bluuebunny/crossref_metadata_2025 huggingface dataset:
An Analysis of Occurrence and Prognosis Related Factors of Renal Dysfunction in Patients with Multiple Myeloma.
Authors: Fu Rong , Wang Ting , Shao Zonghong
Published: November 2019

Objective To analysis the occurrence and prognosis related factors in renal dysfunction with multiple myeloma(MM).

Methods Seventy-four cases with MM were enrolled in this study. The risk factors of occurrence and prognosis were analyzed.

Results The incidence of renal dysfunction (RD) with MM was 56.8%, Age, hypertention, hemoglobin, serum ALB and GLO levels, serum β2MG, serum calcium and phosphonium level, the percentage of myeloma cells in bone marrow, types of MM, Durie-Salmon stage were the single factors associated with the incidence of RD with MM. Hypertention, serum β2MG and ALB levels were the multiple factors associated with the incidence of RD with MM. ALB was the protection factor and the other two were risk factors. The renal function recovered rapidly in the patients who received CR or received blood transfusion. The patients with renal dysfunction survived shorter (28±5months) than those with normal renal function (42±6months). Renal dysfunction caused more MM patients death(84.6%) in 3 months.

Conclusion Hypertention and high tumor burden were the risk factors of renal dysfunction in MM, effective chemothemapy and support treatment help renal function recovery.
Clinical Features and Management of Jaw Osteonecrosis in Patients Receiving Bisphosphonate Therapy.
Authors: Alves Fabio , Prado José D. , Rocha André C.
Published: November 2019

Osteonecrosis of the jaws (ONJ) in patients receiving bisphosphonates is currently being discussed in the scientific literature. Oral bisphosphonates such as alendronate are commonly used to treat osteoporosis and have rarely been associated with ONJ in the benign setting. There is a greater number of reported ONJ cases in patients using intravenous bisphosphonates such as pamidronate and zoledronic acid for cancer-related indications. Many authors have associated local factors, such as dental extraction, periodontal disease, abscesses, and other oral problems, with the development of ONJ, and some have also suggested risk factors other than dental irritation. The aim of this study was to describe the clinical data from oncology patients with ONJ from a single institution. A total of 18 ONJ cases were identified among all patients treated at or referred to our institute (Hospital do Cancer A C Camargo, Sao Paulo, Brazil). These patients had a variety of primary malignancies: breast cancer (n = 7), multiple myeloma (n = 4), prostate cancer (n = 3), lung cancer (n = 3), and renal cancer (n = 1). Current bisphosphonate therapy included zoledronic acid (n = 15; 7 [47%] had received prior pamidronate therapy), pamidronate (n = 2), and alendronate (n = 1). The ONJ cases were managed conservatively using local irrigation with 0.12% chlorhexidine gluconate, debridement, and antibiotics. A total of 29 local ONJ lesions were found: 13 patients had 1 lesion, 3 patients had 2 lesions, and 2 patients had 5 lesions. The majority (n = 16 [89%]) of the patients experienced some level of local discomfort/pain, 6 complained of halitosis, and only 2 patients were asymptomatic. Clinically, 62% of the ONJ lesions were associated with dental work and extractions; only 20% occurred spontaneously. The majority (59%) of the ONJ lesions completely healed in 9 patients. Overall, most of the ONJ lesions were associated with dental procedures, and pain was the symptom most frequently reported. Excellent dental hygiene was found to be important for preventing infections and ONJ and also to contribute to lesion healing in the majority of the cases.
Osteonecrosis of the Jaw in Multiple Myeloma Patients (the IFM Registry).
Authors: Fuzibet Jean Gabriel , Viellard Marie Hélène , Rossignol Benoit B.R. , Doyen Chantal C.D. , Hulin Cyril , Garderet Laurent L.G. , Blanc Michel M.B. , Marit Gerald , Facon Thierry T.F. , Moreau Philippe
Published: November 2019

PURPOSE: to describe the incidence, characteristics and risk factors for osteonecrosis of the jaw (ONJ) in multiple myeloma (MM).

PATIENTS AND METHODS : retrospective review of 51 ONJ collected from the IFM centers. For MM, age at diagnosis, isotype, DS staging, nature of chemotherapy and number of Stem-Cell Transplantation were the same that expected.

RESULTS: ONJ occurred predominantly in the mandible (70%). The median time of exposure to biphosphonates (BP) was 45 months (4 to 144 months). BP therapy included: zoledronate (85%), pamidronate (10%), clodronate (5%), all of the patients have received intravenous BP before, two patients have no BP at the time of diagnosis of ONJ.

RISK FACTORS: dental extraction (59%) other dental care (5%), dental infection (24%), others (30%), no risk factor (10%). SYMPTOMS: pain (92%), purulent discharge (37%), presence of exposed bone (60%), fracture (2%).

MANAGEMENT: BP discontinuation (84%), medical treatment (86%), removal of sequestra (51%), extensive surgery (20%).

EVOLUTION: improvement (55%), chronical symptoms (70%).

INCIDENCE: we have not the number of exposed patients but: 46 centers/73 reported no ONJ.The first report was march 2001. In the same period, 1695 patients where included in IFM trials and only 16 ONJ where observed. Year of diagnosis: before 2004: 5 cases; 2004: 11 cases; 2005:26 cases; 2006: 7 cases; 2007(6 months): 2cases.

