Ellen Friedman

Reduced SMAD7 Leads to Overactivation of TGF-β Signaling in MDS that Can Be Reversed by a Specific Inhibitor of TGF-β Receptor I Kinase

Even though myelodysplastic syndromes (MDS) are characterized by ineffective hematopoiesis, the molecular alterations that lead to marrow failure have not been well elucidated. We have previously shown that the myelosuppressive TGF-β pathway is constitutively activated in MDS progenitors. Because there is conflicting data about upregulation of extracellular TGF-β levels in MDS, we wanted to determine the molecular basis of TGF-β pathway overactivation and consequent hematopoietic suppression in this disease. We observed that SMAD7, a negative regulator of TGF-β receptor I (TBRI) kinase, is markedly decreased in a large meta-analysis of gene expression studies from MDS marrow-derived CD34(+) cells. SMAD7 protein was also found to be significantly decreased in MDS marrow progenitors when examined immunohistochemically in a bone marrow tissue microarray. Reduced expression of SMAD7 in hematopoietic cells led to increased TGF-β-mediated gene transcription and enhanced sensitivity to TGF-β-mediated suppressive effects. The increased TGF-β signaling due to SMAD7 reduction could be effectively inhibited by a novel clinically relevant TBRI (ALK5 kinase) inhibitor, LY-2157299. LY-2157299 could inhibit TGF-β-mediated SMAD2 activation and hematopoietic suppression in primary hematopoietic stem cells. Furthermore, in vivo administration of LY-2157299 ameliorated anemia in a TGF-β overexpressing transgenic mouse model of bone marrow failure. Most importantly, treatment with LY-2157199 stimulated hematopoiesis from primary MDS bone marrow specimens. These studies demonstrate that reduction in SMAD7 is a novel molecular alteration in MDS that leads to ineffective hematopoiesis by activating of TGF-β signaling in hematopoietic cells. These studies also illustrate the therapeutic potential of TBRI inhibitors in MDS.

Multiplicative interaction between mean corpuscular volume and red cell distribution width in predicting mortality of elderly patients with and without anemia

Recent studies have shown that an elevated red cell distribution width (RDW) is an important predictor of adverse outcomes. However, the strength of this biomarker has not been tested in a large outpatient elderly population. Also since increased RDW can be due to a variety of etiologies, additional biomarkers are needed to refine the prognostic value of this variable. We assembled a cohort of 36,226 elderly (≥65yo) patients seen at an outpatient facility within the Einstein/Montefiore system from January 1st 1997 to May 1st 2008 who also had a complete blood count performed within 3 months of the initial visit. With a maximum follow‐up of 10 years, we found that an elevated RDW (>16.6) was associated with increased risk of mortality in both non‐anemic (HR = 3.66, p < 0.05) and anemic patients (HR = 1.87, p < 0.05). The effect of RDW on mortality is significantly increased in non‐anemic patients with macrocytosis (HR = 5.22, p < 0.05) compared to those with normocytosis (HR = 3.86, p < 0.05) and microcytosis (HR = 2.46, p < 0.05). When comparing non‐anemic patients with both an elevated RDW and macrocytosis to those with neither, we observed an elevated HR of 7.76 (higher than expected in an additive model). This multiplicative interaction was not observed in anemic patients (HR = 2.23). Lastly, we constructed Kaplan‐Meier curves for each RDW/MCV subgroup and found worsened survival for those with macrocytosis and an elevated RDW in both anemia and non‐anemic patients. Based on our results, the addition of MCV appears to improve the prognostic value of RDW as a predictor of overall survival in elderly patients. Am. J. Heamtol. 88:E245–E249, 2013.

Abnormal platelet count is an independent predictor of mortality in the elderly and is influenced by ethnicity

Even though alterations in platelet counts are presumed to be detrimental, their impact on the survival of patients has not been studied in large cohorts. The prevalence of thrombocytopenia and thrombocytosis was examined in a large inner city outpatient population of 36,262 individuals aged ≥65 years old. A significant association with shorter overall survival was found for both thrombocytopenia (HR=1.45; 95% CI: 1.36–1.56) and thrombocytosis (HR=1.75; 95% CI: 1.56–1.97) when compared to the survival of patients with normal platelet counts. This effect persisted across all ethnic groups. However, African-Americans (non-Hispanic Blacks) with either thrombocytopenia or thrombocytosis were at significantly lower risk compared to non-Hispanic Caucasians (HR=0.82; 95% CI: 0.69–0.96 and HR=0.70; 95% CI: 0.53–0.94, respectively). Furthermore, Hispanics with thrombocytosis were found to have a lower mortality risk compared to non-Hispanic Caucasians with thrombocytosis (HR=0.60; 95% CI: 0.44–0.81). A value of <125,000 platelets per microliter was a better prognostic marker for non-Hispanic Blacks and these subjects with this platelet count had similar overall survival to that of Caucasians with a value of <150,000 per microliter. In conclusion, thrombocytosis and thrombocytopenia are independently associated with shorter overall survival in elderly subjects and this effect is modified by ethnicity. Using different thresholds to define the association of thrombocytopenia and thrombocytosis with overall mortality risk among non-Hispanic Blacks may, therefore, be warranted.

