3.27%) received hypomethylating agents, and the remaining 227 (2.06%) received other therapies such as clinical trials or off-label drugs. Repeat cycles of therapies were also recorded. In terms of growth factor use after AML diagnosis, 132 (11.99%) used EPO, 209 (18.98%) G-CSF, 32 (2.90%) GM-CSF, and 778 (70.66%) used any other growth factor not mentioned. Out of the 139 episodes of thrombosis, 81 (58.3%) were venous, and 58 (41.7%) were arterial. Among the venous, 45 (55.6%) were lower extremities, 21 (25.9%) were upper extremities, and 10 (12.3%) were pulmonary embolus. Among the arterial thrombosis, 27 (46.6) were coronary events, 23 (39.7%) were cerebral events. Only 15 patients had catheter-associated thrombosis events, 11 (73.3%) were PICC, and 2 (13.3%) were Port lines or central lines. In terms of management, 39 patients were on temporary anticoagulation, 60 patients were on indefinite anticoagulation, and 522 were not on any anticoagulation. In terms of complications, 3 patients had minor bleeding, 1 had major bleeding, 2 had HIT and the large majority of 419 had no complications of anticoagulation. CONCLUSIONS: Thrombosis both venous and arterial events manifest during treatment for leukemias. PICCs lines are associated with more catheter associated thrombosis events than ports. Further prospective studies are needed to evaluate the risk of both arterial and venous thrombosis in patients with acute leukemia. Further risk mitigation strategies are needed. Disclosures Komrokji: Novartis: Honoraria, Speakers Bureau; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau; Celgene: Honoraria, Research Funding; Novartis: Honoraria, Speakers Bureau.

The prognosis for patients with mantle cell lymphoma (MCL) is poor, and at present there is no truly effective therapy. Gene translocation-mediated constitutive expression of cyclin D1 seems to play the key role in the pathogenesis of MCL. Here we report that although 3 of 4 MCL cell lines expressed the recently identified, highly oncogenic cyclin D1b isoform, as well as the canonical cyclin D1a, 8 MCL patient samples expressed only the cyclin D1a protein despite expressing detectable cyclin D1b mRNA. Cell lines and tissue samples displayed constitutive activation of the cyclin D1 signaling cascade, as evidenced by strong expression of CDK4, Rb phosphorylation, and cyclin D1/CDK4 coassociation. All MCL cell lines and tissues examined displayed nondetectable to diminished expression of the cyclin D1 inhibitor p16. Novel small molecule CDK4/CDK6 inhibitor PD0332991 profoundly suppressed—at low nanomolar concentrations—Rb phosphorylation, proliferation, and cell cycle progression at the G0/G1 phase of MCL cells. These findings provide evidence that MCL should be very sensitive to targeted therapy aimed at functional inhibition of the cyclin D1/CDK4 complex.

We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5′ of the breakpoint and 3′ sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. Panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes.

Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-β (TGF-β) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-β did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-β type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-β. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-β and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

Bone marrow (BM) cells from a child with an immature (CD3-) acute T lymphoblastic leukemia (T-ALL) bearing no chromosomal abnormalities failed to grow in long-term culture in the presence or absence of recombinant human (rh) growth factors but could be engrafted in severe combined immunodeficient (SCID) mice and induced leukemia. The leukemic cells recovered from the animal tissues could be adapted to grow in vitro in the presence of rh interleukin-2 (IL-2) and give rise to a growth factor-dependent cell line designated TALL-107. This cell line expresses T-cell-specific mature markers (CD2, CD3/T-cell receptor [TCR] alpha beta, CD8, CD56), and its growth can be inhibited by IL-4 of all the cytokines tested. Similar to the original leukemic blasts, TALL-107 cells are clonal, have rearranged TCR-beta, gamma, and delta loci, and a normal 46 XY karyotype. However, unlike the patient's BM cells, the TALL-107 cell line displays potent tumoricidal activity that is not major histocompatibility complex restricted. The magnitude of mRNA expression of perforin and serine esterases and of lytic activity depends on the doses of IL-2 added. TALL-107 cells can also be triggered by CD3- and CD2-specific monoclonal antibodies (MoAbs) to mediate reverse tumor cell lysis. In addition, this cell line produces high levels of interferon gamma and tumor necrosis factor alpha on stimulation with anti-CD3 and/or anti-CD2 MoAb both in the presence or absence of IL-2. The overall data indicate that the SCID mouse is able to support the functional maturation and expansion of a cytotoxic T- cell subset from some types of T-ALL.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL- 249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL- 249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5′ ends and random hexamers at the 3′ ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLLexon 7 or exon 8 with the GMPS (GUANOSINE 5′-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene ofMLL on chromosome 3q and the first gene of this type in leukemia-associated translocations.