CONCLUSION: ONJ is an adverse side effect of amino BP therapy (zoledronate>pamidronate), is time dependant and often after dental extraction. Preventive recommandations applied in 2005 can explain the decreasing incidence of ONJ in our study.
Risk-Adjusted Therapy of Acute Lymphoblastic Leukemia Can Decrease Treatment Burden and Improve Survival: Treatment Results of 2169 Unselected Pediatric and Adolescent Patients Enrolled in the Trial ALL-BFM 95.
Authors: Möricke Anja , Reiter Alfred , Zimmermann Martin , Gadner Helmut , Stanulla Martin , Ludwig Wolf-Dieter , Harbott Jochen , Mann Georg , Niggli Felix , Henze Günter , Welte Karl , Klingebiel Thomas , Niemeyer Charlotte , Zintl Felix , Riehm Hansjörg , Schrappe Martin
Published: November 2019

An unselected patient population of 2169 children with acute lymphoblastic leukemia (ALL) was enrolled in the multicenter trial ALL-BFM 95 of the Berlin-Frankfurt-Münster (BFM) study group. The study was designed to minimize acute and long-term toxicity through well-directed treatment reduction in a large patient subset with expectedly good prognosis. On the other hand extensive therapy intensification was implemented in a subgroup with very poor prognosis. The precondition was the clear definition of distinct risk groups enabled by a new risk stratification: patients (pts) with translocation t(9;22) and t(4;11), poor response to the cytoreductive prednisone prephase and/or to induction treatment were allocated to the high-risk (HR) group. Non-HR pts were stratified by age, white blood cell count (WBC) and immunophenotype to standard risk (SR, age 1–5 years, WBC <20 Gp/L, and no T-immunology) or medium risk (MR, all other pts). Compared to the previous trial ALL-BFM 90, the following treatment modifications were made in ALL-BFM 95: 1. reduction of the daunorubicin dose by 50% in induction in SR pts; 2. extension of maintenance by 12 months in SR boys; 3. in the MR group, randomized evaluation of intermediate-dose (ID) cytarabine in addition to high-dose methotrexate in the extracompartment/consolidation phase; 4. omission of preventive cranial radiotherapy (pCRT) in all MR pts with Non-T-ALL; 5. intensification of consolidation/reinduction in HR pts by dose increase in the block elements and reintroduction of protocol II as element providing a continuous drug exposure. Except for the randomized question, event-free survival was analyzed in comparison to the matching subsets of the historical control group treated in ALL-BFM 90. Estimated 6-year event-free survival (6y-pEFS) for the total group was 80±1% (median follow-up 7.3 years). The large SR group (35% of pts) achieved excellent 6y-pEFS of 89±1% despite significant reduction of the daunorubicin dose. The elongation of maintenance in boys yielded no significant relapse reduction. In MR (53% of pts), 6y-pEFS was 80±1%; no improvement was accomplished by the use of additional ID-cytarabine. Omission of pCRT in non-T-ALL MR pts was possible without reduction of EFS though the 6-year cumulative incidence of CNS relapses increased from 2.0±0.5% to 4.5±0.8%%. In the small HR group (12% of pts), treatment intensification led to considerable improvement over the previous ALL-BFM trials yielding a 6y-pEFS of 49±3%. Compared with the previous trial ALL-BFM 90, favorable results in SR and MR were firmly established despite significant treatment reduction in the majority of these patients.
Derivation and Validation of a Prediction Tool for Venous Thromboembolism: A VERITY Registry Study.
Authors: Arya Roopen , Paneesha Shankaranarayana , McManus Aidan , Parsons Nick , Scriven Nicholas , Farren Tim , Nokes Tim , O’Shaughnessy Denise , Rose Peter
Published: November 2019

Accurate estimation of risk for venous thromboembolism (VTE) may help clinicians assess prophylaxis needs. Only empirical algorithms and risk scores have been described; an empirical risk score (‘Kucher’) based on 8 VTE risk factors (cancer, prior VTE, hypercoagulability, surgery, age>75 yrs, BMI>29, bed rest, hormonal factor) using electronic alerts improved hospitalized patient outcome (NEJM2005;352:969–77). We wished to develop a multivariate regression model for VTE risk, based on Kucher, and validate its performance. The initial derivation cohort consisted of patients enrolled in ‘VERITY’, a multicentre VTE treatment registry for whom the endpoint of VTE and all 8 risk factors were known. Initial univariate analysis (n=5928; 32.4% with diagnosis of VTE) suggested VTE risk was not accounted for by the 8 factors; an additional 3 were added (leg paralysis, smoking, IV drug use [IVD]). The final derivation cohort was 5241 patients (32.0% with VTE) with complete risk data. The validation cohort (n=915) was derived from a database of 928 consecutively enrolled patients at a single DVT clinic. Model parameters were estimated using the statistical package ‘R’ using a stepwise selection procedure to choose the optimal number of main effects and pair-wise interactions. This showed that advanced age (estimated odds ratio [OR]=2.8, p<0.001); inpatient (OR=3.0, p<0.001); surgery (OR=3.1, p<0.001); prior VTE (OR=2.9, p<0.001); leg paralysis (OR=3.8, p<0.001); cancer (OR=5.3, p<0.001); IVD (OR=14.3, p<0.001); smoking (OR=1.2, p=0.009); and thrombophilia (OR=2.8; p<0.001) increased the risk of VTE. Obesity (OR=0.7; p<0.001) increased the VTE risk only in patients with a hormonal factor (OR=2.0, p=0.007). Backward stepwise regression showed prior VTE as the most important factor followed by cancer, IVD, surgery, inpatient, age, leg paralysis, hormonal factor, obesity, thrombophilia and smoking. Expressing the parameter estimates in terms of probabilities defines a risk score model for VTE. Using the model, the receiver operating characteristic (ROC) curve (see figure) area under the curve (AUC) was estimated as 0.720 (95% CI, 0.705–0.735) for the model (dashed line), indicating a good diagnostic test significantly better (p<0.001) than Kucher (AUC=0.617, 95% CI, 0.599–0.634)(solid line). For the validation cohort, AUC was estimated as 0.678 (95% CI, 0.635–0.721) for the model, which was not significantly different from AUC for the full dataset used for model development, and was 0.587 (95% CI, 0.542–0.632) for Kucher. This model to predict individual patient risk of VTE may contribute to decision making regarding prophylaxis in clinical practice.