Ruxolitinib Withdrawal Syndrome Leading to Tumor Lysis

Case Report A 70-year-old woman with a history of JAK2 V617F mutation– positive post–polycythemia vera (PV) myelofibrosis (MF) presented to the emergency department 4 weeks after discontinuing ruxolitinib with abdominal pain and massive splenomegaly, acute renal failure, hyperkalemia, hyperuricemia, hypocalcemia, and hyperphosphatemia. She was diagnosed with PV 10 years before admission. At that time she had a hemoglobin (Hgb) of 18 g/dL, an elevated red cell mass (51.3 mL/kg; reference range [RR], 24-30 mL/kg), and a hypercellular bone marrow (98%) with clustered megakaryocytes without fibrosis. Cytogenetic analysis showed 70% of cells with trisomy 9 and deletion 20q. She carried the JAK2 V617F mutation, and reverse transcriptase polymer chain reaction was negative for the BCR/ABL transcript. Her WBC count was 25.5 10/ L (RR 4.8-10.8 10/ L), with a platelet count of 210 10/ L (RR, 150-400 10/ L). Abdominal ultrasound showed hepatosplenomegaly, and she was managed with phlebotomy. Six years later she complained of weight loss, night sweats without fever, fatigue, and left upper quadrant abdominal pain resulting from worsening splenomegaly. She was subsequently treated with pegylated interferon, which improved her symptoms and spleen size. Symptoms returned 2.5 years into the course of pegylated interferon and a CBC showed worsening anemia (Hgb, 9.9 g/dL) and thrombocytopenia (platelets, 76 10/ L). A bone marrow biopsy showed hypercellular (100%) marrow with 2/4 reticulin and no significant increase in blasts (Fig 1A, reticulin stain, 40). Cytogenetic analysis revealed no clonal evolution. She subsequently responded to low-dose hydroxyurea, but had recrudescence of symptoms after only 8 months. Red cell morphology was markedly abnormal with poikilocytosis, anisocytosis, and teardrop cells (Fig 2, 100). She was started on low-dose ruxolitinib (10 mg twice daily). Within 5 weeks she experienced improvement in her systemic symptoms and splenomegaly, but had a progressive fall in her Hgb and platelet count with new bruises. Examination of her bone marrow showed marked myelofibrosis with 70% to 80% cellularity (Fig 1B, reticulin stain, 40x), and a new clone with trisomy 8 in addition to the previously seen trisomy 9 and deletion 20q. Again there was no significant increase in blasts. Seven and half weeks after treatment was started, her Hgb dropped to 8.0 g/dL, and platelets decreased to 28 10/ L. The dose of ruxolitinib was reduced to 5 mg twice daily. Despite the low-dose of ruxolitinib, blood counts further deteriorated (Hgb, 7.9 g/dL; platelets, 18 10/ L) and ruxolitinib was stopped after only 9 weeks. Over the next 3 weeks she developed recurrent fever, worsening shortness of breath, diarrhea, and accelerated splenomegaly. She had a gout attack and was treated with prednisone 40 mg daily. A diagnosis of ruxolitinib withdrawal syndrome was considered. Her symptoms deteriorated and she was referred to the emergency department for urgent evaluation. There she was found to be tachycardic with massive splenomegaly and pitting bilateral lower extremity edema. Laboratory tests revealed acute kidney injury (serum creatinine, 2.2 mg/dL; RR, 0.5-1.5 mg/dL; baseline, 1.1 mg/dL), hyperkalemia (potassium, 5.8 mg/dL; RR, 3.5-5.0 mEq/L), hypocalcemia (corrected calcium, 6.6 mg/dL; RR, 8.5-10.5 mg/dL), hyperphosphatemia (phosphorus, 5.9 mg/dL; RR, 2.5-4.5 mg/dL), and hyperuricemia (uric acid, 21.4 mg/ dL; RR, 2.5-8.0 mg/dL). Her lactate hydrogenase was increased at 1,291 U/L (RR, 110-210 U/L), higher than her baseline (756-873 U/L). Her CBC was similar to 1 month prior when ruxolitinib had been stopped (Hgb, 7.1 g/dL; WBC, 30.9 10/ L; platelets, 18 10/ L). An electrocardiogram (EKG) showed peaked T waves without PR prolongation; urine analysis was positive for leukocytes, bacteria, and uric acid crystals. Her chest x-ray was negative for focal infiltrates, vascular congestion, or pleural effusions. She was treated for a presumptive diagnosis of tumor lysis syndrome (TLS) with aggressive hydration and rasburicase (0.2 mg/kg); for hyperkalemia with EKG A