A truncated granulocyte-macrophage colony-stimulating factor (GM-CSF) allele on a putative 5q- chromosome of HL-60 cells was cloned and, by comparison with counterpart normal sequences, analyzed for clues to molecular mechanisms facilitating rearrangement and deletion. Within the 17-kilobase (kb) pair locus surrounding the truncated GM-CSF gene remnant, there are no fewer than four rearranged genomic fragments that seemingly derive from chromosome 5 region q21----23. Two of the fragments, which flank the truncated GM-CSF locus on the 5q-, are contiguous on the normal chromosome 5, centrometric to the normal GM- CSF allele, indicating at least one intrachromosomal insertion event, either preceded or followed by further deletion. Insertion and/or deletion was accompanied by juxtaposition of LINE sequences to the 5′ side of the truncated GM-CSF locus within the inserted fragment. The entire rearranged locus is embedded in repetitive sequences, which may have mediated successive insertions or deletions. The extent of such stepwise deletions, resulting in loss of genes such as interleukin-3 (IL-3), IL-4, IL-5, and GM-CSF, whose gene products are critical to differentiation within the lineage of the affected hematopoietic stem cell, may be mirrored in the heterogeneity of symptoms and 5q- deletion sizes observed in myelodysplasias and acute leukemias carrying a 5q- chromosome. Perhaps most significantly, the sequences surrounding the insertion/deletion region are suggestive of recombination signals, including direct repeats and mirrored repeats. The site of insertion of the GM-CSF 3′ region into an upstream (centromeric) locus is flanked by direct repeats; the upstream site into which it is inserted is also flanked by 12 base pair (bp) direct repeats. After insertion, one member of each pair of repeats is lost. The organization of this rearranged locus implies that direct repeats had a role in the intrachromosomal recombination/deletion event.

Introduction The regulation of neutrophil lifespan is critical for a circumscribed immune response. Neutrophils are sensitive to Fas/CD95 death receptor signaling in vitro but it is unknown if Fas regulates neutrophil lifespan in vivo. We hypothesized that FasL-expressing CD8+ T cells, which kill antigen-stimulated T cells during chronic lymphocytic choriomeningitis virus (LCMV) infection, can also induce neutrophil death in tissues during infection. Infection of Fas- and FasL-deficient mice with the enteropathogenic-like mouse pathogen Citrobacter rodentium (C.rodentium) is associated with neutrophil infiltration and severe diarrhoea. We hypothesized that a deficiency of Fas in neutrophils prolongs neutrophil lifespan and contributes to the accumulation of neutrophils in C.rodentium-infected mice. Methods Fas signaling can drive IL-1β and IL-18 production and thereby affect neutrophil accumulation in tissues, so mixed bone marrow chimeras were generated to compare the accumulation of WT and Fas-deficient neutrophils in vivo. For mixed bone-marrow chimeras, irradiated Ly5.1 mice were reconstituted with 2.5 x 106 bone marrow cells from ubiquitin-GFP or Ly5.1/5.2 mice combined with LysM-Cre Fasfl/fl at a 1:1 ratio. For infection studies, mice were infected with LCMV docile or C.rodentium. For in vitro assays, neutrophils were primed for 1 h with GM-CSF prior to treatment with Toll-like receptor (TLR) ligands or IL-18 and FcFasL. Neutrophil viability was measured by live cell imaging and automated image analysis. Results Using LysM-Cre Fasfl/fl mice, which lack Fas expression in macrophages and neutrophils, we show that Fas regulates neutrophil lifespan in the colon during C.rodentium infection, and during LCMV infection in the lung, peripheral blood and spleen. To examine if pathogen-derived molecules can modulate Fas signaling in neutrophils, we primed neutrophils with TLR ligands, which ablated Caspase-8 processing and Fas signaling. Treatment of neutrophils with the TLR ligands Pam3CSK4, Pam2CSK4 or LPS or the MyD88-dependent cytokine IL-18 strongly protected neutrophils against Fas-induced death. Conclusion These data provide the first in vivo genetic evidence that neutrophil lifespan is controlled by death receptor signaling and provides a mechanism to account for neutrophil resistance to Fas stimulation during infection. Disclosures No relevant conflicts of interest to declare.

