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Introduction: Dysphagia is a common side effect following chemoradiation therapy (CRT) in head and neck cancer (HNC) patients. Current dysphagia management includes swallowing therapy and dilation procedures, but these treatments have limitations. While acupuncture has been reported to positively impact swallowing function and quality of life (QOL) in patients with dysphagia, current evidence is inconclusive., Material and Methods: In an ongoing trial, 42 squamous cell carcinoma HNC patients, who are receiving platinum-based CRT with curative intent, are being recruited from a comprehensive cancer center. They are randomized to 12 sessions of either active acupuncture or to sham acupuncture during and following CRT over a 24-week period. Blinded research staff assesses outcomes at baseline, 20 weeks post-CRT (end of acupuncture), and 12 months after baseline (6-month follow-up). The primary outcome is change in M.D. Anderson Dysphagia Inventory score from baseline to 12 months. Secondary outcomes include QOL measures pertaining to HNC patients. In addition, a subset of study patients are tested for salivary flow rates and cytokines, including plasma transforming growth factor-β1 and interleukin 6 (n=10 per arm), to preliminarily explore the biological mechanisms of acupuncture for dysphagia., Discussion: This paper addresses unique challenges related to study design in nonpharmacological, sham-controlled acupuncture trials including development of evidence-based credible verum and sham treatment protocols, blinding, and assuring fidelity of treatment. Results of this study will inform the feasibility of conducting a large scale trial and will provide preliminary evidence regarding the value of acupuncture for dysphagia in HNC patients., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Sulfur mustard (SM [bis-(2-chloroethyl) sulfide]) is a chemical warfare agent that causes skin blisters presumably due to DNA alkylation and cross-links. We recently showed that SM also induces apoptotic death in cultured normal human bronchial/tracheal epithelial (NHBE) cells and small airway epithelial cells (SAEC) in vitro. In this process, caspases-8 and -3, but not caspase-9, were strongly activated; this suggests a death receptor pathway for apoptosis. We now show that rat lungs were induced to undergo apoptosis in vivo following exposure of rats to SM by inhalation. Further study of the mechanism of apoptosis due to SM was performed with cultured NHBE cells and SAEC using tetrapeptide inhibitors of caspases-3, and -8. Inhibition of caspase-8 drastically reduced the activation of caspase-3 and almost eliminated that of caspase-9. Moreover, caspase-3 inhibition markedly reduced the activation of caspase-8 and also almost completely inhibited activation of caspase-9. These results suggest a death receptor pathway of apoptosis that utilizes a feedback amplification mechanism involving an activated death receptor complex that leads to the activation of caspase-9 via a caspase-3 pathway. These results may be important for the design of inhibitors of these pathways for therapeutic intervention to attenuate SM injury in respiratory tract lesions.

End-stage renal disease (ESRD) is a growing public health concern and non-adherence to treatment has been associated with poorer health outcomes in this population. Depression, likely to be the most common psychopathology in such patients, is associated with increased morbidity and mortality. We compared psychological measures and self-reported medication adherence of 94 kidney transplant recipients to those of 65 patients receiving hemodialysis in a major medical center in Brooklyn, New York. Compared to the transplant group, the hemodialysis cohort was significantly more depressed as determined by the Beck Depression Inventory score. They also had a significantly lower adherence to medication as reported on the Medication Therapy Adherence Scale. Using hierarchical multiple regression analysis, the variance in depression was the only statistically significant predictor of medication adherence beyond gender and mode of treatment, accounting for an additional 12% of the variance. Our study strongly suggests that a depressive affect is an important contributor to low medication adherence in patients with ESRD on hemodialysis or kidney transplant recipients.

