HFE Gene Mutation Status Predicts Response to Gemtuzumab Ozogamicin in AML

Abstract BACKROUND: The protein defective in type 1 hereditary hemochromatosis, called HFE, is similar to MHC class I-type proteins, associates with beta2-microglobulin and is implicated in membranous protein recycling. Gemtuzumab ozogamicin (GO), a monoclonal antibody directed against CD33 linked to a cytotoxic agent, has been used with controversial effects in acute myeloid leukemia (AML). Internalization of GO is required for its anti-leukemic effect. Therefore, we hypothesized that H63D or C282Y HFE gene mutations may impair GO activity by preventing its internalization. METHODS: Wild type, C282Y and H63D HFE leukemic cells and primary cell were used to assess effects of GO alone or in combination with cytarabine on cell proliferation and apoptosis. Flow cytometry, confocal and AMNIS stream analysis were used to evaluate GO internalization. HFE mutations were analyzed by PCR analysis on DNA from patients included in clinical studies. Post-hoc subgroup analysis was performed to assess in vivo the role of HFE status on GO efficacy and toxicity among the 280 patients of the ALFA-0701 study as a study cohort (patients aged 50-70 years; GO 3mg/m² on days 1, 4, and 7 of chemotherapy and on day 1 of the first and second induction ; total dose 15 mg/m² ) and then on the GOELAMS-LAM 2006 IR study (patients aged 18-60 years; GO 6 mg/m² on day 4 of chemotherapy during the induction and the first consolidation; total dose 12 mg/m²) and UK NCRI AML17 study (patients aged 18-81 years; GO 3 vs 6 mg/m² on day 1 of chemotherapy but not during consolidation) as validating cohorts. RESULTS: GO induced cell death by apoptosis in AML cell lines and primary cells in a dose-dependent manner and synergistically in combination with cytarabine. However, the IC5O of GO was significantly higher in HFE mutated cells (125 vs 10 ng/mL p<0.001). To further understand this phenomenon, the CD33 internalization was analyzed upon GO or anti-CD33 antibody exposure. In line with the cytotoxic effect, CD33 was significantly less internalized in HFE mutant cells (17.4% vs 65.1% after 1 hour, p<0.01). HFE mutations were screened in 242 of the 280 ALFA-0701 patients with DNA available. There were 155 non-mutated patients (64%), 68 (28%) heterozygous for H63D, 11 (5%) heterozygous for C282Y, and 8 (3%) homozygous for H63D, which is consistent with the prevalence of the various mutations in the French population. Median age was 62 years (50-71) and the M/F ratio was 0.5, equally distributed among the different groups. No significant difference was observed with respect to the diagnosis of various hematological parameters, including white blood cell count, blasts number, cytogenetic subgroups, molecular mutation incidences (FLT3, NPM1, CEBPA, IDH, DNMT3A). In the ALFA-0701 study cohort, HFE wild-type (WT) patients had a higher overall survival (OS) when treated in the GO arm (median, not reached vs 19.5 months, p=0.0193). In contrast, OS was similar among patients with heterozygotes HFE mutations treated in the GO and the control arm (median, 19.9 vs 21.9, p=0.9675). In confirmatory cohorts, GO treatment led to a trend to increased OS in the GOELAMS-LAM 2006 IR cohort only in HFE WT patients (4-year OS with GO 62% vs 48%, p=0.08) but not in mutated patients (4-year OS with GO 73% vs 64%, p=0.45). Furthermore, in this cohort in the FLT3 WT patients subgroup, GO further improve OS in WT patients (4-year OS with GO 72% vs 50%, p=0.017), but not in patients with heterozygous HFE mutation (4-year OS with GO 80% vs 65%, p=0.23) In the UK NCRI AML17 cohort, which used GO only during induction, 245 patients were randomized between GO at 3mg/m2 and 6mg/m2 and evaluated for HFE status. Overall there was no effect of GO dose on outcomes, and no evidence of either any heterogeneity by HFE, nor any subgroup, which showed a differential effect of GO dose. CONCLUSIONS: Future studies should focus on optimising the fractionated schedule for GO at the 3mg/m2. Fractionated high doses (3x3mg/m2) during induction and single dose during consolidation seems to be the best schedule. Most importantly, the effect of GO treatment differed between HFE WT and heterozygote mutated AML patients. GO only increased OS among HFE WT patients. This is likely related to impaired internalization of the CD33 target. Our data suggest that HFE status should be used as a companion test to predict outcome of AML treated with GO. Disclosures No relevant conflicts of interest to declare.