Antisense oligodeoxynucleotide (ODN) drugs might be more effective if their delivery was optimized and they were targeted to short-lived proteins encoded by messenger RNA (mRNA) species with equally short half-lives. To test this hypothesis, an ODN targeted to the c-mybproto-oncogene was developed and used to purge marrow autografts administered to allograft-ineligible chronic myelogenous leukemia patients. CD34+ marrow cells were purged with ODN for either 24 (n = 19) or 72 (n = 5) hours. After purging, Myb mRNA levels declined substantially in approximately 50% of patients. Analysis of bcr/abl expression in long-term culture-initiating cells suggested that purging had been accomplished at a primitive cell level in more than 50% of patients and was ODN dependent. Day-100 cytogenetics were evaluated in surviving patients who engrafted without infusion of unmanipulated “backup” marrow (n = 14). Whereas all patients were approximately 100% Philadelphia chromosome–positive (Ph+) before transplantation, 2 patients had complete cytogenetic remissions; 3 patients had fewer than 33% Ph+ metaphases; and 8 remained 100% Ph+. One patient's marrow yielded no metaphases, but fluorescent in situ hybridization evaluation approximately 18 months after transplantation revealed approximately 45% bcr/abl+ cells, suggesting that 6 of 14 patients had originally obtained a major cytogenetic response. Conclusions regarding clinical efficacy of ODN marrow purging cannot be drawn from this small pilot study. Nevertheless, these results lead to the speculation that enhanced delivery of ODN, targeted to critical proteins of short half-life, might lead to the development of more effective nucleic acid drugs and the enhanced clinical utility of these compounds in the future.

The partner gene of MLL was identified in a patient with treatment-related acute myeloid leukemia in which the karyotype suggested t(3;11)(q25;q23). Prior therapy included the DNA topoisomerase II inhibitors, teniposide and doxorubicin. Southern blot analysis indicated that the MLL gene was involved in the translocation. cDNA panhandle polymerase chain reaction (PCR) was used, which does not require partner gene-specific primers, to identify the chimeric transcript. Reverse-transcription of first-strand cDNAs with oligonucleotides containing known MLL sequence at the 5' ends and random hexamers at the 3' ends generated templates with an intra-strand loop for PCR. In-frame fusions of either MLL exon 7 or exon 8 with the GMPS (GUANOSINE 5'-MONOPHOSPHATE SYNTHETASE) gene from chromosome band 3q24 were detected. The fusion transcript was alternatively spliced. Guanosine monophosphate synthetase is essential for de novo purine synthesis. GMPS is the first partner gene of MLL on chromosome 3q and the first gene of this type in leukemia-associated translocations. (Blood. 2000;96:4360-4362)

We used a new approach called panhandle polymerase chain reaction (PCR) to clone an MLL genomic translocation breakpoint in a case of acute lymphoblastic leukemia of infancy in which karyotype analysis was technically unsuccessful and did not show the translocation partner. Panhandle PCR amplified known MLL sequence 5' of the breakpoint and 3' sequence from the unknown partner gene from a DNA template with an intrastrand loop schematically shaped like a pan with a handle. The 7-kb panhandle PCR product contained the translocation breakpoint in MLL intron 8. The partner DNA included unique nonrepetitive sequences, Alu and mammalian apparent LTR-retrotransposon (MaLR) repetitive sequences, and a region of homology to expressed sequence tags. MaLR sequences have not been found before near leukemia-associated translocation breakpoints. The nonrepetitive sequences were not homologous to known partner genes of MLL. Screening of somatic cell hybrid and radiation hybrid lines by PCR and fluorescence in situ hybridization analysis of normal metaphase chromosomes mapped the partner DNA to chromosome band 4q21. Reverse transcriptase-PCR identified an MLL-AF-4 chimeric mRNA, indicating that panhandle PCR identified a fusion of MLL with a previously uncharacterized AF-4 intronic sequence. Panhandle PCR facilitates cloning translocation breakpoints and identifying unknown partner genes.