Sulfur mustard (SM) causes blisters in the skin through a series of cellular changes that we are beginning to identify. We earlier demonstrated that SM toxicity is the result of induction of both death receptor and mitochondrial pathways of apoptosis in human keratinocytes (KC). Because of its importance in apoptosis in the skin, we tested whether calmodulin (CaM) mediates the mitochondrial apoptotic pathway induced by SM. Of the three human CaM genes, the predominant form expressed in KC was CaM1. RT-PCR and immunoblot analysis revealed upregulation of CaM expression following SM treatment. To delineate the potential role of CaM1 in the regulation of SM-induced apoptosis, retroviral vectors expressing CaM1 RNA in the antisense (AS) orientation were used to transduce and derive stable CaM1 AS cells, which were then exposed to SM and subjected to immunoblot analysis for expression of apoptotic markers. Proteolytic activation of executioner caspases-3, -6, -7, and the upstream caspase-9, as well as caspase-mediated PARP cleavage were markedly inhibited by CaM1 AS expression. CaM1 AS depletion attenuated SM-induced, but not Fas-induced, proteolytic processing and activation of caspase-3. Whereas control KC exhibited a marked increase in apoptotic nuclear fragmentation after SM, CaM1 AS cells exhibited normal nuclear morphology up to 48h after SM, indicating that suppression of apoptosis in CaM1 AS cells increases survival and does not shift to a necrotic death. CaM has been shown to activate the phosphatase calcineurin, which can induce apoptosis by Bad dephosphorylation. Interestingly, whereas SM-treated CaM1-depleted KC expressed the phosphorylated non-apoptotic sequestered form of Bad, Bad was present in the hypophosphorylated apoptotic form in SM-exposed control KC. To determine if pharmacological CaM inhibitors could attenuate SM-induced apoptosis via Bad dephosphorylation, KC were pretreated with the CaM-specific antagonist W-13 or its less active structural analogue W-12. Following SM exposure, KC exhibited Bad dephosphorylation, which was inhibited in the presence of W-13, but not with W-12. Consequently, W-13 but not W-12 markedly suppressed SM-induced proteolytic processing and activation of caspase-3, as well as apoptotic nuclear fragmentation. Finally, while the CaM antagonist W-13 and the calcineurin inhibitor cyclosporin A attenuated SM-induced caspase-3 activation, inhibitors for CaM-dependent protein kinase II (KN62 and KN93) did not. These results indicate that CaM, calcineurin, and Bad also play a role in SM-induced apoptosis, and may therefore be targets for therapeutic intervention to reduce SM injury.

Sulfur mustard is cytotoxic to dermal fibroblasts as well as epidermal keratinocytes. We demonstrated that poly(ADP-ribose) polymerase (PARP) modulates Fas-mediated apoptosis, and other groups and we have shown that PARP plays a role in the modulation of other types of apoptotic and necrotic cell death. We have now utilized primary dermal fibroblasts, immortalized fibroblasts, and keratinocytes derived from PARP(-/-) mice and their wildtype littermates (PARP(+/+)) to determine the contribution of PARP to sulfur mustard toxicity. Following sulfur mustard exposure, primary skin fibroblasts from PARP-deficient mice demonstrated increased internucleosomal DNA cleavage, caspase-3 processing and activity, and annexin V positivity, compared to those derived from PARP(+/+) animals. Conversely, propidium iodide staining, PARP cleavage patterns, and random DNA fragmentation revealed a dose-dependent increase in necrosis in PARP(+/+) but not PARP(-/-) cells. Using immortalized PARP(-/-) fibroblasts stably transfected with the human PARP cDNA or with empty vector alone, we show that PARP inhibits markers of apoptosis in these cells as well. Finally, primary keratinocytes were derived from newborn PARP(+/+) and PARP(-/-) mice and immortalized with the E6 and E7 genes of human papilloma virus. In contrast to fibroblasts, keratinocytes from both PARP(-/-) and PARP(+/+) mice express markers of apoptosis in response to sulfur mustard exposure. The effects of PARP on the mode of cell death in different skin cell types may determine the severity of vesication in vivo, and thus have implications for the design of PARP inhibitors to reduce sulfur mustard pathology.