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Relationship between gene expression of duodenal iron transporters and iron stores in hemochromatosis subjects

AJP Gastrointestinal and Liver Physiology ◽

10.1152/ajpgi.00175.2009 ◽

2010 ◽

Vol 298 (1) ◽

pp. G57-G62 ◽

Cited By ~ 10

Author(s):

James E. Nelson ◽

Virginia R. Mugford ◽

Ellen Kilcourse ◽

Richard S. Wang ◽

Kris V. Kowdley

Keyword(s):

Gene Expression ◽

Iron Overload ◽

Iron Transport ◽

Iron Absorption ◽

Hereditary Hemochromatosis ◽

Phenotypic Expression ◽

Wild Type ◽

Significant Difference ◽

Hfe Mutations ◽

Relative Gene

To test the hypothesis that differences in duodenal iron absorption may explain the variable phenotypic expression among HFE C282Y homozygotes, we have compared relative gene expression of duodenal iron transporters among C282Y homozygotes [hereditary hemochromatosis (HH)] with and without iron overload. Duodenal biopsy samples were analyzed using real-time PCR for expression of DMT1, FPN1, DCYTB, and HEPH relative to GAPDH from 23 C282Y homozygotes, including 5 “nonexpressors” (serum ferritin < upper limit of normal and absence of phenotypic features of hemochromatosis) and 18 “expressors.” Four subjects of wild type for HFE mutations without iron overload or liver disease served as controls. There was a significant difference in expression of DMT1 ( P = 0.03) and DMT1(IRE) ( P = 0.0013) but not FPN1, DCYTB, or HEPH between groups. Expression of DMT1(IRE) was increased among HH subjects after phlebotomy compared with untreated ( P = 0.006) and nonexpressor groups ( P = 0.026). A positive relationship was observed among all HH subjects regardless of phenotype or treatment status between relative expression of FPN1 and DMT1 ( r = 0.5854, P = 0.0021), FPN1, and DCYTB ( r = 0.5554, P = 0.0040), FPN1 and HEPH ( r = 0.5100, P = 0.0092), and DCYTB and HEPH ( r = 0.5400, P = 0.0053). In summary, phlebotomy is associated with upregulation of DMT1(IRE) expression in HH subjects. HFE C282Y homozygotes without phenotypic expression do not have significantly decreased duodenal gene expression of iron transport genes compared with HH subjects with iron overload. There is coordinated regulation between duodenal expression of FPN1 and DMT1, FPN1 and DCYTB, and FPN1 and HEPH and also DCYTB and HEPH in HH subjects regardless of phenotype.

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Precipitating factors of porphyria cutanea tarda in Brazil with emphasis on hemochromatosis gene (HFE) mutations. Study of 60 patients

Anais Brasileiros de Dermatologia ◽

10.1590/abd1806-4841.20132048 ◽

2013 ◽

Vol 88 (4) ◽

pp. 530-540 ◽

Cited By ~ 6

Author(s):

Fatima Mendonca Jorge Vieira ◽

Maria Cristina Nakhle ◽

Clarice Pires Abrantes-Lemos ◽

Eduardo Luiz Rachid Cancado ◽

Vitor Manoel Silva dos Reis

Keyword(s):

Hepatitis C ◽

Alcohol Abuse ◽

Porphyria Cutanea Tarda ◽

Hereditary Hemochromatosis ◽

Control Group ◽

Hfe Gene ◽

Precipitating Factors ◽

Serological Tests ◽

Hfe Mutations ◽

Precipitating Factor

BACKGROUND: Porphyria cutanea tarda is the most common form of porphyria, characterized by the decreased activity of the uroporphyrinogen decarboxylase enzyme. Several reports associated HFE gene mutations of hereditary hemochromatosis with porphyria cutanea tarda worldwide, although up to date only one study has been conducted in Brazil. OBJECTIVES: Investigation of porphyria cutanea tarda association with C282Y and H63D mutations in the HFE gene. Identification of precipitating factors (hepatitis C, HIV, alcoholism and estrogen) and their link with HFE mutations. METHODS: An ambispective study of 60 patients with PCT was conducted during the period from 2003 to 2012. Serological tests for hepatitis C and HIV were performed and histories of alcohol abuse and estrogen intake were investigated. HFE mutations were identified with real-time PCR. RESULTS: Porphyria cutanea tarda predominated in males and alcohol abuse was the main precipitating factor. Estrogen intake was the sole precipitating factor present in 25% of female patients. Hepatitis C was present in 41.7%. All HIV-positive patients (15.3%) had a history of alcohol abuse. Allele frequency for HFE mutations, i.e., C282Y (p = 0.0001) and H63D (p = 0.0004), were significantly higher in porphyria cutanea tarda patients, compared to control group. HFE mutations had no association with the other precipitating factors. CONCLUSIONS: Alcohol abuse, hepatitis C and estrogen intake are prevalent precipitating factors in our porphyria cutanea tarda population; however, hemochromatosis in itself can also contribute to the outbreak of porphyria cutanea tarda, which makes the research for HFE mutations necessary in these patients

