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Introduction: Geriatric assessment (GA) is a multidimensional evaluation of patient health and function that may detect impairments not identified as part of routine care, predict treatment-related morbidity and mortality, and inform treatment plans. Given evidence of these benefits, the National Comprehensive Cancer Network Clinical Practice Guidelines in Oncology (NCCN Guidelines®) recommend GA for older candidates of hematopoietic stem cell transplantation (HCT). However, both older and younger HCT candidates will often receive multiple rounds of chemotherapy before HCT, leading to functional impairments in all age groups. Furthermore, HCT patients often experience a significant gap between when they are first evaluated and actually proceed to transplant (e.g., while a donor search is conducted), creating an opportunity to identify impairments and optimize function prior to transplant. Methods: To address this opportunity, we created a clinical pre-HCT optimization program (C-POP) to evaluate physical function, cognitive function, nutritional status, and mental health in all adults who were deemed potential candidates for allogeneic HCT by a HCT physician. We applied this standard of care program to all adult candidates for HCT, regardless of age, with the goal of identifying functional impairments and then referring patients to services to optimize those impairments prior to HCT. We defined impairments using validated measures and compared results to established norms or scoring, controlling for age and gender where appropriate (e.g., the cut-off for six-minute walk distance was adjusted for age, gender, height, and weight, while the cut-off for falls was any fall regardless of characteristics). Patients with impairments were referred to the appropriate supportive care (e.g., physical function impairment -> referral to physical therapy). Results were prospectively analyzed at new patient evaluation (NPE), which was the first time the patient met a HCT physician and sign-off, which occurred within a week before starting transplant. While the program is ongoing, we present here the results of patients evaluated between October 16th, 2017 and July 1st, 2019. Patients are divided into three pre-specified age groups: =60 years old, with results compared using a chi-squared test. Results: We evaluated 115 patients: 21 (18%) =60 years). There were no differences between the age groups in other demographics (gender, race, and ethnicity). At NPE, 93 (81%) met criteria for at least 1 impairment in physical function, cognitive function, nutritional status, or mental health; 62 (54%) met criteria for impairments in 2 or more areas. Surprisingly, patients =60 years (36/54, 67%) (p=0.03). Of those 115 patients, 52 (45%) proceeded to HCT, including 12 (57%) =60 years (p=0.75); of those patients who have not proceeded to HCT, 40 (35%) will never proceed to HCT (e.g., deemed not a candidate after functional evaluation or died of disease prior to HCT) while 23 (20%) are still awaiting HCT (e.g., donor search ongoing). Patients who proceeded to HCT were less likely to have mental health impairments (2/52, 4% vs. 9/40, 23%, p=0.006). Of the 52 who were seen at new patient evaluation and proceeded to transplant, 40 (77%) were seen at sign off. Of those who had impairments at NPE, 12/23 (52%) improved their physical function to normal limits, 4/9 (44%) improved their cognitive function, and 9/13 (69%) improved their nutritional status by the time of sign-off (of those who were seen at sign-off, none had mental health impairments at NPE). Discussion: These results demonstrate that younger as well as older candidates for HCT exhibit a high degree of functional impairment. However, this impairment could be amenable to improvement prior to HCT. These findings support application of GA to all HCT candidates regardless of age. We will investigate the effect of referred interventions (e.g., physical therapy, seeing a dietician) in improving functional impairments in future studies, as well as look at the effect of these findings on HCT outcomes. Disclosures Wiggins: Incyte, Inc.: Speakers Bureau. Gasparetto:Janssen: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; Celgene: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed ; BMS: Consultancy, Honoraria, Other: Travel, accommodations, or other expenses paid or reimbursed . Rizzieri:Millennium: Speakers Bureau; Novartis: Consultancy; AbbVie: Consultancy; Pfizer: Consultancy; Amgen: Consultancy; Incyte: Consultancy, Speakers Bureau; TEVA: Consultancy; Spectrum: Consultancy; Kite Pharma: Consultancy; Gilead Sciences: Consultancy, Speakers Bureau; Seattle Genetics: Consultancy, Speakers Bureau; Jazz Pharmaceuticals: Speakers Bureau. Horwitz:Abbvie Inc: Membership on an entity's Board of Directors or advisory committees. Sung:Novartis: Research Funding; Merck: Research Funding; Seres: Research Funding.

We have investigated human lactate dehydrogenase (LDH) isoenzymes and human nuclear matrix protein 41/7 (NMP 41/7) as potential serologic markers to monitor the course of human leukemia in severe combined immunodeficient (SCID) mice. Following the transplantation of 10(6) human acute lymphoblastic leukemia (ALL) Nalm-6 cells, human specific LDH isoenzymes were measurable in the serum of SCID mice as early as 7 days after transplantation, although serum total LDH increased in some animals as early as 5 days after transplantation. Human NMP 41/7 was measurable in all animals at day 15 after leukemia cell injection. Serum levels of total LDH, human specific LDH and NMP 41/7 increased progressively over time, reaching total LDH levels as high as 50,000 U/L at day 25 after transplantation. To determine whether the levels of LDH and NMP 41/7 in serum were a reflection of human tumor burden, we studied these serologic markers in SCID mice bearing measurable subcutaneous human neuroblastoma tumors, or compared the serum levels of these markers with the number of human leukemia CD10+ cells in the bone marrow of the SCID mice. The serum levels of total LDH, human specific LDH isoenzymes, and NMP 41/7 correlated well with tumor burden, and they drastically decreased or disappeared from serum after the human leukemia or neuroblastoma cells were selectively killed with a single intravenous (IV) injection of 1 to 3 micrograms diphtheria toxin (DT) (the cellular receptor for DT is present on human cells, but not on mouse cells). Paraplegic mice with central nervous system leukemia completely recovered after DT treatment. We conclude that measurements of serum levels of total LDH, human LDH isoenzymes, and NMP 41/7 are sensitive, quantitative, rapid, and easy to perform serologic methods useful to monitor the engraftment, progression, and treatment response of human leukemia in SCID mice.