The tumor-suppressor p53 undergoes extensive poly(ADP-ribosyl)ation early during apoptosis in human osteosarcoma cells, and degradation of poly(ADP-ribose) (PAR) attached to p53 coincides with poly(ADP-ribose)polymerase-1, (PARP-1) cleavage, and expression of p53 target genes. The mechanism by which poly(ADP-ribosyl)ation may regulate p53 function has now been investigated. Purified wild-type PARP-1 catalyzed the poly(ADP-ribosyl) of full-length p53 in vitro. In gel supershift assays, poly(ADP-ribosyl)ation suppressed p53 binding to its DNA consensus sequence; however, when p53 remained unmodified in the presence of inactive mutant PARP-1, it retained sequence-specific DNA binding activity. Poly(ADP-ribosyl)ation of p53 by PARP-1 during early apoptosis in osteosarcoma cells also inhibited p53 interaction with its DNA consensus sequence; thus, poly(ADP-ribosyl)ation may represent a novel means for regulating transcriptional activation by p53 in vivo.

The observation that 3-aminobenzamide, which inhibits a variety of ADP-ribose transferases, prolongs the gamma-irradiation-induced increase in intracellular p53 concentration suggested that one or more of such enzymes may determine the duration of the p53 response during G1 arrest. The role of poly(ADP-ribose) polymerase (PARP), an abundant nuclear enzyme activated by DNA strand breaks, in the p53 response to y-irradiation was investigated in Burkitt's lymphoma AG876 cells stably transfected with an inducible PARP antisense construct. Immunoblot analysis revealed that the cellular content of PARP was reduced to virtually undetectable levels after incubation of transfected cells for 72 h with the inducer dexamethasone. In noninduced antisense cells, the p53 concentration reached a maximum 2 h after exposure to 6.3 Gy of gamma-radiation and returned to control values by 4 h. In contrast, the p53 response in PARP-depleted antisense cells peaked at 4 h, with the levels of p53 remaining elevated for up to 12 h after y-irradiation. The maximal increase in p53 concentration was similar in both induced and noninduced cells. These results thus indicate that PARP activity, in part, determines the duration, but not the magnitude, of the p53 response to DNA damage.

Sulfur mustard (SM) induces vesication via poorly understood pathways. The blisters that are formed result primarily from the detachment of the epidermis from the dermis at the level of the basement membrane. In addition, there is toxicity to the basal cells, although no careful study has been performed to determine the precise mode of cell death biochemically. We describe here two potential mechanisms by which SM causes basal cell death and detachment: namely, induction of terminal differentiation and apoptosis. In the presence of 100 microM SM, terminal differentiation was rapidly induced in primary human keratinocytes that included the expression of the differentiation-specific markers K1 and K10 and the cross-linking of the cornified envelope precursor protein involucrin. The expression of the attachment protein, fibronectin, was also reduced in a time- and dose-dependent fashion. Features common to both differentiation and apoptosis were also induced in 100 microM SM, including the rapid induction of p53 and the reduction of Bcl-2. At higher concentrations of SM (i.e., 300 microM), formation of the characteristic nucleosome-sized DNA ladders, TUNEL-positive staining of cells, activation of the cysteine protease caspase-3/apopain, and cleavage of the death substrate poly(ADP-ribose) polymerase, were observed both in vivo and in vitro. Both the differentiation and the apoptotic processes appeared to be calmodulin dependent, because the calmodulin inhibitor W-7 blocked the expression of the differentiation-specific markers, as well as the apoptotic response, in a concentration-dependent fashion. In addition, the intracellular Ca2+ chelator, BAPTA-AM, blocked the differentiation response and attenuated the apoptotic response. These results suggest a strategy for designing inhibitors of SM vesication via the Ca2+-calmodulin or caspase-3/PARP pathway.