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Tyrosine Phosphorylation Enhances ITIM-Dependent Internalization of CD33, the Target for the Anti-AML Immunoconjugate, Gemtuzumab Ozogamicin.

Blood ◽

10.1182/blood.v108.11.2581.2581 ◽

2006 ◽

Vol 108 (11) ◽

pp. 2581-2581

Author(s):

Roland B. Walter ◽

Brian W. Raden ◽

Irwin D. Bernstein ◽

Jonathan A. Cooper

Keyword(s):

Cell Surface ◽

Tyrosine Kinase ◽

Gene Silencing ◽

Tyrosine Phosphorylation ◽

Cell Lines ◽

Myeloid Cell ◽

Gemtuzumab Ozogamicin ◽

Dependent Manner ◽

Wild Type ◽

Phosphorylation State

Abstract Background: CD33, the target for the anti-AML immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). We have previously shown that these motifs control uptake of antibody-bound CD33 and GO-induced cytotoxicity. In this study, we determined which phosphorylation state favors uptake of antibody-bound CD33, identified proteins that bind to CD33 in an ITIM-dependent manner, and assessed their importance for CD33 internalization by siRNA-based gene silencing. Methods: Internalization of anti-CD33 antibodies was measured by flow cytometry in the presence or absence of the tyrosine phosphatase inhibitor, pervanadate, in human CD33+ AML cell lines (ML-1, HL-60, NB4, U937, TF-1) and CD33− Jurkat T cells infected with wild-type and mutant CD33. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33, using human myeloid cell lysates. Co-immunoprecipitations were performed with myeloid cell lines expressing HA-tagged wild-type CD33. Lentivirus-based siRNA constructs were generated for gene silencing, and expressed in human CD33+ AML cell lines. Results: Pervanadate significantly increased uptake of anti-CD33 antibodies in human AML cell lines; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2, suggesting that Src family kinase-dependent phosphorylation of the ITIMs critically controls uptake of antibody-bound CD33, possibly by altering which proteins binds to CD33 or by facilitating binding of adaptor-proteins required for endocytosis. We identified several proteins, including the tyrosine phophatases, SHP-1 and SHP-2, and the non-receptor tyrosine kinase, Syk, which bound to phosphorylated wild-type and mutant CD33 in a manner that paralleled the endocytic properties of the corresponding CD33 protein. Since these three proteins have been implicated in endocytic processes of other cell surface proteins, we assessed their role in uptake of antibody-bound CD33 by siRNA-mediated gene silencing. Simultaneous depletion of SHP-1 and SHP-2, but not SHP-1 or SHP-2 alone, significantly increased internalization of antibody-bound CD33 in the two AML cell lines with the highest cell surface expression of CD33, whereas no effect was seen in two other cell lines with lower CD33 expression levels. In contrast, depletion of Syk, whose expression has previously been correlated to the inhibitory effect of anti-CD33 antibodies on AML cell growth, failed to affect antibody internalization in the cell lines assessed. Conclusion: These studies indicate that the phosphorylation status of the ITIMs controls uptake of antibody-bound CD33. In line with this model, SHP-1 and SHP-2, which have been shown to dephosphorylate CD33 in vitro, can affect this endocytic process. Thus, our data imply manipulation of the phosphorylation state of CD33, e.g. by activating Src family kinases or interfering with phosphatases as a novel means to increase uptake of anti-CD33 antibody-based therapeutics such as GO. Finally, the variable effect of SHP-1 and SHP-2 depletion suggests that there are significant cell-type specific differences in the response to anti-CD33 antibody ligation, for example differences in tyrosine phosphorylation levels and/or activation/recruitment or redundancies of tyrosine phosphatases.