Background: Contemporary approaches to non-Hodgkin lymphoma (NHL) and chronic lymphocytic leukemia (CLL) incorporate chemo-immunotherapy, biologic combinations, and immune modulating agents; toxicities in elderly pts (³65 years) receiving these therapies are not well studied. Further, clinical and biologic factors predicting these toxicities in pts receiving biologic therapy remain undefined. Methods: We reviewed data on NHL and/or CLL pts treated prospectively on 14 studies by the Alliance for Clinical Trials in Oncology from 2004-2014 (Table 1). Toxicity was assessed per the NCI CTCAE at the time of trial enrollment, and the probabilities of experiencing grade 3 and grade 4 hematologic (hem) and non-hematologic (non-hem) toxicities were modeled as a function of age (³65 years vs. < 65), time on study, treatment (biologics only vs. biologic + chemotherapy), gender, race, LDH, performance status (PS), stage, and an age-by-treatment interaction using logistic regression. Results: A total of 1199 pts (409 age ³ 65; 790 age < 65; 736=CLL and 463=NHL) were included. Among these patients, 493 received only biologic therapy (166 age ³ 65; 327 age < 65; 104 CLL; 389 NHL), and 706 received biologic + chemotherapy (243 age ³ 65; 463 age < 65; 632 CLL; 74 NHL). Among CLL pts (259 pts ³ 65), the effect of age on the probability of experiencing a grade 3 heme toxicity differed by treatment type (age-by-treatment interaction p = 0.047). Specifically, the adjusted odds ratio (OR) (age ³ 65 vs. < 65) for pts receiving only biologic therapy was 3.075 (95% CI: 1,15-8.25), and that for pts receiving biologic + chemotherapy was 1.044 (95% CI: 0.69 - 1.57). Similar results were seen in CLL pts for grade 4 heme toxicities (age-by-treatment interaction p = 0.033; biologic OR 6.937, 95% CI: 1.76-27.35; biologic + chemo OR 1.484, 95% CI: 1.04-2.13). No such interactions were found in CLL pts for grade 3 and 4 non-hem toxicities; however, older pts had significantly higher odds of experiencing a grade 3 non-hem toxicity than younger pts (adjusted OR 1.40; p = 0.047; 95% CI: 1.004-1.96). No age group differences were found in CLL pts for grade 4 non-hem toxicity. Similar analyses were performed for NHL pts (150 pts ³ 65), but no significant age group differences were found. Among CLL pts, women had significantly higher odds of experiencing a grade 3 heme toxicity than men (OR 2.01; p = 0.0007); no difference in grade 3 non-hem or any grade 4 toxicity was noted. Non-Caucasian CLL pts had higher odds of experiencing a grade 4 non-hem toxicity than Caucasians (OR 2.892; p = 0.0029), but no other toxicity differences were found. Worse PS was associated with increased toxicities (OR 1.871; p = 0.0009: grade 3 heme; OR 1.647; p = 0.0025: grade 3 non-hem; OR 1.410; p = 0.0448: grade 4 heme, and OR 1.931; p = 0.0252: grade 4 non-hem). CLL pts with advanced stage disease had higher odds of experiencing a heme toxicity (grade 3: OR 1.95; p = 0.0007, grade 4: OR 1.451; p = 0.0329), but no stage associations were found for non-hem toxicities. Among NHL pts, men had significantly higher odds of experiencing a grade 4 hematologic toxicity than women (OR 4.351; p = 0.0169), but no other toxicity differences were found. An increase in LDH was associated with significantly higher odds of experiencing grade 3 non-hem and grade 4 heme toxicities (grade 3 non-heme: OR 1.633; p = 0.021, grade 4 heme: OR 2.039, p = 0.0182), but no such effect was found for grade 3 heme or grade 4 non-hem. Worse PS was associated with higher odds of experiencing grade 3 toxicities (heme: OR 2.025; p = 0.0344, non-hem: OR 2.458; p = 0.0013), but no such differences were found for grade 4 toxicities. No significant stage or race effects were found in NHL patients. Conclusion: In pts ³65 years who have CLL or NHL, we identified several clinical and disease-related factors as potential predictors of developing grade 3 and/or 4 heme and non-hem toxicities (Table 2). A prognostic model is being constructed to predict toxicities in these under-studied pts to guide management and monitoring. Further, the impact of these toxicities on outcomes is being analyzed and will be presented at the meeting. Disclosures Hurria: Celgene: Other: Research; Optum Health Care SOlutions: Consultancy, Other: Conference panel, research; Boehringer Ingelheim Pharmaceuticals: Consultancy; Sanofi: Consultancy; Carevive: Consultancy; Novartis: Other: Research; GTx, Inc: Consultancy. Bartlett:Gilead: Consultancy. Cheson:Gilead: Research Funding; Acerta: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pharmacyclics: Membership on an entity's Board of Directors or advisory committees, Research Funding. Nabhan:Infinity: Consultancy; Abbvie: Consultancy; Cardinal Health: Consultancy; Seattle Genetics: Research Funding; Genentech: Consultancy, Research Funding; Astellas: Research Funding; Celgene Corporation: Consultancy, Research Funding.

We evaluated event-free survival (EFS) and leukemia-free interval (LFI) of children treated for acute lymphoblastic leukemia (ALL). Patients were randomized to receive either a low dose or high dose of methotrexate (MTX) as a single agent at the time of diagnosis. Five days later, multidrug therapy was begun. We assessed the early antileukemic efficacy of the two doses of MTX, as well as toxicity and long-term efficacy. An increase in cell kill, as indicated by a larger decrease in the percentage of viable cells in the bone marrow between days 0 and 5, was observed for the high-dose MTX group when compared with the low-dose MTX group (P = .04). At 7.1 years of median follow- up, the 38 children randomized to receive high-dose MTX had a better EFS and LFI compared with the 39 patients randomized to receive low- dose MTX. The 7-year percentages (+/- SE) for EFS were 82% +/- 6% for high-dose MTX and 69% +/- 7% for low-dose MTX (P = .13). The 7-year percentages for LFI were 91% +/- 5% and 69% +/- 7%, respectively (P = .01). We recommend that high-dose MTX be considered as an effective addition to induction therapy in childhood ALL.

Erythrocyte CR1, a C3b/C4b-binding complement-regulatory protein, is sensitive to proteolysis in vitro. To test the hypothesis that in vivo erythrocyte CR1 reduction results from intravascular proteinase activities, we used enzyme-linked immunosorbent assays to measure gamma- crosslinked fibrin degradation products (D-dimers) as indicators of coagulation/fibrinolytic activity, and complexes of neutrophil elastase with alpha 1 proteinase inhibitor (E/A) as indicators of neutrophil enzyme release in malignant and inflammatory disorders. Erythrocyte CR1, measured by monoclonal anti-CR1 antibody binding, was inversely related to disease activity and blood proteinase markers. Levels of erythrocyte CR1 were significantly lower for patients with active versus remittent squamous and small cell lung cancers, Hodgkin's and diffuse large cell lymphomas, and acute myelogenous leukemias. In patients with active thoracic cancers, elevated D-dimer levels correlated with reduction of CR1. In patients with rheumatoid arthritis, CR1 reduction was correlated with elevated levels of elastase complexes. Our findings substantiate the relationship of acquired CR1 reduction to the activity of certain diseases and provide circumstantial support for the hypothesis that erythrocyte CR1 is lost to proteolysis in vivo. Although heritable differences in CR1 expression reduce the interpretability of single measurements of erythrocyte CR1 levels, disease-associated CR1 reduction may be a useful indicator of disorders with chronically increased blood proteinase activity.

