ALBERT

Description: Abstract 2542 Background: Patients with high-risk chronic lymphocytic leukemia (CLL) uniformly relapse after conventional chemo-immunotherapy, but roughly half achieve long-term disease-free survival (DFS) following allogeneic hematopoietic cell transplantation (allo-HCT). Quantification of minimal residual disease (MRD) following allo-HCT predicts post-transplant relapse (PTR) when CLL disease burden remains greater than 10e-4 (ie, 1 leukemic cell in 10,000 peripheral blood mononuclear cells [PBMC]) when quantified by allele-specific oligonucleotide quantitative polymerase chain reaction (ASO-PCR) or flow cytometry. We previously demonstrated the feasibility of MRD quantification using consensus primers to amplify all immunoglobulin heavy chain (IGH) genes in a mixture of PBMC, followed by high-throughput sequencing (HTS) and clonotypic quantification. In our prior work, we used 454 pyrosequencing technology which enabled 10e-5 sensitivity. Here, we report 10e-6 MRD sensitivity using novel Illumina-based HTS that provides better prediction of disease recurrence than ASO-PCR. Methods: We amplified IGH loci from genomic DNA extracted from PBMC (median input 2.4×10e6 cells; range 1.1–23.7×10e6) using V and J segment consensus primers. Amplified IGH molecules were then sequenced with one million or more dedicated reads using Illumina HiSeq and clones were quantified using Sequenta HTS bioinformatics. To verify 10e-6 sensitivity using this system, a clonal B cell population was diluted to 10e-6 in PBMC from a healthy donor with successful clonal detection. Disease-bearing samples (either pre-treatment or after PTR) were sequenced to verify applicability of consensus primers for each patient and to determine each patient's unique clonal IGH sequence. Thirty-seven PBMC samples from 14 patients which were either negative (n=30) by ASO-PCR or detectable below the linear limit of detection (n=7) were subjected to MRD quantification by HTS. The integrity of all samples was determined by preliminary IGH quantitative PCR. Results: CLL-specific IGH clonotypes from all 14 patients amplified successfully from samples with known disease burden, confirming the acceptability of consensus primers for all patients. Concordant MRD negativity by ASO-PCR and IGH-HTS was only observed in 14/37 samples (38%), while 16 samples (43%) were negative by ASO-PCR but detectable at the 10e-6 level using IGH-HTS (range 0.1–11 CLL IGH sequences per 10e6 PBMC genomes). Two of 37 samples (5%) exhibited concordant low-level positivity in the 10e-5 range. 4/37 samples (11%) were concordantly positive, but quantified more than or equal to 0.5log higher with ASO-PCR than HTS. One sample was positive below the linear limit of detection by ASO-PCR but negative by HTS. With median clinical follow-up of 1072 days (range 522–1986 days), one of 7 patients (14%) who exhibited MRD negativity by both ASO-PCR and IGH-HTS relapsed. All 5 patients found to have MRD negativity by ASO-PCR with concurrent MRD positivity using IGH-HTS relapsed. The association between IGH-HTS negativity and long-term DFS was highly significant (p=0.005), whereas ASO-PCR negativity was not significantly associated with DFS (p=0.47). In patients found to be MRD negative by ASO-PCR but positive by IGH-HTS, the HTS result predicted clinical relapse by a median 321 days (range 38–644 days). Conclusions: Massively parallel immunoglobulin gene sequencing using Illumina HiSeq provides a heretofore unachievable level of MRD sensitivity in peripheral blood samples from patients with CLL. Samples found to be negative or below the linear limits of detection for CLL MRD using ASO-PCR were more accurately quantified using IGH-HTS. Quantification of CLL MRD using IGH-HTS has tremendous prognostic value since achievement of MRD negativity with 10e-6 sensitivity is highly associated with long-term DFS. To further validate the performance of Illumina-based HTS, we are currently sequencing 289 archived post-transplant PBMC from 42 CLL patients. This scalable and cost-effective platform for ultra-sensitive MRD quantification using consensus primers will broadly expand the availability and utility of post-transplant MRD assessment. Disclosures: Faham: Sequenta, Inc.: Employment, Equity Ownership. Carlton:Sequenta, Inc.: Employment, Equity Ownership. Zheng:Sequenta, Inc.: Employment, Equity Ownership. Moorhead:Sequenta, Inc.: Employment, Equity Ownership. Willis:Sequenta, Inc.: Employment, Equity Ownership.