Figure Figure
A Negative D-dimer Excludes Recurrent Deep Vein Thrombosis: Results of a Multicentre Management Study.
Authors: Bates Shannon , Kearon Clive , Kahn Susan , Julian Jim A. , Crowther Mark A. , Gerdes Victor , Geerts William , Kovacs Michael J. , Weise-Kelly Lorraine , Ginsberg Jeffrey S.
Published: November 2019

The high frequency of residual radiologic abnormalities after initial deep vein thrombosis (DVT) makes management of patients with suspected recurrence difficult. D-dimer (DD) and serial compression ultrasonography (CUS) of the proximal veins have a high sensitivity and negative predictive value (NPV) in suspected first DVT. We hypothesized that it would be safe to withhold anticoagulation in patients with suspected recurrence who had a negative sensitive DD or negative serial CUS when DD testing was positive. In a multicentre prospective cohort study, patients underwent DD testing with an immunoturbidometric assay (MDA DD). If the DD was negative (<0.5 ug fibrinogen equivalent units [FEU]/mL), patients had no further testing. If the DD was positive, CUS was performed and, if normal, repeated after 1–3 and 7–10 days. Patients with a positive DD and abnormal CUS at presentation were managed as per their treating physician. Patients were followed for 3 months to detect venous thromboembolism (VTE) and suspected VTE were adjudicated centrally. Of the 504 patients enrolled in this study, 14 were subsequently deemed ineligible and 2 patients were lost to follow-up. The overall prevalence of confirmed recurrent DVT at presentation or during follow-up was 17%. 230 patients had a negative DD at presentation and, of the 227 evaluable patients, 4 had definite confirmed VTE (NPV of DD = 98%; 95% Confidence Interval [CI], 96–99%). Of the 135 patients with a positive DD and normal initial CUS, serial CUS was negative in 129 cases. Of these patients, 3 had definite VTE during follow-up (NPV of serial CUS in patients with positive DD = 98%; 95% CI, 93–99%). These results suggest that a negative MDA DD result excludes clinically significant recurrent DVT and that anticoagulants can also be safely withheld in patients with negative serial CUS, even if their DD is positive. This simple diagnostic approach can be used to safely manage approximately 70% of patients with suspected recurrent DVT.
High Plasma Levels of Soluble P-Selectin Are Predictive for Venous Thromboembolism in Cancer Patients - Results from the Vienna Cancer and Thrombosis Study (CATS).
Authors: Ay Cihan , Simanek Ralph , Vormittag Rainer , Dunkler Daniela , Alguel Guelay , Koder Silvia , Wagner Oswald , Zielinski Christoph , Pabinger Ingrid
Published: November 2019

Cancer patients are at high risk for venous thromboembolism (VTE), which represents an additional burden and a frequent cause of increased mortality. Laboratory parameters with a predictive value for VTE could help to assign a patient to a high or low risk group. In recent studies the cell adhesion molecule P-selectin was identified to be a strong risk factor for VTE. However, the role of soluble P-selectin (sP-selectin) in cancer-associated VTE is not known because up to now clinical data are not available. Therefore, we assessed sP-selectin plasma levels in cancer patients as a risk predictor for VTE and provide a report from the ongoing prospective observational CATS. Patients with newly diagnosed cancer or progression of disease who had no chemotherapy within the last three months were enrolled from October 2003 to October 2006 and followed prospectively via phone and mail. Occurrence of VTE and information on the patientś anti-cancer-treatment within the follow up period were recorded. VTE has always been confirmed by imaging. sP-selectin levels were measured with a highly sensitive ELISA. Kaplan Meier and Cox regression analysis were applied for statistical calculation. We included 687 patients (319 female/368 male, median age [IQR]: 62 [54–68] yrs) with malignant disease and followed them for a median observation period of 415 days. Main tumour entities were malignancies of the breast (n=125), lung (n=86), upper (n=30) and lower gastrointestinal tract (n=100), pancreas (n=42), kidney (n=19) and prostate (n=72). Furthermore, 80 patients had high-grade glioma, 73 lymphomas, 18 multiple myeloma and 42 other tumour types. Distant metastases were found in 268 patients at the time of recruitment. During the observation period VTE occurred in 45 patients (21 female/24 male, median age [IQR]: 62 [48–66] yrs). Elevated plasma levels of sP-selectin (cut-off level 53.1 ng/mL representing the 75th percentile of the total study population, 173 patients) [hazard ratio (HR): 2.5, 95% CI 1.4 – 4.6], surgery [HR: 3.9, 95% CI 1.8 – 8.5] and radiotherapy [HR: 3.2, 95% CI 1.6 – 6.4] were statistically significant risk factors for VTE in multivariable analysis including sP-selectin, age, sex, surgery, chemotherapy and radiotherapy. The cumulative probability of VTE after 6 months was 11.9% in patients with sP-selectin plasma levels above and 3.7% in those below the 75th percentile. In conclusion, high plasma levels of sP-selectin independently predict VTE in cancer patients. Measurement of sP-selectin at diagnosis of cancer would help to identify patients at increased risk for VTE.
Efficacy of Dasatinib in Patients with Chronic-Phase Chronic Myelogenous Leukemia with Resistance or Intolerance to Imatinib: 2-Year Follow-Up Data from START-C (CA180-013).
Authors: Stone Richard M. , Kantarjian Hagop M. , Baccarani Michele , Lipton Jeffrey H. , Hughes Timothy , Ezzeddine Rana , Bleickardt Eric , Hochhaus Andreas
Published: November 2019