LK, SS Equal contributions VG, JTP Equal credit as senior authors Prolyl hydroxylase 2 (encoded by EGLN1), the principle negative regulator of HIF-1 and HIF-2 (encoded by EPAS1), is an iron dependent enzyme (Kaelin WG et al. Mol Cell, 2008); thus iron deficiency can augment hypoxic responses (Zhang X et al Blood Cells Mol Dis, 2014). Because EPAS1 mRNA has a 5' iron regulatory element, iron deficiency may inhibit the translation of HIF-2α and downregulate its target genes (Sanchez M et al Nat Struct Mol Biol, 2007). The Partnership for Anemia: Clinical and Translational Trials in the Elderly conducted a trial to determine the efficacy of IV iron in patients with borderline iron deficiency (Price E et al. Blood Cells Mol Dis, 2014). We measured expression levels of HIF1A, EPAS1, EGLN1 and HIF target genes in platelets and granulocytes. Nineteen patients were treated with 200 mg IV iron sucrose weekly for 5 weeks. Blood was obtained at screening, one week after the second dose of IV iron and 8 weeks after completion of iron therapy. Granulocytes and platelets were isolated and their RNA reverse transcribed. Transcripts were quantified by real-time qT-PCR, and normalized to HPRT gene. The threshold cycle for the expression of each gene was calculated and compared to 5 healthy controls. We found a positive correlation between granulocyte and platelet mRNA of HIF1A, EPAS1 and EGLN1, and these relationships achieved a one-sided significance level of 0.1 or stronger for most data points for HIF1A and EPAS1 (Table 1). There was also a positive correlation for granulocyte and platelet mRNA of 5 of 7 hypoxia response genes (P of 0.1 or stronger in many comparisons; the strongest being for VEGF and PDK1). Table 1. Spearman correlation rho between granulocyte and platelet mRNAs (N = 17). Gene Screen TV2 FU2 Regulators of the hypoxic response HIF1A .52** .21 .40* EPAS1 .40* .54** .67** EGLN1 .28 .30 .09 Responders to the hypoxic response GLUT1 .44** .00 .07 HK1 .25 .20 .36* PDK1 .24 .55** .41* VEGF .68** .33* .62** FOXO3 -.10 .22 .45** BNIP3 .03 -.19 -.08 BNIP3L .15 -.05 .20 *one-sided P There were positive relationships in the expression of the three hypoxic response regulating genes with lower iron stores, as reflected in three measures, and these tended to be strongest for the TfR to log ferritin index (Table 2). These relationships also tended to hold for HK1, PDK1, VEGF and BNIP3L in both types of cells, but the relationships were stronger in platelets. Table 2. Spearman correlation for relationship between iron status and gene expression at screening. A negative correlation with ferritin and positive correlation with TfR and TfR/log ftn indicates increased gene expression with lower iron stores. Granulocytes Platelets Ferritin TfR TfR/log ftn Ferritin TfR TfR/log ftn Regulators of the hypoxic response HIF1A -.04 .05 .15 -.26 .41* .52** EPAS1 -.31 .090 .23 -.016 .15 .09 EGLN1 -.18 .26 .31 -.33* .57** .66** Responders to the hypoxic response GLUT1 .39* -.33 -.38* .27 .35* .23 HK1 -.20 .016 .07 -.22 .23 .23 PDK1 -.22 .31 .39* -.38* .16 .24 VEGF -.22 .12 .30 -.22 .48** .52** FOXO3 .10 -.097 -.061] -.19 -.15 -.14 BNIP3 -.04 -.10 -.007 -.18 -.30 -.25 BNIP3L -.08 .13 .21 -.36* .43** .50** Trends for change in gene expression with iron therapy were also observed. Iron therapy led to decreased expression of HIF1A and EGLN1 in both cell types but increased expression of EPAS1; these relationships were stronger in granulocytes, where they achieved a one-sided significance of P Expression of hypoxic response regulating genes tended to correlate between platelets and granulocytes, as did expression of one-half of hypoxic response genes tested. Furthermore, expected relationships with iron status were observed for the expression of HIF1A, EPAS1 and EGLIN1. However, variable responses were found in the expression of certain HIF target genes in response to iron therapy that may be related to a balance of decreased HIF-a protein with enhanced translation of HIF-2a. Transcripts of these HIF regulated and HIF pathway genes are being compared to normals and to non-anemic elderly subjects to determine if anemia in the elderly is associated with a blunted hypoxic response. Disclosures No relevant conflicts of interest to declare.

The Dana-Farber Cancer Institute (DFCI) Childhood ALL Consortium Protocol 95-01 was designed to minimize therapy-related morbidity for children with newly diagnosed ALL without compromising efficacy. Patients participated in randomized comparisons of (1) doxorubicin given with or without dexrazoxane, a cardioprotectant (high-risk patients), (2) intensive intrathecal chemotherapy and cranial radiation (standard-risk patients), and (3) Erwinia and Escherichia coli asparaginase (all patients). Between 1996 and 2000, 491 patients (aged 0-18 years) were enrolled (272 standard risk and 219 high risk). With a median of 5.7 years of follow-up, the estimated 5-year event-free survival (EFS) for all patients was 82% ± 2%. Dexrazoxane did not have a significant impact on the 5-year EFS of high-risk patients (P = .99), and there was no significant difference in outcome of standard-risk patients based on type of central nervous system (CNS) treatment (P = .26). Compared with E coli asparaginase, Erwinia asparaginase was associated with a lower incidence of toxicity (10% versus 24%), but also an inferior 5-year EFS (78% ± 4% versus 89% ± 3%, P = .01). We conclude that (1) dexrazoxane does not interfere with the antileukemic effect of doxorubicin, (2) intensive intrathecal chemotherapy is as effective as cranial radiation in preventing CNS relapse in standard-risk patients, and (3) once-weekly Erwinia is less toxic than E coli asparaginase, but also less efficacious.