Chronic lymphocytic leukemia (CLL) is the most common leukemia of the western world and is characterized by a slowly progressing accumulation of clonal CD5+ B cells. Our laboratory has investigated the role of transforming growth factor-beta (TGF-beta) and interleukin-4 (IL-4) in the pathogenesis of B-cell expansion in CLL. In vitro addition of TGF-beta did not increase spontaneous apoptosis of B cells from most CLL patients, as determined using the TUNEL method, compared with a twofold increase observed in cultures of normal B cells. There was similar expression of TGF-beta type II receptors on both CLL B cells and normal B cells. In contrast to apoptosis, CLL B-cell proliferation was variably inhibited with addition of TGF-beta. In vitro addition of IL-4, previously reported to promote CLL B-cell survival, dramatically reduced spontaneous apoptosis of CLL B cells compared with normal B cells. CLL B-cell expression of IL-4 receptors was increased compared to normal B cells. Thus, our results show aberrant apoptotic responses of CLL B cells to TGF-beta and IL-4, perhaps contributing to the relative expansion of the neoplastic clone.

Translocations involving chromosome 8 at band q24 and one of the Ig loci on chromosomes 14q32, 22q11, and 2p11 are the hallmark of Burkitt's lymphoma (BL). It has been previously observed that the exact localization of the breakpoints at chromosome 8q24 can vary significantly from patient to patient, scattering over a distance of more than 300 kb upstream of c-myc and about 300 kb downstream of c-myc. To generate probes for fluorescence in situ hybridization (FISH) that detect most c-myc translocations, we screened a yeast artificial chromosome (YAC) library from normal human lymphocytes by colony hybridization, using three markers surrounding the c-myc gene as probes. We obtained 10 YAC clones ranging in size between 500 and 200 kb. Two nonchimeric clones were used for FISH on several BL cell lines and patient samples with different breakpoints at 8q24. Our results show that the YAC clones detected translocations scattered along approximately 200 kb in both metaphase chromosomes and interphase nuclei. The sensitivity, rapidity, and feasibility in nondividing cells render FISH an important diagnostic tool. Furthermore, the use of large DNA fragments such as YACs greatly simplifies the detection of translocations with widely scattered breakpoints such as these seen in BL.

Abstract 1213 The inflammasome is an innate immune complex that recognizes a wide range of pathogen, cellular stress and damage signals. It triggers the Caspase-1-dependent production of inflammatory cytokines including IL-1β and IL-18. Sensors for the inflammasome include the nucleotide-binding oligomerisation domain, leucine-rich repeat (NLR) proteins. The best-characterised of these is NLRP3, which mediates antibacterial, viral, fungal and parasitic immune responses, and is mutated in a spectrum of autoinflammatory diseases. Activation of mammalian NLRs can also result in a Caspase-1-dependent form of cell death termed pyroptosis, the importance of which in disease states remains unclear. For example, it is known that NLRP3 activating mutations in humans can be effectively treated by neutralising IL-1β, suggesting that cell death induced by NLRP3 activation does not play a significant role in pathology. Similarly, the NLRP1b inflammasome is activated by anthrax lethal toxin to cause macrophage pyroptosis, but this does not play a role in anthrax sensitivity in vivo. Here we define a role for NLRP1 in autoinflammatory disease and the pyroptotic death of hematopoietic progenitor cells. We began by generating two mouse models, one carrying a point mutation in NLRP1a that results in constitutive activation of the protein, and another harbouring a deletion of the entire NLRP1 locus. Animals homozygous for the NRLP1a activating mutation developed a multi-organ neutrophilic inflammatory disease characterised by meningitis, hepatitis, pneumonitis, pancreatitis, pulmonary peri-arteritis, myocarditis and inflammatory bowel disease. Mean survival was approximately 3 months of age. Genetic crosses established that this inflammatory disease was driven by Caspase-1 and IL-1β, but was independent of ASC and Caspase-11, and ameliorated by IL-18. Surprisingly, in the absence of IL-1β-driven inflammation, constitutively active NLRP1a triggered the Caspase-1-dependent death of hematopoietic progenitor cells resulting in leukopenia at steady state. To evaluate the effect of activated NLRP1a during hematopoietic stress, IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation were challenged with 5-fluorouracil. Strikingly, these animals succumbed shortly after the nadir of leukopenia at 12 days post-injection. They exhibited hypoplastic bone marrow, lymphopenia, monocytopenia and a deficit of reticulocytes consistent with a functional deficiency in hematopoietic progenitor cells. Conversely, in mice lacking NLRP1, we observed improved recovery of the hematopoietic compartment following hemoablative chemotherapy, with increases in the numbers of platelets, lymphocytes and monocytes relative to littermate controls. Severe infections are commonly associated with a range of cytopenias including anemia, lymphopenia, neutropenia and thrombocytopenia, however, the etiological triggers for these conditions have not been elucidated. We therefore infected IL-1 receptor-deficient mice homozygous for the NRLP1a activating mutation with lymphocytic choriomeningitis virus (LCMV). Relative to controls, more severe cytopenia and bone marrow hypoplasia was observed. In contrast, NLRP1-deficient animals showed enhanced recovery from LCMV. Our results suggest that activation of the NLRP1 inflammasome in hematopoietic progenitors may contribute to cytopenias induced by hematopoietic stresses such as chemotherapy or infection. Disclosures: No relevant conflicts of interest to declare.