Dasatinib (SPRYCEL®) is 325-fold more potent than imatinib against BCR-ABL in vitro and binds to BCR-ABL in both the inactive and active, oncogenic conformations. Dasatinib has been shown to be an effective treatment option for patients with imatinib-resistant or -intolerant chronic-phase chronic myelogenous leukemia (CP-CML). Here we report the extended follow-up of START-C, a 75-center, international study of dasatinib in 387 patients with CP-CML with resistance (n=288) or intolerance (n=99) to imatinib. Recruitment took place from February to July 2005. Dasatinib was administered on a 70-mg BID regimen; dose escalation (90 mg BID) or reduction (50 or 40 mg BID) were allowed for lack of response or toxicity, respectively. Median time from diagnosis of CML was 61 mo (range 32–50). Prior therapy included interferon-α in 65% of patients and stem-cell transplantation in 10%; 55% had received prior imatinib doses >600 mg and 53% treatment with imatinib for >3 years. Best response to prior imatinib therapy was complete hematologic response (CHR) in 82%, and complete (CCyR) and partial cytogenetic response (PCyR) in 19% and 18%, respectively. With a median follow-up of 15.2 mo, CHR was attained in 91% of patients (95% CI 87–93%), major cytogenetic response (MCyR) in 59% (95% CI 54–64%) (52% imatinib-resistant, 80% imatinib-intolerant), and CCyR in 49% (40% imatinib-resistant; 75% imatinib-intolerant). For patients with no prior MCyR to imatinib, 42% achieved a MCyR with dasatinib. A MCyR rate of 59% was recorded for patients with baseline BCR-ABL mutations; responses were seen across all mutations with the exception of T315I. MCyRs were durable, with only 7 of the 230 patients who had achieved a MCyR with dasatinib losing this response. Major molecular response rate (ie, a BCR-ABL/ABL ratio of <0.1% according to the international scale by RQ-PCR) at 12 mo was 25%. Progression-free survival at 15 mo was 90% while overall survival was 96%. Dose interruptions were required for 87% of patients and dose reduction for 73%; the average daily dose administered was 101 mg (range 11–171). Reports of grade 3–4 thrombocytopenia and neutropenia were documented for 48% and 49% of patients, respectively. Non-hematologic toxicity consisted primarily of diarrhea (37%), headache (32%), fatigue (31%), and dyspnea (30%). Pleural effusion was experienced by 27% of patients; this was categorised as grade 1–2 in 21% and grade 3–4 in 6%. Dasatinib-induced cytogenetic responses remain durable in patients with CP-CML resistant or intolerant to imatinib. Updated analyses corresponding to a minimum follow-up of 2 years on all patients will be presented.
B-CLL Antibodies Encoded by Stereotypic VH1-69, D3-16, and JH3 Rearrangements Immunoprecipitate Non-Muscle Myosin Heavy Chain IIA.
Authors: Chu Charles C. , Zhang Lu , Hatzi Katerina , Catera Rosa , Allen Steven L. , Rai Kanti R. , Chiorazzi Nicholas
Published: November 2019

B-cell chronic lymphocytic leukemia (B-CLL) patient clinical outcomes can be segregated based on the degree of mutation of immunoglobulin (Ig) variable region genes. This observation suggests a functional role for antigen binding by the Ig B-cell receptor in B-CLL. Furthermore, over 20% of B-CLL patients share antibodies with very similar Ig heavy chain (H) variable region CDR3 sequences, suggesting common antigen reactivity. For example, sixteen unrelated B-CLL patients have been reported to express an unmutated Ig heavy chain encoded by VH1-69, D3-16, and JH3 with nearly identical HCDR3 sequences. The probability that these patients developed B-CLL with identical IgH variable regions by random chance is extremely small. This stereotypic set of antibodies strongly bind cytoplasmic structures in HEp-2 cells as observed by immunofluorescence. Therefore, HEp-2 cell extracts were used as the source of material to isolate the cellular antigen recognized by these antibodies. These B-CLL antibodies were recombinantly overexpressed and purified as human IgG1 molecules. Recombinant antibody was bound to protein G agarose beads and incubated with cell extracts overnight. Bound cellular antigen was immunoprecipitated, washed, eluted, run on SDS-PAGE, and stained with Coomassie Blue. B-CLL antibodies from this stereotypic set immunoprecipitated protein bands at approximately 45 and 225 kDa in size. These bands were isolated and identified by mass spectrometry analysis of trypsin-digested peptides. The 45 kDa and 225 kDa proteins are cytoplasmic actin and non-muscle myosin heavy chain IIA (MYHIIA), respectively. The VH1-69, D3-16, and JH3 encoded stereotypic set of antibodies has been reported to be polyreactive against multiple autoantigens, such as DNA, Ig, and actin. However, MYHIIA has not been previously reported as an antigen recognized by this stereotypic set of antibodies. Therefore, we confirmed that MYHIIA was recognized by this stereotypic antibody set by probing B-CLL antibody immunoprecipitates by Western blot with anti-MYHIIA antibody. Furthermore, anti-MYHIIA antibody immunofluorescent staining of HEp-2 cells co-localized with this B-CLL stereotypic antibody immunofluorescent staining. Thus, stereotypic VH1-69, D3-16, and JH3 encoded B-CLL antibodies are polyreactive with common cellular proteins, including the newly identified autoantigen MYHIIA. The cellular antigens recognized by these stereotypic antibodies may be important in the pathogenesis, diagnosis, and treatment of B-CLL.
Does Imatinib Change the Outcome in Philapdelphia Chromosome Positive Acute Lymphoblastic Leukaemia in Adults? Data from the UKALLXII/ECOG2993 Study.
Authors: Fielding Adele K. , Richards Susan M. , Lazarus Hillard M. , Litzow Mark R. , Luger Selina M. , Marks David I. , McMillan Andrew K. , Rowe Jacob M. , Tallman Martin S. , Goldstone Anthony H.
Published: November 2019

The UKALLXII/ECOG2993 international study for adults with acute lymphoblastic leukeamia (ALL) included a separate, single-arm study (still open) to evaluate the role of etoposide/TBI conditioned allogeneic bone marrow transplantation (BMT) in patients with Philadelphia chromosome positive (Ph pos) disease. Patients received 2 phases of induction, intensification with high dose methotrexate and BMT using unrelated donor stem cells where a matched sibling was not available. 267 patients with Ph pos ALL were treated from 1993 onwards, in the pre-imatinib era. The study was amended in 2003 to add imatinib 600mg per day following induction and after BMT for 2 years or until relapse. A subsequent amendment at the end of 2005, added imatinib 600mg per day also to phase 2 of induction. 153 patients have now been treated on the imatinib-containing protocol − 89 received imatinib following induction and 64 started imatinib with phase 2 induction.