Background Anemia is a common problem in older adults. Approximately one third of anemic adults 65 years and older have unexplained anemia of the elderly (UAE) as no clear cause can be identified. The Partnership for Anemia: Clinical and Translational Trials in the Elderly (PACTTE), an NIH funded consortium to perform interventional trials in this population, conducted a randomized prospective wait-list control study of the value of IV iron sucrose (IS). PACTTE consists of eight clinical sites (seven university based and one community based) and one data coordinating center. We hypothesized that UAE patients (pts) with moderate serum ferritin levels and no bleeding would benefit from iron repletion. Methods Pts 65 years and older with UAE, hemoglobin (Hb) values between 9 g/dL and 11.5 g/dL (in women) and 12.7 g/dL (in men) inclusive, serum ferritin levels between 20 and 200 ng/mL, and negative stool cards for occult blood, were randomized to receive 5 weekly doses of 200 mg of intravenous iron sucrose (Venofer®, Luitpold Pharmaceuticals), either immediately (immediate group) or to watch and wait (wait list group) for a 12 week waiting period. After 12 weeks, the wait-list control received IV Iron. The primary outcome was the difference in change in 6 minute walk test (6MWT) distance, from baseline to week 12 between the two groups. Secondary outcome measures included erythroid response, cognitive function, self-report function (FACT-An), quality of life (SF-36), and frailty (Fried Frailty Index). We targeted enrollment of 84 patients to provide 85% power to detect 50 meters difference in 6MWT distance. Results 43 pts were formally screened and 19 pts enrolled with a mean age of 78.5 years. 9 pts were randomized to the immediate group and 10 to the wait list group. The study was stopped due to poor accrual both related to strict eligibility and problems identifying pts to be screened. Baseline hematologic and demographic characteristics of the immediate and wait-list groups were similar except for a few variables such as lower serum iron (55.3 vs 84.5 mcg/dL, p=0.006) and transferrin saturation (12.4% vs 27.4%, p=0.015) in the immediate group. However, serum ferritin was similar (79 ng/mL in immediate and 53 ng/mL in wait-list, p=0.21). There was no significant difference in the primary outcome: at week 12, the immediate intervention group had a mean increase of 8.05± 55.48 meters on the 6MWT and the wait list group had a mean decline of 11.45±49.46 meters walked (p=0.44). Hb rose 0.39 g/dL ± 0.46 in the immediate intervention group compared to a mean decline of 0.39 ± 0.85 in the wait list group (p=0.026). Anemia symptoms improved by the FACT-An in the immediate group compared to the wait list group (+7.3 ±10.4 vs. -1.3 ±8.2, p=0.03). There were no significant differences in other secondary outcomes. Two patients in the immediate intervention group and 1 patient in the wait list group experienced grade 1 or grade 2 possibly or probably related treatment adverse events. One patient in each group experienced a serious adverse event, considered unlikely to be related to the study drug. Discussion The study was hampered by difficulty in recruitment. Future studies of older adults with UAE likely will require more sites, less stringent eligibility, and methods to screen and stimulate interest among eligible community dwelling subjects and their physicians. Nevertheless, the improvements in symptoms and hemoglobin from IV iron, in this limited sample justify further evaluation of IV iron in UAE with low to normal iron stores if recruitment barriers can be overcome. Disclosures: Off Label Use: IV iron with venofer is off label and given for unexplained anemia with low to normal iron stores.

Idiopathic neutropenia in children is marked by low neutrophil counts ( Patient number Chronicity Age FasL (pg/ml) Anti-neutrophil Ab Fold increase in neutrophil death over control plasma Fold increase in apoptosis over control plasma 1 chronic 2 yrs 7±1 positive 6.5±0.7 8.5±0.8 2 chronic 18 mo. 6±1 positive 7.3±0.5 8.1±0.2 3 chronic 23 mo. 5±0.5 wk positive/neg 7.8±0.6 8.5±0.4 4 chronic 1 yr 5±2 positive 6.5±0.5 7.3±0.6 5 chronic 5 yrs 7±1 wk positive 6.1±0.9 2.7±0.2 6 chronic 20 mo. 10±1 negative 11.3±1.4 2.1±0.5 7 chronic 11 mo. 6±1 negative 6.1±0.8 5.7±0.6 8 acute 2 yrs 16±2 N/A 18.7±1.3 22.4±2.9 9 acute 2 yrs 18±3 negative 12.0±2.1 19.1±2.5 10 acute 5 yrs 4±1 negative 2.5±0.5 2.6±0.4 11 acute 11 mo. 3±0.6 N/A 1.5±0.3 2.1±0.2 12 acute 3 yrs 17±1 negative 15.3±1.2 19.2±0.8 13 acute 2 yrs 40±3 negative 22.9±1.4 38.4±2.4 14 acute 2 yrs 21±2 N/A 14.3±1.0 20.5±1.2 15 acute 8 mo. 10±0.9 N/A 9.6±0.8 12.0±1.6 16 acute 17 mo. 19±3 negative 21.3±2.2 26.7±3.5 17 acute 6 yrs 8±1 N/A 6.7±1.3 11.5±2.1 18 acute 3 yrs 9±2 N/A 7.2±2.6 8.8±1.5 19 acute 11 yrs 12±2 negative 14.8±0.9 17.4±1.9 20 acute 5 yrs 6±1 negative 3.4±0.6 5.2±0.7 21 acute 4 mo. 8±2 N/A 6.2±1.0 8.1±2.2 22 acute 3 mo. 27±3 N/A 21.4±3.5 39.4±2.4 23 acute 11 yrs 22±1 negative 17.2±3.0 24.3±2.8 24 acute 2 yrs 18±2 positive 15.8±1.2 25.0±1.7 Representative control N/A 4 yrs 1.3±0.4 N/A 1.2±0.3 0.8±0.4

The WHO definition of anemia, Hgb71 and evaluated functional and cognitive status at baseline and 4 years later, and mortality for 8 years. Using WHO criteria, the prevalence of anemia was 24%. It was age related; 17% (65–74), 25% (75–79), 32% (>80) p

Expression of differentiation markers in common acute lymphoblastic leukemia (cALL) cells from 25 patients was compared with subpopulations of normal common ALL antigen (CALLA) (CD10)-positive bone marrow lymphoid cells (cBMLs). In cBML, CD10 intensity is positively correlated with CD34 (MY10) and terminal transferase (TdT) expression and inversely correlated with common leukocyte antigen (CLA), CD20 (B1), and ctyoplasmic mu chain (Cmu) expression. In cALL, CD10 density was inversely correlated with CLA and Cmu expression and strongly correlated with CD34 expression as in cBML. In contrast to cBML, TdT and CD20 expression were not related to CD10 density in cALL. Furthermore, cALL TdT intensity measured by enzyme immunoassay was not related to expression of CD10, CLA, or CD34, but was positively correlated with CD20 expression. Cmu expression in cALL was inversely correlated with expression of CD34 and positively correlated with CLA as in cBML, but showed no association with TdT intensity or CD20 expression in contrast to the relationship found in cBMLs. Analysis of TdT intensity and Cmu expression in sorted subpopulations of cells from individual patients that were positive or negative for CD34, CD20, or CD10 was consistent with the data obtained by comparison of cells from different patients. These results indicate that from patient to patient and within individual patients, cALL cells express the markers CD34, CLA, CD10, and Cmu in a coordinated fashion similar to cBMLs, but demonstrate differences in expression of TdT and CD20 with respect to the marrow cells considered their normal counterparts. The cALL cells that are CD34 positive show increased expression of CD10 and are less likely to be CLA or Cmu positive, suggesting that they may represent a phenotypically less differentiated form of cALL than does CD34-negative cALL.

A patient with severe arsenic poisoning that resulted in marked peripheral blood and bone marrow abnormalities, including megaloblastic erythropoiesis experienced many of the previously reported hematologic complications of arsenic poisoning: leukopenia, granulocytopenia, absolute eosinophilia, and profound anemia. In this study we report an ultrastructural and electron-probe analysis of the bone marrow. Although megaloblastic anemia associated with arsenic poisoning has been described rarely, the presence of arsenic in the local bone marrow milieu has not been demonstrated previously. The ultrastructural features of arsenic-induced bone marrow toxicity are similar to those described in other dyserythropoietic states and include marked nuclear aberrations involving shape, chromatin distribution, and nuclear envelope. Using the technique of energy-dispersive x-ray analysis (electron probe) we demonstrated arsenic in bone marrow spicules; this supports the contention that arsenic can cause megaloblastic anemia. We suggest that this technique may be a useful tool in further studies that attempt to explore the mechanism of arsenic-induced hematologic toxicity. Finally, we suggest that arsenic has a direct toxic effect on DNA synthesis that results in marked disturbances of nuclear division. We recommend that the most appropriate screening procedure to evaluate possible arsenic poisoning is tissue arsenic measurements (hair and nails) rather than 24-hr urinary measurements.