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Phenobarbital for Long-Term Management of Marked Hyperbilirubinemia.

Blood ◽

10.1182/blood.v114.22.5089.5089 ◽

2009 ◽

Vol 114 (22) ◽

pp. 5089-5089

Author(s):

A. Majid Shojania

Keyword(s):

Hemolytic Anemia ◽

Skin Color ◽

Conflicts Of Interest ◽

Hereditary Hemochromatosis ◽

High Serum ◽

Hfe Gene ◽

Therapeutic Trial ◽

Uridine Diphosphate ◽

Gilbert’S Syndrome ◽

Gilbert's Syndrome

Abstract 5089 Gilbert's syndrome (GS) is associated with a mild chronic unconjugated hyperbilirubinemia, due to partial deficiency of bilirubin uridine diphosphate glucuronyl transferase (UDPGT). Phenobarbital is a known inducer of hepatic UDPGT and has been used in hyperbilirubinemia of newborns. It has also been used as a test for support of the diagnosis of GS. However, because hyperbilirubinemia of GS is mild and harmless, phenobarbital is not used for treatment of hyperbilirubinemia in adults. I report a case of a 46-year-old woman who, because of having chronic hereditary hemolytic anemia and GS, had marked hyperbilirubinemia with psychosocial problems, as the result of her hyperbilirubinemia and her skin color, which responded well to chronic phenobarbital treatment. Case report- CH was diagnosed to have hereditary high phosphatidylcholine hemolytic anemia (HHPCHA) at the age of 23. She was seen again at the age of 30 because of very high serum ferritin and iron saturation which seemed disproportionally high for the degree of her mild hemolytic anemia (51Cr RBC survival T½ of 16.5 days). Further investigation revealed that she had hereditary hemochromatosis due to homozygosity for H63D HFE gene. She was started on phlebotomies initially weekly and later on every 2-3 months to control her iron overload. During the follow-up it was noted that her serum unconjugated bilirubin (SUB) was persistently much higher than is expected from her mild hemolytic anemia (up to 288 μmol/L). Since she had no abnormality of liver function tests, I suspected that she also has Gilbert's syndrome. In September 2008 her blood was sent for genetic testing which showed that she has an additional TA repeat [(TA)7/(TA)7], confirming the diagnosis of GS. On January 21, 2009 when her SUB was 149 μmol/L, she expressed concern that her friends and coworkers keep making fun of her, because of the orange color of her face and sclera. She was started on phenobarbital 30 mg daily for a month and then 60 mg daily. This therapy rapidly brought her bilirubin down and changed the color of her face to normal, making her very happy. Her SUB on February 20, March 20 and June 30, 2009 were 103, 63 and 37 μmol/L, respectively. Conclusion Gilbert's syndrome is a common hereditary disorder that can aggravate hyperbilirubinemia of chronic hemolytic anemia. However, this association is often unrecognized, because many physicians attribute the hyperbilirubinemia to hemolysis and do not look for associated GS. In chronic hemolytic anemias, if hyperbilirubinemia is more than expected, the possibility of an associated GS should be considered. If such association exists, small daily doses of phenobarbital can markedly reduce this hyperbilirubinemia and improve the psychosocial effects of hyperbilirubinemia. Furthermore, marked reduction of bilirubin, following the therapeutic trial of Phenobarbital, will confirm the association of GS with hemolytic anemia. Disclosures No relevant conflicts of interest to declare.

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Glutaminase Inhibition Selectively Slows the Growth of Primary Acute Myeloid Leukemia (AML) Cells with Isocitrate Dehydrogenase (IDH) Mutations.

Blood ◽

10.1182/blood.v120.21.2624.2624 ◽

2012 ◽

Vol 120 (21) ◽

pp. 2624-2624

Author(s):

Ashkan Emadi ◽

Sung Ah Jun ◽

Takashi Tsukamoto ◽

Amir T Fathi ◽

Mark D. Minden ◽

...