Complete remission (CR) rate at the end of 2 phases of induction was 91% with imatinib started in induction, 81% with imatinib started in consolidation only & 83% in the pre imatinib era. 128 of the 153 patients treated with any imatinib were eligible for BMT. Of the 98 patients with over 6 months follow-up, 57 (58%) actually received BMT. This transplant rate does not differ from the 62% in the pre-imatinib era although the data pertain mostly to those whose imatinib started later during therapy. Overall survival of the imatinib-treated group as a whole is 23% at 3 years, which does not differ from the 26% pre-imatinib rate as shown in the figure. Hence, imatinib started post-induction does not appear to improve survival. Imatinib given with induction may improve the CR rate, but there is insufficient follow up to assess transplant or survival rates. Earlier imatinib may be optimal but overall, our study does not provide evidence yet that imatinib alters the outcome of this disease.

Figure Figure Patient characteristics Pre-imatinib (N=267) Post-imatinib (N=153) Gender: Male 150 (56%) 96 (63%) WBC (x 10 ^9/l) (2 unknown): <30 141(53%) 86 (56%) ≥ 30 124 (47%) 67 (44%) Median (range) 26.8 (1.5–438) 21.0 (0.5–491) Age: median (range) 40 (15–60) 42 (16–64) CNS disease (82 unknown) 14 (8%) 4 (3%)
Identification of Novel, Non-Synonymous Sequence Changes in the Tyrosine Kinase Genes of Patients with Acute Myeloid Leukemia.
Authors: Xiang Zhifu , Zhao Yu , Mitaksov Vesselin , Fremont Daved H. , Kasai Yumi , Molitoris AnnaLynn , Ries Rhonda E. , Miner Tracie , McLellan Michael D. , DiPersio John F. , Link Daniel C. , Payton Jacqueline E. , Graubert Timothy A. , Watson Mark , Shannon William , Heath Sharon , Mardis Elaine R. , Wilson Richard K. , Ley Timothy J. , Tomasson Michael H.
Published: November 2019

Activating mutations in receptor tyrosine kinase (RTK) genes (including FLT3 and KIT) occur in more than 30% of newly diagnosed patients with acute myeloid leukemia (AML); we and others have speculated that mutations in other TK genes may be present in the remaining 70%. We therefore examined the expression of all annotated RTK and cytoplasmic tyrosine kinase (CTK) genes to prioritize these genes for sequencing. We performed high-throughput re-sequencing of the kinase domains of 24 TK genes (9 RTK and 15 CTK) using amplified genomic DNA from the bone marrow (tumor) and matched skin biopsy samples (“germline”) from 94 patients with de novo AML, and validated positive findings in an additional 94 AML tumor samples (14.4 million base pairs of double-stranded coverage). In addition to previously reported somatic mutations in FLT3, KIT, and JAK2 (which occurred at expected frequencies), we found novel somatic mutations in four patients in JAK1, NTRK1 and DDR1. Unexpectedly, we also identified novel non-synonymous germline sequence changes in 14 genes, including TYK2. We examined frequencies of known polymorphisms in our patients versus controls. We determined that the previously reported JAK3P132T allele is a germline variant that occurs in 19% of normal African Americans. Even when controlling for race, the TYK2G363S allele was found significantly less frequently in AML samples (12/376 alleles, 3.2%) compared to 147 normal controls (27/294 alleles, 9.2%, p=0.0013). Notably, there was loss of heterozygosity (LOH) at TYK2 in 2 patients. Additional population based studies and biologic validation will be required to define the significance of these sequence changes for AML pathogenesis. Lastly, we compared the expression of RTK and CTK genes in AML samples (n=92) to highly enriched normal human CD34+, promyelocyte, or polymorphonuclear neutrophil populations (n=5 each). We found several RTKs (FLT3, KIT, LTK) and CTKs (FYN, LCK, ITK, HCK and FGR) were tightly regulated in normal hematopoietic development but were dysregulated in many AML samples. Taken together, our data suggest that RTK or CTK mutations are not required for AML development but may be disease modifying events. Our data also suggest that germline variants and dysregulated expression of RTK and CTK genes may play significant roles AML pathogenesis.
Bimodal Degradation of MLL Protein by the Cell Cycle SCFSkp2 and APCCdc20 E3 Lilgases Assures Cell Cycle Execution: A Critical Regulatory Circuit Lost in Leukemogenic MLL-Fusions.
Authors: Liu Han , Chen David , Westergard Todd , Takeda Shugaku , Sasagawa Satoru , Cheng Emily H.-Y. , Hsieh James J.-D.
Published: November 2019