Plasma glutathione peroxidase (GSHPx) (glutathione: H2O2 oxidoreductase) is a unique selenoglycoprotein. Treatment of this enzyme with glycopeptidase F partially deglycosylates it and establishes the presence of N-linked sugar moieties. Antibodies raised in a rabbit against the purified enzyme from plasma were found to be specific, noninhibitory, and capable of precipitating the enzymatic activity. The antibodies precipitated greater than 90% of the GSHPx activity of normal plasma, thus indicating that the selenoenzyme is the main if not the sole GSHPx activity of plasma. The antibodies did not precipitate RBC GSHPx. A slight cross-reactivity of the antibodies was found with rat plasma GSHPx. A GSHPx activity precipitation assay of normal plasma in the presence of selenium (Se)-deficient plasma indicates that no cross-reactive protein in the Se-deficient plasma interferes with the precipitation of the GSHPx activity from normal plasma. Thus, GSHPx protein as well as activity is deficient in plasma in the absence of Se. Antibodies against GSHPx either from RBCs or from plasma were used to specifically immunoprecipitate most of the GSHPx activity from RBCs or plasma, respectively, in healthy individuals to determine the amount of Se associated with the protein. GSHPx accounts for approximately 15% of the Se in RBCs and 12% of the Se in plasma. Thus, in normal individuals, these proteins account for only a fraction of plasma and RBC Se.

Human granulocytes (polymorphonuclear leukocytes, PMN) produce H2O2 and other reactive oxygen species while undergoing phagocytosis. To examine the role of the glutathione cycle in metabolizing H2O2, we incubated PMN with 1,3-bis (2-chloroethyl) nitrosourea (BCNU). Incubation of PMN with BCNU results in a dose-dependent inhibition of PMN glutathione reductase (GRED), with 50% inhibition occurring at approximately 2 micrograms/mL BCNU. PMN hexose monophosphate shunt activity stimulated with an exogenous H2O2-generating system was inhibited only when the GRED activity was reduced to less than 30% of control. BCNU-treated cells contained lower levels of reduced sulfhydryls and reduced glutathione, which decreased even more in the presence of an exogenous H2O2-generating system. The effect of BCNU and exogenous H2O2 on various aspects of phagocytosis were examined. Exposure of BCNU-treated PMN to an H2O2-generating system resulted in an inhibition of chemotactic peptide-induced shape changes and degranulation. The ability of BCNU-treated cells to produce O2- was diminished only when the PMN were incubated with an H2O2-generating system in the presence of cyanide. Ingestion of opsonized bacteria by BCNU-treated PMN was unaffected by incubation in an H2O2-generating system even in the presence of cyanide. We conclude that PMN GRED is inhibited by BCNU, the ability of PMN to metabolize H2O2 is affected only when GRED is reduced more than 70%, this inhibition affects the glutathione content of these cells, and some, but not all of the phagocytic functions of GRED-inhibited PMN are inhibited after exposure to an H2O2-generating system.

Superoxide production by granulocytes is a result of the activation of an NAD(P)H-dependent oxidase present in the plasma membrane. Chlorpromazine (5–50 muM) prolongs the time necessary to activation of the superoxide generating system and inhibits the extent of activation. When chlorpromazine is added after activation, there is an inhibition of further superoxide production. These effects are seen with digitonin, phorbol myristate acetate, and opsonized zymosan stimulated guinea pig and human granulocytes. Other phenothiazines (1–20 muM) and tetracaine (0.1–1.0 muM) produce similar effects. Lidocaine (1–10 mM) inhibits superoxide production but has no effect on the rate of activation. The effect of chlorpromazine on the rate of activation is reversible, but its effect on extent of activation is unaffected by extensive washing. Incubation of granulocytes with chlorpromazine results in decreased activation of the plasma membrane superoxide generating NADPH oxidase. Chlorpromazine also competes with NADPH for the membrane oxidase. These data and previously published results provide the basis of a model for the activation of the superoxide generating system.

Phagocytic cells generate superoxide (O2) as a part of the respiratory burst of phagocytosis. We studied human granulocyte O2 production in vitro in response to phorbol myristate acetate, a soluble fatty acid ester that stimulates O2 generation. The properties of this compound permit the adaptation of a continuous assay of O2– production to the study of the activity and activation (i.e., the change from the resting to the active state) of the O2 -generating system. The two processes, measured respectively as the rate of O2– production and the lag time for its attainment, respond differently to manipulations involving temperature, pH, phorbol myristate acetate concentration, N-ethyl maleimide inhibition, and energy metabolism. Changes in pH profoundly influence the rate without changing the lag time. Increases in temperature above 37°C depress the rate but continue to shorten the lag time. Thus, the activation and activity of the O2 -generating system appear to have different pH and temperature dependency. In addition, they have different dose-response curves for phorbol myristate acetate stimulation: at high concentrations the rate reaches a plateau but the lag time continues to shorten. Activity of the human O2-generating system is sensitive to metabolic inhibitors, such as N-ethyl maleimide, 2-deoxyglucose, and the combination of 2-deoxyglucose with dinitrophenol or cyanide. The rate of activation (lag time) is unaffected by these compounds. These findings show that the activity of the human O2 -generating system and its activation are separable processes. Furthermore, comparison of the above characteristics of human peripheral blood and guinea pig peritoneal exudate granulocytes reveals several important differences.

We studied the effect of tumor necrosis factor (TNF) and gamma interferon (IFN-gamma), alone and in combination, on the expression of chemotactic peptide receptors, stimulus-induced actin polymerization, hydrogen peroxide production (H2O2), and expression of nonspecific esterase (NSE) positivity in human promyelocytic leukemic cell line HL- 60. These parameters were analyzed following a five-day culture with the cytokines. Chemotactic peptide receptor expression was studied using the fluoresceinated hexapeptide, formyl-norleucyl-leucyl- phenylalanyl-norleucyl-tyrosyl-lysine and flow cytometry. Actin polymerization, an important event required for chemotaxis and phagocytosis, was studied using NBD-phallacidin labeling, following stimulation with the chemotactic peptide formyl-methionyl-leucyl- phenylalanine (FMLP) or phorbol myristate acetate (PMA). TNF increased the expression of chemotactic peptide receptors in a dose-dependent fashion, and there was good correlation between the receptor expression, stimulus-induced actin polymerization, H2O2 production, and NSE positivity. IFN-gamma was less potent in inducing all the parameters studied but exerted a positive cooperative effect when combined with TNF. IFN-gamma at high concentrations induced chemotactic peptide receptors comparable in magnitude to that seen with TNF but failed to prime these cells to undergo actin polymerization in response to FMLP or PMA. Undifferentiated HL-60 cells showed a decrease in F- actin content on stimulation with PMA. This suggests that protein kinase C might have a negative regulatory role in stimulus-induced actin polymerization. The observations reported here indicate that appropriate combinations of different inducing agents with different modes of action might be necessary to duplicate the functional abilities of mature phagocytic cells.