Keyword(s):

Small Molecule ◽

De Novo ◽

Conflicts Of Interest ◽

Myeloproliferative Neoplasms ◽

Growth Curves ◽

Primary Source ◽

Wild Type ◽

Research Groups ◽

Idh Mutations ◽

Significant Difference

Abstract Abstract 2624 Introduction: The incidence of mutations in IDH1 and IDH2 (mIDH) in de novo AML is 10–15%. These mutations are enriched in normal karyotype AML, and their presence carries an unfavorable prognostic factor, according to some studies. Furthermore, mutations in IDH1/2 genes have been identified in approximately 5% of myelodysplastic syndromes and 10% of myeloproliferative neoplasms. Although wild-type IDH in cytosol and mitochondria catalyze the conversion of isocitrate to α-ketoglutarate (α-KG) with the production of NADPH, altered amino acids in mIDH1 and mIDH2 reside in the catalytic pocket and result in a neoenzymatic activity, converting α-KG to 2-hydroxyglutarate with the consumption of NADPH. The primary source for α-KG for these cells is glutamine, which is first converted to glutamate by glutaminase and subsequently to α-KG. Because glutamine is the primary source for α-KG, we hypothesized that cells with mIDH are in essence addicted to glutamine via glutaminase activity, such that depletion of glutamine or interruption of its metabolism would be deleterious to cellular metabolism and survival. The aim of this study was to investigate whether inhibition of glutaminase by a small molecule, BPTES (bis-2-[5-(phenylacetamido)-1,3,4-thiadiazol-2-yl]ethyl sulfide), could selectively kill primary AML cells with mIDH1, but not IDH-wild type AML cells. We and others have previously demonstrated that BPTES inhibits glutaminase effectively. Method: Two independent sets of experiments were performed by two separate research groups. One group was blinded to mutant versus wild type IDH status. The other group was blinded to drug identity including solvent versus BPTES and to various BPTES concentrations. Primary AML cells from patients were cultured in RPMI-1640 medium with 20% fetal bovine serum, 20% 5637 cell-conditioned medium and 1% antibiotics with no drug, DMSO control (0.1% concentration) and 20 or 40 microM BPTES. Cells were counted manually on days 2, 4 and 6. Growth curves were generated for viable cells as assessed by trypan blue exclusion. Experiments were performed in triplicates. Results: Growth curves of primary AML cells (with mutation status indicated) with no drug and with DMSO or BPTES (20 or 40 microM) are shown in Figure 1. Cells #2, #3, #5 and #10 carried IDH1 mutations. Cells #4 and #9 were wild type. On day 4, there was approximately a two-fold decrease in the growth of all IDH-mutant AML cells exposed to 20 microM BPTES compared to DMSO. No significant difference in activity was observed between 20 and 40 microM of BPTES. There was no difference in cell growth between exposure to no drug and to DMSO. The growth of wild type AML cells was not significantly affected by the glutaminase inhibitor. Results were consistent between the two research groups. Conclusions: Although IDH mutations are frequently found in AML, a therapeutic strategy targeted at these mutations has not been reported. To the best of our knowledge, this is the first report of a targeted approach to the treatment of IDH-mutant AML. We found that inhibition of glutaminase by a small molecule, BPTES, preferentially slows the growth of primary AML cells with mutant IDH1 versus those AML cells with wild type IDH. Further investigation in xenograft models and pharmacologic studies are ongoing. Disclosures: No relevant conflicts of interest to declare.

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Radiosensitivity of Fancd2-/- mouse Bone Marrow Stromal Cells Is Altered By Abrogation of TGF-β Signaling

Blood ◽

10.1182/blood.v126.23.4796.4796 ◽

2015 ◽

Vol 126 (23) ◽

pp. 4796-4796

Author(s):

Katherine Chen ◽

Darcy Franicola ◽

Donna Shields ◽

Michael W. Epperly ◽

Xichen Zhang ◽

...

Keyword(s):