Poly(ADP-ribose) polymerase (PADPRP) modifies nuclear proteins in response to DNA-damaging agents. The principal organ subject to exposure to many of these agents is the skin. To understand the role of PADPRP in the maintenance of the epidermis, a model system has been developed in which we have selectively lowered the levels of this enzyme by the use of induced expression of antisense RNA. Human keratinocyte lines were stably transfected with the cDNA for human PADPRP in the antisense orientation under an inducible promoter. Induction of this antisense RNA in cultured cells selectively lowers the levels of PADPRP mRNA, protein, and enzyme activity. Induction of antisense RNA also led to a reduction in the levels of PADPRP in individual cell nuclei, as well as the loss of the ability of cells to synthesize and modify proteins by poly(ADP-ribose) polymer in response to DNA damage. When keratinocyte clones containing the antisense construct or empty vector alone were grafted onto nude mice, they formed histologically normal human skin. The PADPRP antisense construct was also inducible in vivo by the topical application of dexamethasone to the reconstituted epidermis. In addition, poly(ADP-ribose) polymer could be induced and detected in vivo following the topical application of a DNA alkylating agent to the grafted transfected skin layers. Accordingly, a model system has been developed in which the levels of PADPRP can be selectively manipulated in human keratinocytes in cell culture, and potentially in reconstituted epidermis as well. This system will be a useful tool to study the role of PADPRP and DNA repair in general in essential biologic processes in the epidermis.

Epidermal transglutaminase-K is believed to catalyze the covalent linking of loricrin and involucrin to form cross-linked (CE) envelopes. In normal skin, transglutaminase-K is expressed as a band immediately below the stratum corneum, whereas in psoriasis and healing skin its expression is considerably expanded throughout the suprabasal layers. We have investigated whether the hyperproliferative state induced by short-term application of topical retinoic acid is similarly characterized by an increase in transglutaminase-K enzyme activity and immunoreactivity. Retinoic acid (0.1% cream) or vehicle were applied to human skin and occluded for 4 d. Skin biopsies were obtained for measurement of transglutaminase-K and transglutaminase-C activity and immunoreactivity. For comparison, cultured normal human keratinocytes were incubated for 4 d in the presence of 1 microM retinoic acid and the subsequent transglutaminase-K activity and immunoreactivity measured. Transglutaminase-K activity was increased 2.8 times in retinoic acid compared to vehicle-treated skin (p less than 0.005, n = 12) whereas there was no significant difference in transglutaminase-C activity. However, transglutaminase-K mRNA levels were not significantly different between retinoic acid- and vehicle-treated skin. In vehicle-treated skin, transglutaminase-K immunoreactivity was limited to a narrow, substratum corneal band, but was considerably expanded in a diffuse suprabasal pattern in retinoic acid-treated epidermis. In contrast, transglutaminase-K immunostaining was decreased and its enzymatic activity reduced sixfold in retinoic acid-treated keratinocytes (p less than 0.01, n = 4). These results demonstrate that retinoic acid treatment in vivo, in contrast to in vitro, leads to not only increased transglutaminase-K protein expression but also increased enzymatic activity in the absence of detectable increases in mRNA levels. These data, taken with the previously reported lack of in vivo modulation of the differentiation markers keratins 1 and 10 by retinoic acid, indicate that certain aspects of keratinocyte terminal differentiation that are altered in vitro by retinoic acid do not occur in vivo in human skin.

The nonleukemic chronic myeloproliferative disorders, polycythemia vera, essential thrombocytosis, and myeloid metaplasia with myelofibrosis, are clonal disorders with similar but distinct clinical and laboratory findings. This review will discuss the diagnostic criteria for each disease, the variable clinical picture, and the therapeutic modalities, actual and theoretical.