In order to study the effectiveness of the drug combination BCNU, cyclophosphamide, and prednisone (BCP) in myeloma, the Southeastern Cancer Study Group entered 126 previously untreated and 33 previously treated patients in this study. Patients received cyclophosphamide, 400 mg/sq m, BCNU, 75 mg/sq m, and prednisone, 80 mg/sq m, for 7 days, every 4 wk, and were evaluated for response after 6 mo of therapy. Responding patients were continued on therapy and followed for evaluation of survival. Previously untreated patients were evaluated for good and poor risk status as well as clinical stage by calculation of tumor cell mass. These calculated values, as well as the individual components such as blood urea nitrogen (BUN), immunoglobulin (Ig) type, and hemoglobin (Hb), were evaluated for correlation with response and survival. The response rate was approximately 50% for all untreated patients regardless of cell mass or risk status. Toxicity was not excessive, and no case of acute leukemia was seen. Median survival for untreated patients was 27.8 mo; good risk patients, 44 mo; and poor risk, 12 mo. Though cell mass status did not correlate well with survival, individual factors of BUN, IgAλ, and IgGκ protein types had strong predictive value. Using these factors, a model for survival predication was created that had high predictive value in this group of patients. The study indicates that this treatment regimen is effective and could be used as an alternative to standard chemotherapy or in sequential combinations. Since it does not contain melphalan, it might be attractive if melphalan-contain-ing regimens prove to be particularly leuke-mogenic. The survival-predictive model may be useful in assessment of patient prognosis and will be further evaluated in subsequent group studies.

To characterize the defect in “nonsecretory” myeloma, and to relate it to the secretory phenomenon of human myeloma cells, plasma cells from such a patient were studied in vitro and compared with other myeloma cells. The cells had the morphologic appearance of plasma cells, and contained cytoplasmic IgA-λ, but lacked surface membrane Ig and were PAS-negative. The rough endoplasmic reticulum and Golgi apparatus were anatomically intact, but the endoplasmic reticulum was distended with proteinaceous material. Incorporation of 3 H-labeled leucine and carbohydrate precursors into Ig, as determined by immune coprecipitation, was measured for nonsecretory and secretory myeloma cells. Typical myeloma cells synthesized intracellular Ig, then actively secreted the newly synthesized protein into the medium. The nonsecretory cells synthesized Ig, though at a slower rate, but only released a small amount into the medium. The Ig released by these cells had a much higher 3 H-carbohydrate/ 3 H-leucine incorporation ratio than that released by the secretory myeloma cells. The data suggest that these cells have a block at the level of the Golgi apparatus, perhaps related to glycosylation, that prevents secretion. This finding may represent an abnormality induced during the malignant transformation or the clonal proliferation of a cell from a subpopulation of immunocyte precursors present normally in small numbers.

A modified zymosan preparation was used to probe the interaction of particulate stimuli with human neutrophils (PMNs). After extraction with alkali and detergent, the zymosan particles retained their ability to be opsonized in serum and to stimulate PMNs. Serum-treated zymosan (STZ) induced dose-dependent superoxide (O2-) production and membrane potential depolarization in the range of 1 to 10 mg/mL of STZ. The rate and extent of secretion of lysozyme and beta-glucuronidase were also dose-dependent in the range of 1 to 10 mg/mL of STZ. Cytochemical studies using nitroblue tetrazolium, however, showed that 92% of PMNs were stimulated to produce O2- at 0.1 mg/mL of STZ. The dose response of O2- production induced by STZ is therefore due to increasing O2- production by individual PMNs and not to the stimulation of more PMNs to produce O2-. Evidence for O2- production was found only in the area of PMN-zymosan contact, suggesting a mechanism for the graded responses of PMNs treated with particulate stimuli. In order to determine the nature of the dose dependence of depolarization (a measure of PMN activation), PMNs equilibrated with the fluorescent probe 3,3′- dipentyloxacarbocyanine were analyzed by flow cytometry. The results demonstrate that STZ induces a dose-dependent depolarization of the membrane potential of individual PMNs. These results also demonstrate that increasing concentrations of STZ can induce increasing PMN responses even when all of the PMNs have been activated. These results are consistent with the hypothesis that receptor-mediated particulate stimulation of PMNs is a phenomenon that results in graded PMN responses.

The subcellular localization, kinetics of activation, and substrate specificity of the guinea pig granulocyte superoxide (O2-) generating system was investigated. Membrane-enriched particles (podosomes) were made from granulocytes by mild sonication and differential centrifugation. These podosomes are enriched threefold for known plasma membrane markers, 5′-nucleotidase, and adenylate cyclase. Podosomes made from resting granulocytes have very little NAD(P)H-dependent O2- production. Podosomes made from cells stimulated with digitonin are equally enriched for membrane markers but have a 15- to 20-fold increase in NAD(P)H-dependent O2- production. The KmAPP for NADPH is one-tenth that for NADH, but the Vmax is the same. The kinetics of digitonin-stimulated whole-cell O2- production parallel the changes in enzyme activity in these podosomes. Temperature affects both the rate and extent of activation of this enzyme. The pH optimum for the enzyme, the pH optimum for activation, and the pH optimum for whole-cell O2- production are all 7.5. Enzyme activity is increased if the cells are treated with glucose and cyanide, inhibited in cells treated with 2- deoxyglucose (2-DOG), and requires the presence of calcium for activation. These effects are similar to those found for granulocyte O2- production. Thus, the granulocyte O2- generating enzyme system is located on a fraction enriched for plasma membrane markers, and the kinetics of granulocyte production are directly related to the rate and amount of activation of this enzyme.

The stimulation of granulocyte O2- production by concanavalin-A can be reversed with alpha-methylmannoside. Such cells can be reactivated to generate O2- by adding phorbol myristate acetate or N-formyl-methionyl- leucyl-phenylalanine. Opsonized zymosan, however, is not an effective stimulant to these cells. alpha-Methylmannoside prevents, but does not reverse, depolarization of granulocytes by concanavalin-A. Previously activated cells have a shorter lag time for reactivation by phorbol myristate acetate. Incubation in 2-deoxyglucose of cells previously treated with concanavalin-A and alpha-methylmannoside prevents reactivation. EGTA prevents concanavalin-A-stimulated O2- production only when added prior to the stimulant. EGTA has only a slight effect on reactivation. alpha-Methylmannoside prevents concanavalin-A- stimulated release of lysozyme only when added prior to the stimulant. Prior treatment of cells with concanavalin-A and alpha-methylmannoside inhibits subsequent ingestion of complement-coated particles. We conclude that although the O2--generating system can be reversibly activated with concanavalin-A followed by alpha-methylmannoside, these cells are different from untreated cells. Cells treated in such a way do not respond to all stimuli, remain depolarized, have shortened lag times, no longer require calcium for activation, continue to degranulate, and do not ingest well. Thus, although some changes that accompany the interaction of stimuli with granulocytes are reversible, some are not, and the previously activated cell does not return to a true resting state.