Bone Marrow ◽

Cell Line ◽

Cell Lines ◽

Conflicts Of Interest ◽

Survival Curve ◽

Radiation Sensitivity ◽

Mouse Bone Marrow ◽

Wild Type ◽

Increased Risk ◽

Significant Difference

Abstract Both marrow-transplanted and non-transplanted Fanconi Anemia (FA) patients are often radiosensitive. Due to an increased risk of developing secondary malignancies, these patients require dose and volume modification during radiotherapy. To determine whether abrogation of TGF-β signaling alters the radiation sensitivity of Fancd2-/- mice, cell lines derived from double knockout (DKO) (SMAD3-/- Fancd2-/-) mice were compared with those from Fancd2-/-, SMAD3-/-, and wild-type mice for ionizing irradiation sensitivity. Bone marrow stromal cell lines were derived from long-term bone marrow cultures of DKO, Fancd2-/-, SMAD3-/-, and wild-type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 mice. Radiation sensitivity was determined using clonogenic irradiation survival curves. There was no significant difference in radiosensitivity comparing DKO cells (Do = 1.95 ± 0.06 Gy, ň = 4.3 ± 0.7) to the wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell line (Do = 2.00 ± 0.11 Gy, and ň = 5.1 ± 0.7, p = 0.7003 and 0.4820, respectively). The Fancd2-/- cell line was more radiosensitive with a Do of 1.37 ± 0.09 Gy compared to 1.95 ± 0.07 and 2.00 ± 0.11 for DKO and wild type cells (p = 0.0063 and 0.0360, respectively. In contrast, the SMAD3-/- cell line was more radioresistant with an increased shoulder on the irradiation survival curve (ň = 12.1 ± 2.9) compared to the DKO or wild type SMAD3+/+ (129/Sv) X Fancd2+/+ (B6) F1 cell lines (ň = 4.335 ± 0.7 or 5.1 ± 0.7, p = 0.00277 or 0.0426, respectively). This confirms and extends results with SMAD3-/- mouse derived cell lines on another background strain (C57BL/6J) (Epperly, et al., Radiation Research, 165:671-677, 2006). TGF-β signaling was abrogated in both DKO and SMAD3-/- mouse cell lines (measured by TGF-β inhibition of fresh marrow CFU-GEMM in vitro), confirming the phenotype of altered TGF-β signaling. Therefore, radiosensitivity associated with the Fancd2-/- genotype was abrogated by interruption of the TGF-β signaling pathway in the same cells. Supported by research grant NIAID/NIH, U19A168021. Disclosures No relevant conflicts of interest to declare.

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98% IGHV Gene Identity Is Still the Optimal Cutoff to Predict the Prognosis of Chronic Lymphocytic Leukemia Patients in China

Blood ◽

10.1182/blood-2018-99-118141 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 5545-5545

Author(s):

Yi Xia ◽

Ke Shi ◽

Qian Sun ◽

Chun Qiao ◽

Huayuan Zhu ◽

...

Keyword(s):

Prognostic Factor ◽

Chronic Lymphocytic Leukemia ◽

Conflicts Of Interest ◽

Somatic Hypermutation ◽

Lymphocytic Leukemia ◽

Study Cohort ◽

Optimal Cutoff ◽

Entire Cohort ◽

Mutational Status ◽

Significant Difference

Abstract Background: Immunoglobulin heavy chain variable region (IGHV) has been an important prognostic factor for chronic lymphocytic leukemia (CLL) for decades. 98% being a cut-off value for IGHV is a mathematical choice and researches on the best cut-off value have never been stopped. Chinese CLL patients are known to differ from Caucasian CLL patients on both clinical and genetical features. However, the optimal cutoff for IGHV mutational status has not yet been studied in this particular ethnic group. Method: We carried out a study on 595 Chinese CLL patients in order to find out whether 98% is the best cut-off value for IGHV in Chinese CLL patients. Genomic DNA from peripheral blood or bone marrow was subjected to PCR amplification following the IGH Somatic Hypermutation Assay v2.0 protocol (InVivoScribe). Sequences were aligned to ImMunoGeneTics/V-QUEry and Standardization (IMGT/-VQUEST) database. Result: 600 sequences were received after IGHV rearrangement sequencing. IGHV3-23, IGHV4-34, IGHV3-7, IGHV4-39 and IGHV1-69 were the most frequently used IGHV genes. 352 (58.7%) cases were IGHV-mutated while 248 (41.3%) cases were IGHV-unmutated if the classical 98% classification by ERIC was used. In order to determine the optimal cut-off value, we used 1% as the interval to divide the entire cohort into 7 groups according to the mutational rate, which were <95%, 95%-95.99%, 96%-96.99%, 97%-97.99%, 98%-98.99%, 99%-99.99% and 100% respectively. Binet A patients had a relatively indolent course of disease and cases with different IGHV mutational rates had no significant differences in time to first treatment (TTFT) apart from truly unmutated (100%) cases. For the whole study cohort, significant difference appeared at 98% interval (P<0.001 and P=0.005 for TTFT and OS respectively) while intervals less than 98% had no significant difference compared with the <95% group. Similarly, there was no clear dissimilarities among 98%, 99% and 100% intervals (Table 1a and b). All the other prognostic factors including del(17p), del(11q), TP53 mutation, MYD88 mutation, NOTCH1 mutation, SF3B1 mutation, CD38, ZAP-70, Binet staging, gender, β2-microglobulin and EBV-DNA were differently distributed between group <98% and group ³98%, but not among subgroups in ³98%. In multivariate analysis, the 98% IGHV was also an independent prognostic factor for TTFT and OS. Conclusion: 98% is the optimal cutoff value for IGHV mutational status to predict the prognosis of CLL patients in China. Table 1. Table 1. Disclosures No relevant conflicts of interest to declare.