Description: Abstract 315 Introduction: Dysregulated JAK-STAT signaling in chronic myeloproliferative neoplasms (MPNs) has primarily been attributed to activating mutations in tyrosine kinases. However, JAK-STAT activation can be demonstrated in some patients lacking JAK2 or MPL mutations, suggesting alteration of other regulatory elements in this pathway. One regulator of JAK-STAT signaling is LNK (SH2B3), an adapter protein that contains a proline-rich N-terminal dimerization domain (Pro/DD), a pleckstrin homology (PH) domain (plasma membrane localization), and an SH2 domain. LNK binds to cytokine receptors (e.g. MPL, EPOR) and JAK2 via its SH2 domain, inhibiting downstream STAT activation and providing critical negative feedback regulation. LNK-/- mice exhibit features consistent with an MPN phenotype. We recently reported the first human disease-related LNK mutations in two JAK2 V617F-negative MPN patients (Oh et al, Blood, Aug 12, 2010). One patient with primary myelofibrosis (PMF) exhibited a 5 base-pair (bp) deletion and missense mutation (DEL) leading to a premature stop codon and loss of the PH and SH2 domains. A second patient with essential thrombocythemia (ET) was found to have a missense mutation (E208Q) in the PH domain. Both mutations conferred aberrant JAK-STAT signaling in cell lines and primary patient samples, indicating that loss of LNK negative feedback regulation contributes to MPN pathogenesis. We now report the results of a comprehensive screen of a large cohort of MPN, overlap myelodysplastic syndrome (MDS)/MPN, and post-MDS/MPN acute myeloid leukemia (AML) patients for LNK mutations. Methods: A total of 341 samples were sequenced (Table 1; polycythemia vera (PV)=34, erythrocytosis=7, ET=61, PMF=75, post-PV/ET MF=25, MPN-U=7, chronic myelomonocytic leukemia (CMML)=71, juvenile myelomonocytic leukemia=20, MDS/MPN=8, MDS with fibrosis=2, refractory anemia with ring sideroblasts and thrombocytosis=4, idiopathic hypereosinophilic syndrome/chronic eosinophilic leukemia=4, systemic mastocytosis=4, and post MDS/MPN AML=19). A deep sequencing approach (Illumina multiplexing system) was used to evaluate 84 samples, in which all exons of LNK were sequenced. For the remainder of the samples, direct sequencing was performed on exon 2, the region containing the previously reported DEL and E208Q mutations. Results: After excluding variants previously reported in SNP databases, a total of 11/341 (3.2%) patients were found to have non-synonymous mutations, including 3/61 (4.9%) ET, 3/75 (4.0%) PMF, and 5/71 (7.0%) CMML patients (Table 1). Each of the mutations localized to exon 2 of LNK, implicating this region as a possible mutational hotspot. This included the aforementioned patients with the DEL and E208Q mutations, which were confirmed by deep sequencing. In two other patients, sequencing of DNA from cultured skin fibroblasts DNA indicated that the mutations were germline. For the remaining seven patients, germline analysis is currently ongoing. In one patient with CMML, a 1 bp deletion leading to a frameshift and premature stop codon was identified (Q72fs). This mutation localized to the Pro/DD, likely resulting in a complete loss of LNK function. Interestingly, this patient who is wild type for the JAK2 and RAS genes, also carries a heterozygous CBL mutation (C396Y), suggesting that LNK and CBL mutations may have cooperative effects. Four patients (one with PMF, three with CMML) were found to have a missense mutation (S186I) at a highly conserved residue in the Pro/DD. The previously reported E208Q mutation was also found in one patient with ET and one patient with CMML. None of the 81 patients known to be JAK2 V617F-positive exhibited somatic LNK mutations, suggesting that LNK mutations may provide an alternative basis for JAK-STAT activation in the absence of JAK2 V617F. Conclusion: Missense and deletion mutations of the LNK gene occur at a low frequency in MPNs and MDS/MPNs and segregate predominantly in exon 2. Further analysis of post-MPN AML samples (represented at a low frequency in the current cohort) and other subtypes of acute and chronic myeloid malignancies is warranted to better characterize the disease spectrum of LNK mutations and whether they are mutually exclusive of JAK2 V617F. We are currently investigating whether loss of negative feedback regulation of JAK-STAT signaling is related to haploinsufficiency of LNK or dominant negative effects of the mutant protein. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 3086 Background: Hereditary thrombocythemia (HT) is a rare autosomal dominant disorder related to mutations in either the gene encoding thrombopoietin (THPO), or its receptor MPL. In some cases of HT, mutational involvement of the THPO or MPL genes has been excluded. To date, five HT families with three distinct THPO mutations have been published, including Dutch, Japanese, and Polish pedigrees. In all cases, the mechanism of overproduction of platelets is related to alteration of the 5′ untranslated region (5′ UTR) of the THPO gene which results in enhanced translation of thrombopoietin (TPO) mRNA. We present our analysis of a Philippine family with a novel THPO gene mutation which segregates with the thrombocytosis phenotype. Methods: Informed consent was obtained from research participants. Peripheral blood was obtained from 6 family members, and serum was collected from 10 healthy adults. DNA was extracted with the Puregene DNA Isolation Kit (Qiagen). For the human TPO ELISA assay, the serum TPO concentrations of the family members as well as the healthy controls were measured with the human TPO Quantikine kit (R&D Systems). The exon 2 to intron 3 region of THPO gene covering the 3 previously reported mutations was amplified and purified PCR products were sequenced. To examine the functional effects of increased TPO levels on downstream JAK-STAT signaling, freshly obtained PB samples were incubated for 15 minutes at 37C, followed by fixation with paraformaldehyde and lysis of red blood cells. Fixed cells were permeabilized with methanol and stained with a combination of surface marker and intracellular phospho-specific antibodies, followed by analysis on a LSRII flow cytometer. Data analysis was performed with FlowJo (Tree Star). Results: The proband (see Figure 1a, pedigree subject II-1, arrow) is a 39 year-old Philippine woman who was informed at age 19 that she had an elevated platelet count. Although placed on hydroxyurea, anagrelide, and interferon-α in the distant past, she was currently maintained on only low-dose aspirin. At the time of her presentation at Stanford Cancer Center in March 2010, her complete blood count revealed a platelet count of 1,015,000/mm3 without other abnormalities. The peripheral blood smear revealed occasional large, hypogranular platelets. Reactive causes of thrombocytosis were excluded and her examination was normal. JAK2 V617F mutation analysis was negative. Routine CBC evaluation of her two children revealed thrombocytosis in her 11 year-old daughter (subject III-1; platelet count 550,000/mm3) and 9-year old son (subject III-2; platelet count 759,000/mm3). None of the three family members with thrombocytosis experienced a history arterial or venous thrombosis nor vasomotor symptoms. As shown in Figure 1b, DNA sequencing revealed a novel heterozygous mutation of T〉C at the splice donor site of intron 2 in the proband (II-1) and her two children (III-1, III-2), while the proband's parents (I-1, I-2) and husband (II-2)were wild type at this site. In Figure 1a, the pedigree of the six family members with serum TPO concentrations (pg/mL) (1st row) and platelet counts (/mm3)(2nd row) is shown. Figure 1c demonstrates that the serum TPO concentration of family members with the THPO mutation was significantly higher than those family members without the mutation (un-paired t-test, p=0.0378), or healthy controls (p=0.0003). The TPO level in non-affected family members showed no statistical difference with healthy controls (p=0.98). In Figure 1d, basal phosphorylated STAT5 (pSTAT5) levels in CD3−/CD66−/CD14− progenitors were evaluated, and the 95th percentile is presented in the mean with SEM format. An un-paired t-test revealed that pSTAT5 levels were significantly higher in the affected group (p = 0.024, denoted by *). Conclusion: In our Philippine family, we have identified a novel heterozygous mutation at the conserved T in the invariant donor splice site of intron 2 of the THPO gene. Mutations at this position result in splicing errors, leading to exon skipping, intron retention or exposure of cryptic splice sites. Loss of the inhibitory wild-type 5′UTR has been shown to result in enhanced THPO transcription. This autosomal dominant gain of function germline mutation is associated with thrombocytosis, increased TPO levels, and constitutive activation of JAK-STAT signaling. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 2863 Background: JAK2 V617F is a recurrent, activating mutation in patients (pts) with BCR-ABL1-negative MPNs. Mutations in codon 515 of MPL occur in 1–5% and 5–10% of ET and PMF pts, respectively, and similar to JAK2 V617F, lead to constitutive JAK-STAT signaling. The acquisition of multiple mutational events affecting the JAK-STAT axis or components of the epigenetic machinery is common in MPNs and likely contributes to phenotypic diversity, including progression to acute myeloid leukemia. Mutated genes thus far implicated in MPN initiation and/or progression include TET2, CBL, SH2B3, ASXL1, DNMT3A, IDH1/2, IKZF1, EZH2, SRSF2, and TP53. In order to identify novel somatic mutations associated with classic BCR-ABL1-negative MPNs, we performed whole genome sequencing of DNA extracted from peripheral blood granulocytes and cultured skin fibroblasts from a patient with PMF and a known MPL W515K mutation. Methods: Whole genome sequencing (WGS) was undertaken in a 55 year-old man with untreated PMF four years after initial diagnosis. His DIPSS Plus risk group was low (score 0). His karyotype was normal, and molecular testing revealed wild-type JAK2 in addition to the MPL W515K mutation. WGS of purified granulocytes and paired cultured skin fibroblasts was performed using both Illumina HiSeq and Complete Genomics (CGI) platforms. The resulting data were analyzed using multiple independent aligners and variant callers. Amplicon-based targeted resequencing with the Illumina MiSeq platform was used to evaluate additional patient samples for recurrent mutations. Stanford institutional review board approval and informed pt consent was obtained for these analyses. Results: The PMF genome was sequenced to 88X (Illumina) and 128X (CGI) average fold coverage, and the cultured skin fibroblast genome was sequenced to 47X (Illumina) and 126X (CGI). The PMF genome had a low somatic mutation rate, consistent with that observed for other sequenced hematopoietic tumor genomes, with a low number of true somatic mutation calls. To definitively identify true mutations among various sequencing artefacts and germline variants, we use cultured skin fibroblasts which can be prepared with no contamination by neoplastic cells. In addition to re-identification of the MPL W515K mutation, this approach identified six additional somatic mutations that alter gene coding regions, splice sites, or known regulatory regions: a nonsense mutation in CARD6, implicated in modulation of NF-kappaB activation; a splice-site mutation in CAP2; three nonsynonymous point mutations in KIAA0355, SOX30, MFRP; and a 19-base pair (bp) deletion involving a regulatory region in the 5'-untranslated region (5'-UTR) of BRD2, a bromodomain-containing protein implicated in transcriptional regulation (Table). CARD6, BRD2, and KIAA0355, an uncharacterized protein, are expressed by the granulocytes derived from this patient, supporting a potential role in the development of PMF in this pt. Using massively parallel sequencing, we are currently examining the transcribed region of BRD2 and the coding region of the other five genes in additional pt samples. Analysis of the first 87 samples (MF=47, PV=20, ET=20) out of a cohort of 180 MPN pts has thus far not identified recurrent somatic mutations in these six genes. Conclusion: High-coverage genome sequencing of neoplastic and germline cells from a patient with MPL-mutated PMF identified six additional somatic mutations of potential functional significance. Work is ongoing to determine if somatic mutations in these genes are found in other patients with BCR-ABL1-negative MPNs, and their pathogenetic relevance to MPN biology. Disclosures: Snyder: Illumina: Consultancy; GenapSys: Membership on an entity's Board of Directors or advisory committees; Personalis: Consultancy, Founder Other.