A patient with plasma cell myeloma producing only Bence Jones lambda protein was found to have pale intranuclear inclusions in the majority of the bone marrow plasma cells. These inclusions, previously undescribed in myeloma patients producing only Bence Jones protein, contained Bence Jones lambda protein, were non-electron dense, bound by a single membrane,and contained no cytoplasmic structures. Intracytoplasmic inclusions were not present, and the perinuclear cistern was not dilated. Thus, the inclusions may represent intranuclear protein synthesis with anomalous release in the abnormal cells.

Stimulation of granulocyte (PMN) superoxide (O2-) production by concanavalin-A (Con-A) can be monitored continuously in the spectrophotometer. Both the rate of activation and final activity of the O2--generating system is dependent on the concentration of Con-A. Alpha methylmannoside (alpha MM) can prevent Con-A, but not phorbol myristate acetate (PMA) or zymosan, induced O2- production. Alpha MM inhibits both the rate of activation and the final rate of O2- production. When alpha MM is added after the attainment of a maximal rate of O2- production with Con-A, O2- production continues for another minute before it ceases. When PMA is added to such treated cells, it restores O2- production. Although the inhibition of O2- production by alpha MM on previously activated cells requires time, most of the bound concanavalin-A is removed immediately after the addition of alpha MM. Treatment of cells with L-1-tosylamido-2-phenylethyl-chloromethyl ketone (TPCK) prevents activation of PMN by Con-A to a greater extent than it does for either PMA or zymosan. TPCK has no effect on the binding of Con-A. TPCK, when added after Con-A, will inactivate O2- production by the cells. The addition of PMA after TPCK treatment restores O2--generating activity. Membrane-enriched particles from PMN activated with Con-A, alpha MM, and PMA demonstrate that the change in O2- production seen by whole cells is due to an alteration of the activity of the NADPH oxidase. Thus, Con-A stimulation of human PMN O2- production can be prevented and reversed by the addition of either alpha MM or TPCK and that PMA can reactivate Con-A and either alpha MM- or TPCK-treated cells. The activation, inactivation, and reactivation occur as a result of changes in the plasma membrane NADPH-dependent O2-- generating enzyme.

Phagocytic cells generate superoxide in response to stimulation by opsonized particles. A continuous assay for opsonized zymosan- stimulated granulocyte superoxide production shows that there is a lag time between the addition of particles and the onset of detectable superoxide production. Superoxide production is preceded by membrane potential depolarization. Neither superoxide production nor membrane depolarization occurs in granulocytes from patients with chronic granulomatous disease. The extent of activation by opsonized zymosan is affected by the dose of zymosan from 0.5 to 4.5 mg/ml, but the time necessary for activation (lag time) is not. Similarly, the extent of depolarization but not the time necessary for attaining maximum depolarization is concentration-dependent. Effects of temperature, divalent cations, 2-deoxyglucose, cyanide, and N-ethyl maleimide on superoxide production are similar for granulocytes treated with soluble stimuli and with opsonized zymosan. Thus, zymosan stimulates granulocytes to generate superoxide and undergo membrane depolarization in a manner similar to that elected by soluble stimuli.

The effect of concanavalin A (Con A) on the concentration of ionized intracellular calcium [( Cai++]) in human granulocytes (PMN) was monitored using the fluorescent calcium indicator and chelator, Quin2. The addition of Con A to PMN resulted in a rise in [Cai++] that was markedly enhanced by the presence of Ca++ in the external buffer. The onset of the increment in [Cai++] preceded the onset of O2- production. The rise in [Cai++] induced by Con A is not transient, with a new, higher steady-state level of [Cai++] being attained within five minutes. The addition of alpha-methylmannoside (alpha-MM) one minute after Con A resulted in the return of [Cai++] to the original baseline level and the cessation of O2- production. The addition of a second stimulus (such as arachidonic acid) to these cells resulted in a second increment in [Cai++] and the return of O2- production. Thus the rise in [Cai++] induced by Con A is tightly coupled to the activation, inactivation, and reactivation of the O2- generating system by Con A. Further experiments were undertaken to assess the possible requirement for the rise in [Cai++] in the activation of PMN by Con A. PMN could be depleted of Cai++ by loading with Quin2 in the absence of extracellular Ca++. These Ca++- depleted PMN can be induced to produce O2- after treatment with PMA but not with Con A. The addition of Ca++ to Ca++ - depleted PMN results in a return of [Cai++] to the normal resting level of Ca++ -replete PMN. The time required to return to baseline is a function of the concentration of intracellular Quin2. The addition of Ca++ to Ca++ -depleted, Con A-treated PMN results in the elevation of [Cai++] and the production of O2-. Over a tenfold range of intracellular Quin2 concentration, the onset of O2- production always occurred at [Cai++] that were less than the normal resting level. Thus, activation of the O2- generating system by Con A can occur at [Cai++] which are much lower than the incremental level induced by Con A in Ca++ -replete PMN. Supporting this is the observation that only a very small increment in [Cai++] is induced by Con A in PMN cytoplasts, even though Con A could induce O2- production in the Quin2-loaded cytoplasts.

Previous studies of germ-free and conventional irradiated mice led to the hypothesis that the increased levels of serum colony-stimulating factor (CSF) observed after whole-body irradiation (WBI) might be due to endotoxemia. Other investigations showed that CF1 mice preinjected with Salmonella typhosa endotoxin became tolerant or resistant to the CSF-elevating effect of endotoxin. In the current studies, tolerant CF1 mice were subjected to 1000 rads WBI; they developed serum CSF elevations comparable to those seen in control irradiated mice. Tolerance was not abolished by 1000 rads WBI or reversed by infection with gram-negative or gram-positive bacteria. Changes in inhibitor levels did not explain these results. These data indicated that serum CSF elevations observed after WBI may not be due solely to endotoxemia, despite the observation that limulus-reactive material, presumably endotoxin, was detected in 61.5% of CF1 mice 1-12 days after 1000 rads. In these studies, 29.4% of normal pooled plasma samples also were limulus positive, suggesting that transient endotoxemia may occur in some strains of normal mice. Infection of CF1 mice with gram-negative or gram-positive bacteria caused serum CSF elevations 6-24 hr after infection; levels returned to baseline by 48-144 hr in all groups. The return to normal in serum CSF levels in mice infected with gram-negative or gram-positive bacteria coincided with the development of tolerance to the CSF-elevating effect of endotoxin. Infected mice developed marked increases in marrow granulopoiesis and continued to show evidence of accelerated marrow granulocyte release 6-144 hr after infection.