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Lysosomal Degradation of CD33, the Target for the Anti-Leukemia Immunoconjugate, Gemtuzumab Ozogamicin: Evidence for Cbl-Mediated Monoubiquitination.

Blood ◽

10.1182/blood.v106.11.2469.2469 ◽

2005 ◽

Vol 106 (11) ◽

pp. 2469-2469

Author(s):

Roland B. Walter ◽

Brian W. Raden ◽

Darren M. Kamikura ◽

Irwin D. Bernstein ◽

Jonathan A. Cooper

Keyword(s):

Tyrosine Phosphorylation ◽

Kinase Inhibitor ◽

Lentiviral Vector ◽

Gemtuzumab Ozogamicin ◽

Jurkat Cells ◽

Dependent Manner ◽

Wild Type ◽

Src Family Kinase ◽

Lysosomal Degradation ◽

Src Tyrosine Kinase

Abstract Background: CD33, the target for the anti-leukemia immunoconjugate, gemtuzumab ozogamicin (GO; Mylotarg™), is a transmembrane glycoprotein that contains two cytoplasmic immunoreceptor tyrosine-based inhibitory motifs (ITIMs). Although we have previously shown that disruption of the ITIMs prevents effective uptake of antibody-bound CD33 and significantly reduces GO-induced cytotoxicity, the mechanisms underlying this uptake and intracellular trafficking of the antibody-CD33 complex are not known. In this study, we tested whether CD33 is a target for monoubiquitination, a posttranslational modification that marks proteins for lysosomal degradation, and how this modification relates to tyrosine phosphorylation and endocytosis. Methods: CD33− Jurkat and 32D cells transduced with a lentiviral vector expressing either wild-type or mutant CD33 were analyzed for CD33 expression as well as internalization of anti-CD33 antibody by flow cytometry. Pull-down experiments were performed with glutathione S-transferase (GST) proteins fused to phosphorylated cytoplasmic tails of CD33 or TKB domain of Cbl/Cbl-b, using human myeloid cell lysates. For co-immunoprecipitation experiments, constructs encoding wild-type and mutant CD33, ubiquitin, Cbl/Cbl-b, and wild-type Fyn were transfected into HEK293T cells. Results: In engineered Jurkat cells, treatment with either anti-CD33 antibody or the tyrosine phosphatase inhibitor pervandate increased tyrosine phosphorylation of CD33. Pervanadate enhanced uptake of antibody-bound CD33; this effect was dependent upon the integrity of the ITIMs and was prevented by co-treatment with the Src tyrosine kinase inhibitor PP2. CD33 interacted with the TKB domains of Cbl and Cbl-b in GST fusion protein pulldown assays. Similarly, Cbl and Cbl-b were co-immunoprecipitated with CD33 in transfected 293T cells. Experiments in 293T cells further showed that CD33 is monoubiquitinated in an ITIM-dependent manner. Cbl or Cbl-b significantly increased the amount of CD33-associated ubiquitination; Cbl/Cbl-b-dependent ubiquitination could further be enhanced by co-transfected Fyn. Finally, 32D cells expressing lysine-to-arginine mutants of CD33 displayed much higher levels of surface CD33 but had reduced internalization of antibody-bound CD33 compared to cells expressing wild-type CD33, consistent with a reduced internalization/degradation of the lysine-to-arginine mutants. Conclusion: These studies indicate that Src-family kinase dependent phosphorylation favors internalization of antibody-bound CD33 and identify Cbl family proteins as potential binding partners of CD33. Importantly, CD33 is a target for ITIM-dependent monoubiquitination, and Cbl family proteins can act as an E3 ligase in this reaction. Our data therefore suggest a model where lysosomal routing and degradation of antibody-bound CD33 is secondary to Src-family kinase-induced phosphorylation of CD33 with subsequent phosphotyrosine-dependent binding of Cbl to and monoubiquitination of CD33.

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Alpha-Catenin Is Dispensable for Normal Hematopoietic Stem Cell Function.