Histologic and immunocytochemical analyses were performed on cutaneous biopsies from 10 patients treated with retinoic acid under occlusion for 4 d compared to biopsies from 19 patients treated nightly for 16 weeks. Acute application of RA caused epidermal thickening (9 of 10 samples), stratum granulosum thickening (7 of 10), parakeratosis (4 of 10), a marked increase in the number of cell layers expressing epidermal transglutaminase (7 of 10), and focal expression of two non-epidermal keratins, K6 (8 of 10) and K13 (2 of 10), changes also observed with chronic treatment. Involucrin, filaggrin, and loricrin were also altered in samples from both acute and chronic treatment. An increased number of cell layers expressed both involucrin and filaggrin from both the acute (7 of 10) and chronic (14 of 19) treatment groups. In the acute group, loricrin expression was significantly reduced or absent in some regions of the epidermis (5 of 10), whereas most chronic samples showed an increased number of cell layers expressing loricrin (12 of 19). The pattern of expression of three major epidermal differentiation products, keratins K1, K10, and K14, was not significantly altered in any of the acute or chronic samples, although there was a slight reduction in the detection of K10 in two of the acute samples. Thus, acute topical RA treatment under occlusion caused substantial changes in the epidermis, and reproduced most, but not all of the effects of chronic treatment.

Although topical applications of retinoids on rodents and humans have been shown to cause epidermal hyperplasia, a detailed study of the influence of retinoids on epidermal differentiation in vivo has not been performed. In order to assess the pharmacologic effects of chronic topical tretinoin application used to improve the appearance of patients with photoaged skin, cutaneous biopsies from 25 patients in a controlled clinical study were examined histologically and immunocytochemically. Chronic application of tretinoin causes epidermal thickening (25 of 25 samples), stratum granulosum thickening (15 of 25), parakeratosis (13 of 25), a marked increase in the number of cell layers expressing epidermal transglutaminase (13 of 25), and focal expression of two keratins, K6 (12 of 25) and K13 (8 of 25), not normally expressed in the epidermis. The morphologic changes correlated with immunohistochemical abnormalities; neither of these correlated with the subjective cosmetic response. Three major epidermal differentiation products, keratins K1, K10, and K14 were not altered, within the limits of the methods used. Thus, chronic topical tretinoin reprograms some, but not all, aspects of human epidermal differentiation in vivo.

A transplantable myelogenous leukemia of an inbred Wistar/Furth rat has been established in tissue culture and cloned. The resulting transplantable leukemia line demonstrates in vitro doubling time of 20 hr, colony-forming efficiency of 5% in liquid and methylcellulos-containing medium, and a saturation density of 3.0 x 106 cells/sq cm in liquid medium. Following intraperitoneal inoculation, newborn rats developed solid tumors, ascities, and leukemia with ld50 of5 x 103 cells and mean latency of 60 days. The tumor cell morphology was consistent with that of acute myelogenous leukemia. Histochemical staining for myeloid enzymes revealed no evidence of myeloperoxidase, esterase, or leukocyte alkaline phosphatase; however, fluorescent antibody staining for lysozyme was markedly positive. Serum, urine, and ascitic fluid from rats with transplanted leukemia also contained elevated levels of lysozyme. There was no detectable type-CRNA virus production by this cell line after as long as 100 days in vitro. This inbred rat myelogenous leukemia should provide a useful model for studies of chemotherapy and immunoltherapy of human acute myelogenous leukemia.

A 68-yr-old male with chronic lymphocytic leukemia (CLL) presented with splenomegaly and skin infiltration but no lymphadenopathy. The peripheral blood WBC cound was 300 x 10(9)/liter, with 95% small mature-appearing lymphocytes that were E-rosette positive and EAC-rosette negative. Further characterization of the patient's cells was performed using antisera with known lymphoid sub-population specificity. Anti-p23,30, which reacts with normal circulating B cells but not with T cells or thymocytes, was unreactive with the patient's cells. Anti-311, which reacts with both thymocytes and circulating T cells, was reactive with the patient's cells. Anti-Bk, which reacts only with thymocytes and not with circulating T-cells, failed to react with the patient's cells. The enzyme terminal deoxynucleotidyl transferase, present in thymocytes but absent for circulating T-cells, was also absent from the patient's lymphoid cells. Multimarker analysis therefore showed a mature T-lymphocyte phenotype on this patient's leukemia cells. Further functional analysis will probably show that such cells represent clonal expansion of a mature T-cell subpopulation, analogous to the B-cell clonality of common-variant CLL.