The MLL (mixed lineage leukemia) gene encodes a highly conserved 3,969 aa histone H3 K4 methyl transferase, of which chromosome translocations result in poor prognostic infant and therapy-related leukemias. Mysteriously, the pathognomonic MLL leukemia fusions consist of a common amino(N)-terminal ∼1400 aa of MLL fused in frame with more than 60 different partners with no shared characteristics. The most well known genetic function of MLL is to antagonize polycomb group of proteins for proper Hox gene expression. Consequently deregulated Hox genes caused by MLL translocations contribute to the MLL leukemogenesis. Besides Hox genes, it remains largely undetermined whether MLL participates in other biological processes. The 500kD MLL precursor undergoes evolutionarily conserved proteolytic maturation mediated by Taspase1 (Hsieh et al. 2003, MCB, 23, 186–194; Hsieh et al. 2003, Cell, 115, 293–303). Our recent studies on Taspase1 knockout cells established an MLL-E2F axis in orchestrating core cell cycle gene expression including Cyclins and possibly Cdk inhibitors (Takeda et al. 2006, Genes & Development, 20, 2397–2409). As MLL actively participates in the cell cycle regulation, we investigated the regulation of MLL through cell cycle transition. We uncovered a unique biphasic expression of MLL conferred by defined windows of degradation mediated by specialized cell cycle E3 ligases. Specifically, SCFSkp2 and APCCdc20 mark MLL for degradation at S phase and late M phase, respectively. Abolished peak expression of MLL incurs corresponding defects in G1/S transition and M phase progression. Conversely, over-expression of MLL blocks S phase progression. Remarkably, MLL degradation initiates at its N-terminal ∼1400 aa that is retained in all MLL leukemia fusions. We examined prevalent MLL-fusions, including MLL-AF4, MLL-AF9, MLL-ELL and MLL-ELL, and observed their increased resistance to degradation. Furthermore, the same resistance was observed with the leukemogenic MLL-lacZ but not the non-leukemogenic MLL-Myc tag fusion. Thus, non-oscillating expression of MLL-fusions through the cell cycle, resulted from impaired degradation, likely constitutes the universal mechanism underlying all MLL leukemias. Our data conclude an essential post-translational regulation of MLL by the cell cycle ubiquitin/proteasome system (UPS) to assure the temporal necessity of MLL in coordinating cell cycle progression. Future studies aim at providing a comprehensive analysis on the cell cycle consequences associated with MLL-fusions using genetically modified cells derived from mice carrying various MLL-fusion knockin alleles, including MLL-AF4, MLL-AF9, and MLL-CBP.
Retrospective Survey of Children Treated for Relapsed Acute Lymphoblastic Leukemia (Complete Remission (CR) Rates): A Report from the Therapeutic Advances in Childhood Leukemia (TACL) Consortium.
Authors: Ko Richard H. , Ji Lingyun , Sposto Richard , Barnette Phillip , Bostrom Bruce , Hutchinson Raymond , Raetz Elizabeth , Seibel Nita , Twist Clare , Eckroth Elena , Gaynon Paul , Loh Mignon
Published: November 2019

Introduction: Event-free survival (EFS) for children with ALL is approximately 80%. Despite substantial success in achieving second and subsequent remissions, survival of patients with relapsed ALL (rALL) remains dismal. We propose that progress depends on identification of novel drug combinations with more activity in rALL than those commonly employed. The literature suggests a 40% CR rate for second and subsequent relapse (Br. J Haematol2005;131:579). However, CR rates depend on the number of prior therapeutic attempts and duration of any prior response, complicating identification of promising regimens. Limited patient numbers and a large number of potential candidate regimens discourage randomized trials. We surveyed local experience with rALL to establish a robust benchmark for evaluation of novel drug combinations.

Methods: The TACL Consortium (www.tacl.us) was formed to develop novel drug combinations for patients with rALL. We initiated a review of all patients with rALL treated between 1995 and 2004 at eight TACL institutions. Detailed data on therapy, response, and duration of response were collected on all patients.

Results: Of 313 rALL patients, 62% were males, 27% were older than 10 years at diagnosis, 26% had initial white blood counts (WBC) at diagnosis >=50,000/uL, and 46% were high-risk by NCI risk criteria. Re-induction attempts ranged between 1 and 9 and most commonly employed combinations of traditional ALL agents. Limiting analyses to patients with marrow involvement, we obtained 86% CR’s for 1st relapse (95% confidence interval 80%–90%), 44% for second relapse (35%–53%), and 30% for third relapse (19%–43%). CR rates declined with the number of prior treatment attempts (see Table, p<0.001). With first relapse, the CR rate trended lower in older patients (<10 y.o., 89%; ≥ 10 y.o., 78%; p=.056). With second relapse, CR rates increased with a longer duration of the prior remission (p<0.005), but this was not true for first relapse. (p=0.33).

Conclusion: Our data support a 40% baseline CR rate for 2nd and subsequent relapse ALL and provide a useful benchmark for comparison with new therapies.

CR Rates by Treatment Attempt and Duration of Any Prior Remission CR Rates (percent) CR status/ duration of previous CR 2nd Attempt 3rd Attempt 4th Attempt CR not achieved with prior attempt 8/8 (100) 3/14 (21) 10/35 (29) CR < 18 months 41/52 (79) 26/ 69 (38) 5/ 22 (23) CR 18–36 months 59/ 66 (89) 14/ 23 (61) 1/ 1(100) CR ≥ 36 months 61/ 68 (90) 7/ 10 (70) 1/ 1(100) CR unknown 16/22 (73) 2/2 (100) 1/ 1(100) Totals 185/216 (86) 52/118 (44) 18/ 60 (30)
Pharmacokinetic and Pharmacodynamic Effects of PEG-Asparaginase in Newly Diagnosed Childhood Acute Lymphoblastic Leukemia: Results from a Single Agent Window Study.
Authors: Appel Inge M. , Kazemier Karin M. , Veerman Anjo J.P. , van Wering Elisabeth , Den Boer Monique L. , Pieters Rob
Published: November 2019