Chronic lymphocytic leukemia of B-cell origin (B-CLL) is generally thought to arise by neoplastic transformation of B lymphocytes, which express CD5 and have features of an early stage of B-cell differentiation. To study isotype-switched B-CLL as a potentially more differentiated variant, we performed genetic and functional immunoglobulin (Ig) gene analysis in two cases of CD5+ B-CLL in which the peripheral blood mononuclear cells (PBMC) secreted predominantly IgA (CLL-249) or IgG (CLL-412) when stimulated with pokeweed mitogen in vitro. By cDNA sequencing and by studies of CLL-heterohybridomas, CLL-249 expresses the heavy chain constant region C alpha as anticipated, while CLL-412 expresses C mu, not C gamma. In CLL-249, the expressed VH gene is 98% homologous to VH26, a germline VH3 gene that occurs frequently in the fetal repertoire, and which has been associated with anti-DNA specificity. The VL gene of CLL-249 is a lambda VL gene for which the germline sequence is not known. In CLL-412, the VH gene is 100% homologous to the VH1 gene of a published anti-DNA antibody (21/28), and is probably a germline gene sequence; the VL gene is 100% homologous to 15AVKI, also a germline gene. The supernatant antibody of the CLL-412 heterohybridoma is an IgM-kappa, which reacts with ssDNA and cardiolipin. The CLL-249 heterohybridoma secreted IgA-lambda, which bound none of the antigens tested, a finding that may be related to amino acid differences from the probable germline V genes. The demonstration of an in vivo isotype-switched variant, such as CLL-249, suggests that B-CLL may be a heterogeneous group of clonal disorders, of which less common variants may have features of more differentiated B-cell stages, such as isotype switching.

To investigate the molecular basis of the autoimmune response to the related i and I carbohydrate antigens, we studied cold agglutinins (CA) from B-cell clones and from the peripheral circulation of patients with lymphoproliferative syndromes. Sequence analyses of expressed variable region genes indicate that both anti-i and anti-I specificities from B- cell clones from two patients are encoded by the VH4.21 or a very closely related VH4 heavy chain gene, whereas the expressed light chain genes differed. The anti-i-secreting B-cells express unmutated germline- encoded VH4.21 and VKI gene sequences. The VH region gene encoding anti- I has the closest homology (97%) to the VH4.21 germline gene and differs at the protein level by only three amino acids. In contrast, while the VL region gene encoding anti-I is most homologous (96%) to the VKIII, kv328 germline gene, there are seven amino acid differences due to nonrandom replacement mutations, which suggests a role for antigen-mediated selection in the anti-I response of this individual. These studies were extended by a structural survey of 20 additional serum CA using antipeptide antibodies specific for determinants in VH and VL regions. All anti-I and anti-i CA were shown to express VH4 heavy chains, and 14 of 17 CA expressed a previously described VH4 second hypervariable region determinant, termed VH4-HV2a. We also found that 13 of 14 anti-I CA used VKIII light chains, while the anti-i CA used light chains from at least three VL families. Taken together, the data show that anti-i and anti-I CA probably both derive from the VH4.21 gene (or a closely related gene). Furthermore, the restricted VH and different VL gene use in anti-i and anti-I CA may reflect the close structural relationship of the i and I antigens.