Bone marrow samples from normal adults and children with nonhematologic malignancies not involving the marrow, acute lymphoblastic leukemia (ALL) in continued remission, and immune cytopenias were studied by two- color immunofluorescence (IF) and flow cytometry to characterize common acute lymphoblastic leukemia antigen (CALLA)-positive marrow lymphoid cells. Marrow was separated by Ficoll/Hypaque centrifugation followed by passage over a monoclonal antibody affinity column to remove myeloid cells prior to IF staining. A higher proportion of CALLA-positive cells was found in the pediatric marrows (mean, 33.7% +/- 6.3% SEM) than in the adult marrows (mean, 4.5% +/- 1.6% SEM). Two subpopulations of CALLA-positive cells identified by cell sorting to be of lymphoid morphology were found in both adult and pediatric marrows. A small subpopulation comprising 12.3% of the total CALLA-positive cells was characterized by a high intensity of CALLA, terminal deoxynucleotidyl transferase (TdT), and MY10 expression, but low B1, common leukocyte antigen, and peanut agglutinin receptor expression. The remainder of the CALLA-positive cells displayed low intensity of CALLA expression, positivity for the common leukocyte antigen, B1 and peanut agglutinin, and negative reaction with TdT and MY10. Both CALLA-positive subpopulations were positive for HLA-DR and the pan-B cell marker B4, but negative for cytoplasmic immunoglobulin, B2 and Leu 1. BrdU labelling studies showed that a similar proportion of cells in each subpopulation was in S phase. A slightly higher proportion of the strongly CALLA-positive cells possessed the morphologic features of high nuclear/cytoplasmic (N/C) ratio and prominent nucleoli. These studies suggest that a discrete maturation step occurs among CALLA- positive marrow lymphoid cells, resulting in the loss of TdT and MY10 expression, but gradual acquisition of the B cell marker B1 and the common leukocyte antigen. The presence of B4 antigen in nearly all CALLA-positive cells suggests that both subpopulations of normal CALLA- positive marrow cells are committed to the B cell lineage.

A randomized controlled trial was initiated in 1972 to compare two chemotherapeutic regimens [1–3-bis (2-chloroethyl) 1-nitrosourea (BCNU), cyclophosphamide, and prednisone versus melphalan and prednisone], to determine whether the two regimens are cross-resistant, and to evaluate the effectiveness of sodium fluoride, vitamin D, calcium gluconate, and fluoxymesterone in the promotion of bone healing. Initial responses (50%) and survival (36 mo median) for patients treated with the two chemotherapeutic regimens were the same. Patients on either regimen who failed to respond after 6 mo had a very low response rate to the alternative regimen (approximately 10%). Initially responding patients were randomly assigned to either an active drug regimen (sodium fluoride, vitamin D, calcium gluconate, fluoxymesterone) or placebo tablets. There was no significant difference in the low percentage of patients demonstrating bone improvement. Thus, the BCNU, cyclophosphamide, prednisone regimen is as effective as melphalan and prednisone. Fluoride, calcium, vitamin D, and androgenic steroids should not be routinely recommended in myeloma, as they seem to add little to effective chemotherapy and may contribute to morbidity.

Selenium-deficient humans and animals are known to be deficient in glutathione peroxidase (GSHPx) activity in their cells and plasma. To determine the relationship between enzyme activity and protein content, the enzyme was purified from human erythrocytes, and polyclonal antibodies were made against the purified protein in rabbits. These antibodies were found to be monospecific, noninhibitory, and capable of precipitating the enzymatic activity. All the GSHPx activity in erythrocytes and almost all the activity in neutrophils and platelets was precipitated by these antibodies. None of the plasma enzyme was precipitated by these antibodies, indicating that the plasma enzyme activity was attributable to a different selenium dependent protein moiety. Utilizing a radioimmunoassay, we were able to determine that there was a direct relationship between GSHPx activity and protein content in the erythrocytes of both normal and selenium-deficient individuals, and a similar relationship between control and selenium- deficient rat erythrocytes and liver cells. Thus, the ability to examine GSHPx as a protein resulted in two new observations concerning the selenium-dependent GSHPx. The first is that the plasma enzyme is antigenically distinct from the erythrocyte enzyme, and the second is that in the absence of selenium, there is a concomitant decrease in GSHPx protein.

The pharmacokinetics of continuous subcutaneous cytosine arabinoside (ara-C) infusions were compared with continuous intravenous infusions. Steady-state serum ara-C levels and myelosuppression were similar with both routes of administration. CSF/serum ara-C ratios ranged from 0.14 to 0.91 (mean, 0.58). Continuous subcutaneous ara-C infusions were a convenient and reliable alternative to intravenous infusions.

The characteristics of human lymphocyte motility and its relationship to the redistribution of surface membrane antigens (capping) are poorly defined. Since chronic lymphocytic leukemia (CLL) cells cap poorly when compared with normal human lymphocytes, this study was undertaken to compare the motility of these two cell types. A modification of the Boyden chamber system was employed to quantify lymphocyte motility by placing lymphocyte suspensions on 8-mum convoluted-pore nitrocellulose filters and measuring the depth of migration of the cells into the filter at 37 degrees C. After 3 hr of incubation, CLL cells migrated significantly less into the filter than normal cells. Incubation in the presence of sodium azide or at 4 degrees C abolished all motility, indicating the active nature of the process. The relative motility of individual CLL patients' cells correlated best with the proportion of abnormal cells present as determined by surface receptor assays. The possibility that decreased cell motility in CLL was a reflection of enrichment by a “bone marrow-derived” (B cell) population was eliminated by the finding that normal B cells purified by gradient separation of rosetted cells migrated faster than normal T cells and considerably faster than CLL cells. Motility of normal and CLL lymphocytes was decreased by cytochalasin B and increased by colchicine, vincristine, and vinblastine. Thus, human lymphocyte motility appears to be dependent on microfilament integrity but not to require the colchicine-sensitive cytoskeleton. The decreased motility of CLL cells is the result of an intrinsic cell abnormality, but this finding cannot fully explain the decreased capping, since in human lymphocytes the latter is not prevented by an inhibitor of motility.

Hairy cell leukemia (HCL) is caused by the proliferation and accumulation of morphologically characteristic cells whose cell line of origin (lymphocyte versus monocyte) has not been determined. In an attempt to determine the cell line from which these cells are derived, we studied the cells from the peripheral blood and spleens of 6 patients with HCL for their cytochemical, immunologic, surface, and functional properties. Clinically the patients were similar to others reported with HCL, with some beneficial responses to splenectomy. Both the peripheral blood cells and spleen cells from these patients had tartrate-resistant acid phosphatase and lacked nonspecific esterase. They failed to rosette with sheep red blood cells (SRBC), C3-coated RBC, or anti-D-coated SRBC. Intense surface fluorescence was produced by fluoresceinlabeled anti-immunoglobulin reagents, and capping rapidly occurred. Reincubation following capping or trypsinization and restaining with anti-immunoglobulin reagents showed the reappearance of monoclonal immunoglobulin. The pattern of immunoglobulin synthesis and release was demonstrated directly by 3H-leucine incorporation into specific immune coprecipitable immunoglobulin and was similar to that of normal B lymphocytes. The cells had a low labeling index but had modest phagocytic ability. The unequivocal demonstration of endogenous immunoglobulin synthesis establishes these cells as B-lymphoid in nature. However, they may represent a particular B-lymphocyte subset originating in the spleen and having phagocytic ability.