L-Asparaginase is an effective drug for treatment of children with acute lymphoblastic leukemia. The effectiveness is generally thought to result from a rapid depletion of asparagine in serum and cells. Several studies have shown that in vitro resistance to this drug is an independent prognostic factor in ALL. We investigated the clinical response of one in vivo dose of 1000 IU/m2 PEG-Asparaginase and its pharmacokinetic and pharmacodynamic effects in children with newly diagnosed ALL before the start of combination chemotherapy. 57 children (36M / 21F) were enrolled in the study: 2 pro B-ALL, 38 common/ pre B-ALL and 17 T-ALL. Genotyping of precursor B-ALL revealed 11 hyperdiploid, 8 TELAML1 positive, 2 BCRABL positive, no MLL rearrangement, 8 normal, 11 others. The clinical response to PEG-Asparaginase on day 0 (5 days after the PEG-Asparaginase infusion) was defined as good when the number of leukemic cells of peripheral blood was < 1 × 109/L, as intermediate when leukemic cells were 1-10 × 109/L, and as poor when leukemic cells were > 10 × 109/L. The in vivo window response was significantly related to immunophenotype and genotype: 26/38 common / pre B-ALL cases, especially those with hyperdiploidy and TELAML1 rearrangement, demonstrated a good clinical response compared to 8/17 T-ALL (p=0.01). Both BCRABL positive ALL cases showed a poor response (p=0.04). A poor in vivo clinical window response was related to in vitro resistance to L-Asparaginase (p=0.02) and both in vitro as well as in vivo response were prognostic factors for long-term event-free survival (Hazard ratio 6.4; p=0.004, and Hazard ratio 3.7; p=0.01, respectively). The L-Asparaginase activity in the serum was >100 IU/L for at least 15 days. The asparagine levels remained below the detection limit of 0.2 mM for at least 26 days with a concomitant rise in serum aspartate and glutamate. These findings confirm that PEG-Asparaginase will yield its pharmacodynamic effects for 2-4 weeks. After administration of one in vivo dose of 1000 IU/m2 PEG-Asparaginase no changes in apoptotic parameters or changes in intracellular levels of twenty amino acids in leukemic cells could be measured, in contradiction to the changes found after in vitro exposure. This may be explained by the rapid removal of apoptotic cells from the circulation in vivo. Otherwise it is possible that in vivo mesenchymal cells from the bone marrow supply leukemic blasts with asparagine in response to treatment with L-Asparaginase.

Conclusion: The clinical response to one dose of 1000 IU/m2 PEG-Asparaginase intravenously is related to phenotype and genotype and predicts outcome. These results suggest that children with ALL with a poor clinical response to PEG-Asparaginase might benefit from a more intensive antileukemic therapy.
Different FLT3 Inhibitors Display Limited, Non-Overlapping Profiles of Specific FLT3-ITD Resistance Mutations in a Cell-Based Screen.
Authors: von Bubnoff Nikolas , Saenger Jana , Peschel Christian , Duyster Justus
Published: November 2019

Mutations causing resistance to therapeutic kinase inhibition can be identified in target kinases in various malignant diseases, such as Bcr-Abl in CML, FLT3-ITD in AML, cKit in GIST, EGFR in NSCLC, and FIP1L1-PDGFRalpha in HES/CEL. Thus, mutations in target kinases constitute a general mechanism of resistance to therapeutic kinase inhibiton. It has been shown that sensitivity toward tyrosine kinase inhibitors varies between different activating mutations of the FLT3 receptor. We therefore intended to determine, whether different FLT3 inhibitors would produce distinct profiles of secondary, FLT3 resistance mutations. Using a cell-based screening approach, we generated FLT3-ITD expressing cell lines resistant to the FLT3 inhibitors su5614 and PKC412. The frequency of resistant clones per million cells in the presence of inhibitor at 10 times the IC50 was 0.17 for su5614 (1,5?M) and 0 with PKC412 (100nM). When FLT3-ITD transformed cells were mutagenized with ENU (ethylnitrosourea) prior to addition of inhibitors, the frequency of resistant clones increased to 2.33 with su5614, and to 0.34 with PKC412. When we analyzed FLT3-ITD TK1 and TK2 for mutations that may confer inhibitor resistance, we found mutations in 127 out of 179 cell clones (71%) with su5614. Nine different exchanges affecting six positions in TK2 were identified, within or shortly behind the FLT3-ITD A-loop, with exchanges of D835 predominating. We did not detect exchanges affecting TK1. With PKC412, four out of 26 resistant clones (15%) contained mutations. In contrast to su5614, no mutations of TK2 were identified. However, three different amino acid exchanges emerging with PKC412 exclusively affected N676, which is located in TK1 of the FLT3-ITD split kinase domain. This is in line with the observation of a FLT3-ITD N676K exchange identified in a single AML patient with clinical resistance to PKC412 (Heidel et al, 2006). When expressed in cell lines, the identified TK2 A-loop mutations shifted dose-response curves to higher concentrations of su5614, but did not affect response to PKC412, su11248 or sorafenib. However, Y842D caused a strong resistance towards sorafenib, but not to su11248 or PKC412. Recent results also demonstrate a differential response of TK1 N676 exchanges towards different FLT3 inhibitors. We are currently analyzing su11248- and sorafenib- resistant FLT3-ITD clones for specific resistance mutations. Our results demonstrate that cell-based resistance screening is a useful and valid tool for prediction of resistance mutations to kinase inhibitors. In contrast to Bcr-Abl kinase inhibitors such as imatinib, nilotinib and dasatinib, which display highly overlapping resistance profiles, FLT3 kinase inhibitors may generate distinct, non-overlapping profiles. This opens the possibility of using combined FLT3 inhibitor therapy in AML to prevent resistance due to FLT3-ITD kinase domain mutations.
Overexpression of APC Reduces Stem Cell Capacity and Leukemogenic Potential of PLZF/RAR-Positive Leukemic Stem Cells.
Authors: Seshire Anita , Beissert Tim , Oancea Claudia , Hoelzer Dieter , Ruthardt Martin , Puccetti Elena
Published: November 2019