We performed a phase I/II study of the administration of recombinant human granulocyte-macrophage colony-stimulating factor (GM-CSF) to patients with aplastic anemia or myelodysplastic syndrome. Doses ranging from 15 to 480 micrograms/m2 were administered as a one-hour or four-hour intravenous infusion daily for 7 days or as a 12-hour infusion for 14 days. Temporary improvements were seen in granulocyte counts, monocyte counts, and reticulocyte counts in six of eight patients with aplastic anemia and five of seven patients with myelodysplastic syndromes. The patients with myelodysplastic syndromes had larger increases in granulocyte, monocyte, and reticulocyte counts than did those with aplastic anemia, and they also had increases in the numbers of eosinophils (two of seven), immature myeloid cells (two of seven), and myeloblasts (two of seven) that were not observed in patients with aplastic anemia. There was no reduction in erythrocyte transfusion requirements, and no effect was observed on platelet counts. There was only minimal toxicity consisting of transient low-back discomfort, anorexia, myalgias/arthralgias, and low-grade fever. Our data suggest that GM-CSF is well tolerated and is more likely to result in elevations of blood counts in patients with myelodysplasia than in patients with aplastic anemia, but the role of GM-CSF therapy in these disorders remains to be determined.

We designed a protocol (VAPA) that featured 14 mo of intensive postremission induction chemotherapy in an effort to improve remission durations for patients with acute myelogenous leukemia (AML). One hundred and seven patients under 50 yr of age were entered into this study. The rate of complete remission is 70%. A Kaplan-Meier analysis of patients entering remission predicts that 56% +/- 7% (+/-SE) of patients less than 18 yr and 45% +/- 9% of patients aged 18-50 yr will remain in remission at 3 yr (median follow-up is 43 mo). Patients with the monocytic subtype had a statistically significant shorter duration of remission (2-sided p less than 0.05). There was a high incidence of primary CNS relapse in children. Thirty-one of 41 patients who completed the regimen remain in remission without maintenance therapy. We conclude that the VAPA protocol continues to offer a promising approach to treatment of AML.

Cytochemical identification of T lymphocytes on the basis of alpha-naphthyl acetate esterase (NAE) activity was compared with immunologic markers for cell suspensions and/or cryostat sections of 113 specimens. Nonneoplastic tissues (peripheral blood, lymph nodes, spleens, tonsils, thymus, and pleural fluid) and specimens from various lymphoproliferative disorders, including acute and chronic lymphocytic leukemia, lymphosarcoma cell leukemia, hairy cell leukemia, non-Hodgkin's lymphomas of B-and T-cell types, and Hodgkin's disease, were evaluated. T (E-rosetting) cells demonstrated several patterns of NAE reactivity: 1) a strong globular reaction product, the most specific pattern for T-cell identification, 2) granular cytoplasmic staining, or 3) no reactivity. B lymphocytes revealed a granular pattern of NAE staining, were devoid of enzyme, or, in rare instances, exhibited strong NAE activity. Percentages of lymphoid cells with strong (globular) NAE activity closely paralleled T-cell (E-rosette) values in the majority of cases, with the best correlations observed for peripheral blood studies. However, discordant results were noted for some neoplastic and nonneoplastic tissues, including cases of T-cell lymphoma or leukemia. Markedly discrepant results were noted for thymic lymphocytes, most of which revealed E-rosette formation and weak or absent NAE activity. Lymph nodes involved by Hodgkin's disease demonstrated a heterogeneous pattern of staining in E-rosetting cells and in Reed-Sternberg variants. Cryostat section studies of reactive lymph nodes and nodular lymphomas demonstrated strong NAE staining in lymphoid cells of T-cell (interfollicular, internodular) areas, with little or no positivity in follicles or nodules (B-cell areas). NAE staining patterns further suggested that T cells comprise part of the follicular cuff and possibly represent a minor population of some neoplastic nodules. Although NAE determinations do not represent a consistently reliable alternative to immunologic methods for T-cell identification, this easily applicable cytochemical marker is complementary to other techniques in assessing neoplastic or nonneoplastic tissues, particularly cryostat sections. (Am J Pathol 97:17--42, 1979).