Cells from 10 cases of childhood acute T-lymphoblastic leukemia (T-ALL) were cultured in the presence of recombinant human interleukins (rhIL) or colony-stimulating factors (CSF) to analyze their growth factor requirements and differentiative potential. Although cells from most leukemic samples displayed a short-term proliferative response to several hematopoietic growth factors, only the ones featuring chromosomal translocations could be established as permanent cell lines. Two cell lines could be initiated only in the presence of IL-3 (TALL-103 and TALL-106), one in granulocyte-macrophage CSF (GM-CSF) (TALL-101), and one in IL-2 (TALL-104); only one cell line (TALL-105) was originated in the absence of growth factors. The TALL-101 and TALL-103 cell lines, derived from very immature T-ALL cases, underwent growth factor-dependent phenotypic conversion (lymphoid to myeloid). However, the T-cell receptor rearrangement and karyotype of the original leukemic clones were retained. In contrast, the TALL-104, -105, and -106 cell lines which originated from more mature T-ALL cases, maintained a T-lymphoblastic phenotype regardless of the growth factors in which they were expanded. These data demonstrate in vitro the aggressive nature of T-ALL cases bearing chromosomal abnormalities, and indicate that the lineage commitment of the malignant clone depends on its stage of maturation in T-cell ontogeny.

Background: Imatinib has become standard front-line therapy for CML. In ~3% of patients treated with imatinib, abnormalities including trisomy 8 and/or monosomy 7 occur in Ph’ negative subclones and dysplasia occasionally is present, but transformation to AML is rare. We describe the first known case of AML with an MLL translocation during an imatinib-induced molecular remission of CML. Methods: The Ph’ and the t(11;19)(q23;p13.1) were detected and monitored in sequential marrows using cytogenetic and FISH analyses. The BCR-ABL1 fusion transcript was traced by RT-PCR. The MLL translocation was identified and characterized in the AML blasts by Southern blot analysis, panhandle PCR-based methods and conventional PCR. Clinical History and Results: The patient presented with chronic phase CML at age 53 and was treated with hydroxyurea for 6 weeks, IFN-α for 20 months and imatinib for 32 months. Complete cytogenetic response was achieved at 10 months. Molecular remission, as indicated by absence of the BCR-ABL1 transcript, occurred after 15 months of imatinib (37 months from CML diagnosis). Mild dysgranulopoiesis was noted 6 months after imatinib was started and increased on subsequent studies. After 28 months of imatinib (50 months from CML diagnosis), there was marked trilineage dysplasia and the karyotype showed t(11;19)(q23;p13.1) in cells that were Ph’ negative and negative for the BCR-ABL1 fusion transcript. FAB M5b AML was diagnosed four months later. The patient succumbed to AML despite aggressive management with chemotherapy and allogeneic stem cell transplantation. Southern blot analysis of the AML revealed two MLL bcr rearrangements. The reciprocal breakpoint junctions on the der (11) and der (19) chromosomes indicated a translocation of intron 8 of MLL and intron 1 of the known MLL partner gene ELL, which encodes a transcription elongation factor. The involved region of MLL was more 5′ than the secondary leukemia translocation breakpoint hotspot. The der (11) fusion transcripts joined MLL exon 7 to ELL exon 2, which is consistent with alternative splicing, and the der (19) fusion transcript joined ELL exon 1 to MLL exon 9. Conclusions: Secondary leukemias with MLL translocations have been associated with topoisomerase II poisons, but not with the agents administered to this patient. The entity that we describe is distinct from blast crisis, in which Ph’ positive subclones evolve to acquire additional alterations. This case establishes that persistent clonal abnormalities and/or dysplasia in Ph’ negative cells following imatinib therapy may not be benign and may herald AML transformation. With effective and selective molecular eradication of the Ph’ positive clone, the emergence of leukemia with independent abnormalities may become more common. This is a highly concerning clinical complication to consider with BCR-ABL targeted agents.

To the Editor: We read with great interest the recent reports on hepatosplenic γδ T-cell lymphoma (γδ HSTCL)[1][1] and T-cell posttransplant lymphoproliferative disorders (T-PTLD).[2][2] Since 1991 we have encountered 5 patients with γδ HSTCL: 2 occurred in chronic renal transplant patients (