Hereditary spherocytosis in the elderly

Five patients with hereditary spherocytosis diagnosed in their seventh to ninth decades of life are presented. These patients are remarkable for absent or mild clinical manifestations of disease. Splenectomy is the recommended treatment for hereditary spherocytosis to avoid the complications of aplastic or hemolytic crisis. When the diagnosis is made in the elderly, the treatment of choice may be careful observation with folic acid supplementation rather than splenectomy. This recommendation is based on the incidence of complications of splenectomy in the elderly in comparison to the severity and incidence of complications from the disease itself.

Safe and successful bone marrow biopsy: an anatomical and CT-based cadaver study.

Bone marrow biopsy is generally a safe procedure. However, infrequently the procedure is associated with serious injuries that are attributed to inadvertent needle penetration of the iliac bones inner cortex. An evidence‐based approach to needle orientation during iliac crest biopsy does not exist. In our study, the posterior to anterior path of the bone marrow needle from the posterior superior iliac spine (PSIS) was studied in human cadavers in two orientations: (1) perpendicularly to the coronal plane (the perpendicular approach) and (2) laterally toward the ipsilateral anterior superior iliac spine (ASIS) (the lateral approach). The biopsy needle was deliberately advanced through the inner ilial cortex in both approaches. Dissections and imaging studies were done to identify the relationship of the penetrating needle to internal structures. Both approaches begin with a perpendicular puncture of the outer cortex at the PSIS. The perpendicular approach proceeds anteriorly whereas in the lateral approach the needle is reoriented toward the ipsilateral ASIS before advancing. The lateral approach caused less damage to neurovascular structures and avoided the sacroiliac joint compared to the perpendicular approach. This procedure is best done in the lateral decubitus position. Proper use of the lateral approach should obviate many of the complications reported in the literature. Am. J. Hematol. 89:943–946, 2014.

Analysis of chronic myelogenous leukemia in an underserved, inner-city cohort shows a significant five year overall survival that is not affected by choice of tyrosine kinase inhibitor

Tyrosine kinase inhibitors (TKIs) have become the firstline treatment of choice for chronic myelogenous leukemia (CML) after imatinib was shown to offer improved and durable responses.[1,2] Second generation TKIs such as nilotinib [3,4] and dasatinib [5,6] have shown superior efficacy in achieving faster remissions in the first-line setting. Patients who achieve a complete cytogenetic response at two years have a life span similar to that of the general population as long as they receive adequate therapy and adhere to treatment.[7] Despite efficacy, the cost of TKIs created a significant financial burden which led leaders in the field to speculate on whether such prices are justifiable.[8] Individuals with ‘adequate’ healthcare coverage are not immune and may have a 20% out of pocket co-payment. Adherence to therapy is essential to achieve adequate responses.[9] Higher socioeconomic status was linked to better imatinib adherence [10] and patients with higher copayments were more likely to have poorer adherence.[11] To examine the impact of cost of TKIs on efficacy, we conducted a retrospective analysis to determine the treatment patterns in an inner-city population comprised predominantly of ethnic minorities with low socioeconomic status. We aimed to study access to different generations of TKI, reasons for switching TKIs as well as overall survival (OS). We included CML cases that presented to Montefiore Medical Center between 1997 and 2014 in the Bronx, New York. Cases were identified by a data-mining software (Clinical Looking Glass , CLG) used to search by ICD-9 diagnosis code. Records were manually reviewed to confirm diagnosis and to collect relevant demographic and CML-specific data. The population number (n value) was adjusted to reflect the number of records with available data pertaining to each variable. As such, the number of evaluable records for phase at diagnosis, first-line therapy, line of therapy at the conclusion of the study, and line of therapy at expiration was 77, 118, 125, and 20 patients, respectively. All chart review was conducted by one of the study authors and discrepancies were reviewed by at least two authors. This research was approved by our institutional review board and ethics committee. For comparison, we obtained data from the Surveillance, Epidemiology and End Results (SEER) program database. SEER collects cancer incidence, treatment, and survival information from 18 geographic areas in the United States, representing 28% of the entire population. We used direct case listings extracted by SEER*Stat software (version 8.1.5, released March 31, 2014) and included patients with a diagnosis of CML with the International Classification of Disease for Oncology, third edition, ICD-0-3 histology code 9863, 9875, 9876, 9945, 9946 until the latest follow-up recorded in the SEER submission. For analysis of categorical variables, we reported proportions and p values calculated with Pearson chi square or Fisher exact test as appropriate. Kaplan–Meier curves were used to compare survival and significance was examined using the log rank test. Statistical analyses were performed with computer software (SPSS 18, SPSS, Inc., Chicago, IL) and a two-tailed alpha of 0.05 was used to denote significance.