Description: Background: PTLDs are typically B-cell neoplasms occurring as uncommon but serious complications of reduced T-cell immune surveillance associated with organ transplantation. RIS benefits only a subset of PTLD patients and cytotoxic therapy may be poorly tolerated. Therefore, in October 1998 we initiated a prospective study of rituximab in patients who failed or were unable to receive RIS and now report mature results. Methods: Patients with CD20+ PTLD were eligible if they had failed to completely respond to RIS or RIS was contraindicated and had Karnofsky performance status 〉60, age 3–70 years (y), measurable disease, and no change in immunosuppression for at least 2 weeks and no cytotoxic therapy within 4 weeks. Rituximab was given as 375 mg/m2 weekly x 4 with disease evaluation at 1, 3, 6, 9, 12 and 18 months. Response data, survival curves, and the impact of clinical and pathological factors were evaluated. Results: 24 of 26 enrolled pt were eligible and evaluable. Median age was 42y with 5

Description: Diffuse large B-cell lymphoma (DLBCL) is characterized by a marked degree of morphologic and clinical heterogeneity. Establishment of parameters that can predict outcome could help to identify patients who may benefit from risk-adjusted therapies. BCL-6 is a proto-oncogene commonly implicated in DLBCL pathogenesis. A real-time reverse transcription–polymerase chain reaction assay was established for accurate and reproducible determination of BCL-6 mRNA expression. The method was applied to evaluate the prognostic significance ofBCL-6 expression in DLBCL. BCL-6 mRNA expression was assessed in tumor specimens obtained at the time of diagnosis from 22 patients with primary DLBCL. All patients were subsequently treated with anthracycline-based chemotherapy regimens. These patients could be divided into 2 DLBCL subgroups, one with high BCL-6 gene expression whose median overall survival (OS) time was 171 months and the other with low BCL-6 gene expression whose median OS was 24 months (P = .007). BCL-6 gene expression also predicted OS in an independent validation set of 39 patients with primary DLBCL (P = .01). BCL-6 protein expression, assessed by immunohistochemistry, also predicted longer OS in patients with DLBCL. BCL-6 gene expression was an independent survival predicting factor in multivariate analysis together with the elements of the International Prognostic Index (IPI) (P = .038). By contrast, the aggregate IPI score did not add further prognostic information to the patients' stratification byBCL-6 gene expression. High BCL-6 mRNA expression should be considered a new favorable prognostic factor in DLBCL and should be used in the stratification and the design of risk-adjusted therapies for patients with DLBCL.

Description: B cells are involved in the pathogenesis of chronic GVHD (cGVHD). We hypothesized that prophylactic anti–B-cell therapy delivered 2 months after transplantation would decrease allogeneic donor B-cell immunity and possibly the incidence of cGVHD. Therefore, in the present study, patients with high-risk chronic lymphocytic leukemia (n = 22) and mantle-cell lymphoma (n = 13) received a total lymphoid irradiation of 80 cGy for 10 days and antithymocyte globulin 1.5 mg/kg/d for 5 days. Rituximab (375 mg/m2) was infused weekly on days 56, 63, 70, and 77 after transplantation. The incidence of acute GVHD was 6%. The cumulative incidence of cGVHD was 20%. Nonrelapse mortality was 3%. Rituximab treatment after allogeneic transplantation significantly reduced B-cell allogeneic immunity, with complete prevention of alloreactive H-Y Ab development in male patients with female donors (P = .01). Overall survival and freedom from progression at 4 years for chronic lymphocytic leukemia patients were 73% and 47%, respectively; for mantle-cell lymphoma patients, they were 69% and 53%, respectively. This study is registered at www.clinicaltrials.gov as NCT00186628.

Description: The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband’s FV activity ranges from 2–14%. Two siblings are unaffected. His father’s FV activity is 50% and his mother’s is 70%. We performed DNA sequencing spanning the entire coding region of the proband’s FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient’s FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.