Acute myeloid leukemia (AML)-associated fusion proteins (AAFP), such as PML/RAR, PLZF/RAR or AML1/ETO have leukemogenic potential in vivo which is related to their capacity to induce a differentiation block and aberrant self renewal in early hematopoietic progenitors. The potential of the AAFP to induce the leukemic phenotype is strictly related to their capacity to oligomerize and to form high molecular weight complexes (HMW). Furthermore we recently showed that the deregulation of the Wnt-signaling seems to be a key event for the leukemogenesis by the above mentioned AAFP because it is indispensable for the aberrant self renewal of leukemic stem cells (LSC). A „tandem affinity purification” (TAP) screen of KG1 cells expressing PLZF/RAR, PML/RAR or AML-1/ETO for elucidating the composition of the related HMW revealed the „adenomatous polyposis coli” protein (APC) as an interaction partner of PLZF/RAR. APC is a key inhibitory regulator of the Wnt-signaling and is frequently inactivated by mutations in colon carcinomas. Therefore the role of the interaction between APC and PLZF/RAR for the leukemogenesis was further investigated. Here we report that i.) only PLZF/RAR strongly interacted with APC as confirmed by co-immunoprecipitation experiments; ii.) the overexpression of APC reverted the aberrant activation of the Wnt-signaling by PLZF/RAR as revealed by a reduction of the TCF/LEF mediated transcriptional activity; iii.) the overexpression of APC also reduced the self renewal potential of PLZF/RAR-positive HSC as revealed by a reduced replating efficiency of these cells in semi solid medium, as well as by a reduction of the colony number in colony forming units spleen (CFU-S) assays; iv.) APC was able to revert the leukemogenic potential of PLZF/RAR-positive LSC as revealed by the retroviral overexpression of APC in bone marrow isolated from mice with PLZF/RAR-induced AML which were inoculated into secondary recipients. In fact until now the presence of APC significantly increased the survival of these secondary recipients with respect to mock-infected controls. Taken together these data strongly suggest that the direct interaction with PLZF/RAR is an important mechanism for the functional inactivation of APC which contributes to the aberrant activation of the Wnt-signaling in PLZF/RAR-positive cells. Furthermore our data provide further evidence that the aberrantly activated Wnt-signaling pathway represents a valid target for molecular therapy approaches.
The Structure of the GM-CSF Receptor Ternary Complex Reveals a New Mode of Cytokine Receptor Activation.
Authors: Lopez Angel , Hercus Timothy , Hansen Guido , Woodcock Joanna , McClure Barbara , Stomski Frank , McKinstry William , Parker Michael Parker
Published: November 2019

Granulocyte-macrophage colony stimulating factor (GM-CSF) is a pleiotropic cytokine that controls the production and function of blood cells, is deregulated in multiple diseases such as asthma, arthritis and leukaemia yet offers therapeutic value in Crohn’s disease and as an adjuvant in anti-cancer therapy. Its receptors are heterodimers consisting of a ligand-specific alpha subunit and a beta subunit which is critical for signalling and is shared with the interleukin (IL)-3 and IL-5 receptors. The latter have also been implicated in pathologies such as acute myeloid leukaemia and allergic inflammation respectively. Despite the clear involvement of this family of receptors in human diseases their three dimensional structures in complex with the cognate ligand is not known and the mechanism by which they signal has remained an enigma. In particular it has baffled scientists that the membrane proximal domains of the beta subunit are 120A apart, too big a distance to allow transphosphorylation of the receptors by their associated JAK-2 kinases and receptor activation. We report here the structure of the human GM-CSF/GM-CSF receptor ternary complex and its assembly into hexamers (comprising 2 molecules of GM-CSF, alpha subunits and beta subunits each) and into unexpected dodecamers or higher order complexes. The dodecamer arrangement allows the interaction of two hexamers through a distinct site 4 which is composed of both alpha and beta subunits. Importantly, mutagenesis of the GM-CSF receptor at the dodecamer interface revealed that whilst site 4 is not involved in high or low affinity binding of ligand it is critical for receptor activation and signalling. This novel form of receptor assembly likely applies also to the IL-3 and IL-5 receptors, providing a structural basis for understanding cytokine receptor activation and for the development of novel therapeutics.
The t(6;9) Associated DEK/CAN Fusion Protein Does Not Block Differentiation of Early Hematopoietic Stem Cells but Increases Their Stem Cell Capacity.
Authors: Oancea Claudia , Puccetti Elena , Henschler Reinhard , Ruthardt Martin , Bug Gesine
Published: November 2019

Leukemia-specific translocations such as t(15;17), t(11;17), or t(8;21) lead to the expression of aberrant fusion proteins (FP) such as PML/RAR, PLZF/RAR and AML-1/ETO. These FP induce and maintain the leukemic phenotype by blocking terminal differentiation of early hematopoietic progenitors and by interfering with the biology of the leukemic counterpart of the hematopoietic stem cells. The t(6;9) with its DEK/CAN FP is of particular interest because it is more frequent in young patients and associated with a poor prognosis. The t(6;9)-DEK/CAN fusion occurs with an incidence of 1–5% in adult patients with AML and in most of the cases t(6;9)-positive AML is classified as FAB-M2 or M4. In contrast to other FP the overexpression of DEK/CAN does not block VitD3-induced differentiation in U937 cells. These findings called in question the leukemogenic potential of DEK/CAN. To further disclose the role of DEK/CAN in leukemogenesis we investigated its effect on the biology of early hematopoietic stem cells (HSC) in comparison to PML/RAR. Therefore we retrovirally transduced Sca1+/linmurine HSC (SL cells) with DEK/CAN and PML/RAR and performed both differentiation and stem cells assays (morphology, surface marker expression, replating efficiency, colony forming unit - spleen - CFU-S). Here we show that i.) DEK/CAN in contrast to PML/RAR was apparently unable to block terminal differentiation of SL cells as revealed by morphology and the expression of differentiation-specific surface markers; ii.) DEK/CAN slightly increased the replating efficiency of SL cells, but did not reach the level of PML/RAR with a reduced number of either colony forming units as plating rounds with respect to PML/RAR; iii.) the increased replating efficiency was related to an increased stem cell capacity as revealed by the fact that in contrast to mock infected control cells DEK/CAN-positive cells gave origin to a positive CFU-S assay even after one or two plating rounds in semi-solid medium similar to PML/RAR. Apparently DEK/CAN seems to share with other FP the capacity to increase the self renewal of HSC, but not that to block terminal differentiation. These findings strongly suggest that the effect of DEK/CAN is limited to a very small subset of cells within the stem cell compartment which might represent the origin of a DEK/CAN-positive leukemia. Actually the capacity of DEK/CAN to give origin to leukemia in vivo is under investigation.