Visualization of the bone marrow biopsy needle track

REFERENCES [1] DeLoughery TG. Microcytic anemia. N Engl J Med. 2014;371(14): 1324–1331. [2] Gunshin H, Mackenzie B, Berger UV, et al. Cloning and characterization of a mammalian proton-coupled metal-ion transporter. Nature. 1997;388(6641):482–488. [3] Wolff NA, Ghio AJ, Garrick LM, et al. Evidence for mitochondrial localization of divalent metal transporter 1 (DMT1). FASEB J. 2014; 28(5):2134–2145. [4] Iolascon A, Camaschella C, Pospisilova D, Piscopo C, Tchernia G, Beaumont C. Natural history of recessive inheritance of DMT1 mutations. J Pediatr. 2008;152(1):136–139. [5] Van Allen EM, Wagle N, Sucker A, et al. The genetic landscape of clinical resistance to RAF inhibition in metastatic melanoma. Cancer Discov. 2014;4(1):94–109. [6] Wang CY, Knutson MD. Hepatocyte divalent metal-ion transporter-1 is dispensable for hepatic iron accumulation and non-transferrinbound iron uptake in mice. Hepatology. 2013;58(2):788–798.

Analysis of overall survival in a large multiethnic cohort reveals absolute neutrophil count of 1,100 as a novel prognostic cutoff in African Americans

Although absolute neutrophil counts (ANC) below 1.5x103/uL are used to define neutropenia as a marker of increased susceptibility to infections, their relationship with survival has not been examined. Since low counts trigger extensive investigations, determining prognostic cutoffs especially for different ethnicities and races is critical. A multiethnic cohort of 27,760 subjects, 65 years old and above, was utilized to evaluate the association of neutropenia with overall survival in different ethnicities and races. The mean ANC was 4.6±1.51x103/uL in non-Hispanic whites, 3.6±1.57x103/uL in non-Hispanic blacks and 4.3±1.54x103/uL in Hispanics (p<0.001). An ANC below 1.5x103/uL was associated with significantly shorter overall survival among whites (HR 1.74; 95% CI 1.18 - 2.58; p<0.001), but not in blacks (HR 0.89; 95% CI 0.86 - 1.17; p=0.40) or Hispanics (HR 1.04; 95% CI 0.76 - 1.46; p=0.82), after adjustment for age, sex, comorbidities, anemia and thrombocytopenia. Using Cox regression multivariable models, an ANC below 1.1x103/uL in blacks was found to be associated with increased mortality (HR 1.86; 95%CI 1.21 - 2.87; p<0.01). We found no association between neutropenia and mortality at any ANC cutoff in elderly Hispanics. In conclusion, neutropenia was found to be an independent prognostic variable in the elderly, when determined in race-specific manner. Most importantly, a cutoff of 1.1x103 neutrophils/uL may be a more prognostically relevant marker in elderly blacks and could serve as a novel threshold for further evaluation and intervention in this population.

Feasibility and safety of targeting the anterior superior iliac spine to perform a bone marrow procedure: a prospective, clinical study

Aims The bone marrow procedure (BMP) has been performed worldwide for years. Nonetheless, no generally accepted standards or guidelines for the performance of the BMP exist. Recent studies suggested that the lateral angulation technique (LAT), targeting the anterior superior iliac spine (ASIS) after penetration of the posterior superior iliac spine, yields longer biopsy cores and is safer for patients. We assessed the feasibility and safety of targeting the ASIS in the prone and lateral decubitus positions. Methods We first observed the BMP needle tracks on cadavers. Our cadaver study revealed that the LAT is feasible and safe but requires different operator techniques. Next, we studied 25 adult haematology patients undergoing elective BMP via the LAT approach. Patients returned 5 days after the BMP for a haemoglobin assessment, pain questionnaire and low-dose non-contract CT. Results 8% of patients reported persistent pain. No fall in haemoglobin and no pelvic haematomas or neurovascular injuries were detected. 88% of BMPs were successfully accomplished by targeting the ASIS. 12% required a back-up traditional angulation technique (TAT), directing the needle straight in, perpendicular to the coronal plane of the back. All three demonstrated inadvertent, but asymptomatic, penetration of the sacrum. Biopsy lengths were compared with a historical TAT control demonstrating that specimens obtained by LAT are significantly longer. Imaging studies showed that a seven-degree change in needle direction can convert a TAT to a LAT. Conclusion The LAT approach is feasible, safe and more productive than the TAT, and may be the preferred standard for training haematologists. Trial registration number NCT02524613.