Blood ◽

10.1182/blood.v114.22.1438.1438 ◽

2009 ◽

Vol 114 (22) ◽

pp. 1438-1438

Author(s):

Natallia Mikhalkevich ◽

Michael W. Becker

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Conflicts Of Interest ◽

Fusion Product ◽

Wild Type ◽

Hematopoietic Stem ◽

Myeloid Neoplasms ◽

Increased Risk ◽

Significant Difference ◽

The Impact

Abstract Abstract 1438 Poster Board I-461 We previously demonstrated the loss of expression of alpha-E-Catenin, the product of the CTNNA1 gene, in primary leukemic stem cells isolated from patients with advanced Myelodysplastic Syndrome (MDS) and Acute Myeloid Leukemia (AML) associated with loss of all or part of the long arm of chromosome 5. To formally assess the impact of loss of Ctnna1 expression on hematopoiesis, we employed a murine model for the hematopoietic specific conditional loss of Ctnna1 expression. We demonstrate that Ctnna1 deficiency is associated with normal hematopoietic maturation and proliferation as assessed by peripheral blood examination and methycellulose colony assays. We assessed stem cell and early progenitor frequencies using both flow cytometry and functional assays. Ctnna1 deficiency was associated with equivalent frequencies of Sca1+C-Kit+CD135-Lineage- HSCs in both experimental animals and controls. Short term HSC and MPP frequencies were likewise unaltered. We assessed HSC function using transplantation studies. In competitive repopulation experiments, HSCs deficient for Ctnna1 maintained stable engraftment of recipient mice for up to 1 year. Limiting dilution analyses detected no significant difference in HSC frequency between wild type and Ctnna1 deficient mice. We examined the potential role of Ctnna1 deficient hematopoietic stem cells in two murine models for myeloid neoplasms 1.) exposure to mutagen ENU and 2.) a model for murine AML driven by the HoxA9-Nup98 fusion product. Following exposure of HSCs to ENU, loss of Ctnna1 was not associated with an increased risk of development of a myeloid neoplasm. Expression of the HoxA9-Nup98 fusion product by retroviral infection of Ctnna1 deficient and wild type Sca1+C-Kit+Lineage- cells resulted in no difference in time to development of the previously characterized myeloproliferative disorder or acute leukemia. Taken together, these data demonstrate that in the absence of specific genetic abnormalities, loss of Ctnna1 expression in primary murine HSCs is not associated with aberrant HSC function or the development of myeloid neoplasms. Further studies are necessary to define a role for of loss of Ctnna1 expression in human myeloid malignancies. Disclosures No relevant conflicts of interest to declare.

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Multiplex Allele-Specific PCR for Simultaneous Detection of H63D and C282Y HFE Mutations in Hereditary Hemochromatosis

The Journal of Applied Laboratory Medicine ◽

10.1373/jalm.2017.024984 ◽

2018 ◽

Vol 3 (1) ◽

pp. 10-17

Author(s):

Heleen H Arts ◽

Barry Eng ◽

John S Waye

Keyword(s):

Hereditary Hemochromatosis ◽

Simultaneous Detection ◽

Turnaround Time ◽

Molecular Diagnostic ◽

Hfe Gene ◽

Specific Pcr ◽

Liver And Pancreas ◽

Allele Specific ◽

Hfe Mutations ◽

Allele Specific Pcr

Abstract Background Hereditary hemochromatosis (HH) is characterized by excessive iron absorption in the intestine, which can lead to failure of vital organs such as the heart, liver, and pancreas. Among northern Europeans, HH is most often associated with the C282Y and H63D mutations of the HFE gene. We developed a test that allows screening for both mutations in a single reaction. Methods A multiplex allele-specific PCR was developed for simultaneous genotyping of the H63D and C282Y HFE mutations. PCR fragments were designed such that the resulting PCR product can be analyzed in a single polyacrylamide gel lane. Results Test results from our multiplex assay were concordant with genotypes of 55 Canadian patients with suspected hemochromatosis, which had previously been established by allele-specific PCRs that targeted H63D and C282Y in separate reactions. Conclusions Molecular diagnostic detection of H63D and C282Y mutations can be achieved by a variety of methods, but these are not necessarily time-efficient or economical. Multiplex allele-specific PCR is an excellent tool for molecular diagnostic screening for H63D and C282Y mutations in patients with suspected hemochromatosis. This method is inexpensive, accurate, and highly efficient in terms of labor, throughput, and turnaround time.

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