Long-term follow-up of 18 patients with hairy cell leukemia seen at a single institution is reported: diagnostic and therapeutic modalities are described. Although the clinical features of this disease are homogeneous, it is frequently misdiagnosed initially. Bone marrow biopsy was diagnostic in all 17 patients studied prior to splenectomy. The presence of circulating "hairy" cells is greatly variable. The cause of pancytopenia is unclear: splenic sequestration was not demonstrated by 51Cr red cell studies in three of four patients. In most patients, splenectomy resulted in amelioration of pancytopenia. No clinical features prior to splenectomy could predict response to surgery. The results of chemotherapy and splenic irradiation were variable and unpredictable. Although bone marrow infiltration persists following splenectomy, this remains the only established therapy in hairy cell leukemia and is usually followed by long, uncomplicated survival.

Seventy-three patients with advanced non-Hodgkin lymphoma were treated with bleomycin, Adriamycin, cyclophosphamide, vincristine (Oncovin) and prednisone (BACOP), administered intensively during a 7-wk induction course followed by intermittent cycles every 3 wk for a total of 28 wk. The objective response in 44 evaluable nonleukemic patients with diffuse histology was 86%, with 66% achieving a complete remission (CR), varying from 80% for diffuse poorly differentiated lymphocytic (DPDL) to 56% for diffuse histiocytic (DH) lymphoma. In patients with nodular histology 89% (8/9) achieved a CR with a projected 75% of patiients in CR at 14 mo. Median follow-up from time of CR for nodular histology was 17 mo. The projected median duration of CR in diffuse histology was 14 mo. with median survival 14 mo. Patients with a partial response survived a median of 7 mo, compared to 3 mo for nonresponders. Of 29 patients with diffuse histology, 17 (59%) have remained disease free for 5-34 mo with a median follow-up of 12 mo. Survival beyond 20 mo has been projected for 42% of patients with diffuse histology (58% with DPDL and 32% with DH). The central nervous system (CNS) was involved in a total of 11/44 (25%) patients with diffuse histology, including 5 with primary CNS relapse. BACOP resulted in a higher CR rate and longer survival than a previous three-drug program (COP), especially in patients with diffuse histology.

The role of heparin in the treatment of the disseminated intravascular coagulation (DIC) associated with acute promyelocytic leukemia (APL) remains unclear. Between 1974 and 1985, we treated 27 patients with APL using four different chemotherapeutic regimens; 23/27 (85%) had evidence of DIC either at presentation or following the initiation of induction chemotherapy. The coagulopathy was treated primarily with fresh frozen plasma and platelet transfusions; only 2/27 (7%) patients received heparin. Twenty of 27 patients (74%) entered complete remission. Major bleeding or thrombotic complications occurred in 5/27 patients (19%), but 2 of these 5 patients presented after hemorrhage had already occurred. None of the 5 patients with bleeding or thrombosis entered complete remission. All of the hemorrhagic complications due to DIC in our study occurred before 1979, which may reflect changes in the management of leukemic patients. This observation emphasizes the risks inherent in the use of historical controls in this population. In conclusion the DIC associated with APL can be successfully treated with intensive blood product support without the use of heparin.

One hundred and seventeen consecutive patients with refractory dysmyelopoietic anemia (RDA) were followed and studied over 6 yr. All RDA cases had at least two marrow cell lines involved with dysplasia and usually all three. Morphologically, the series could be divided into 55 cases that had primarily "erythroid" hyperplasia and 62 cases with primarily "myeloid" dysplasia. There was a significantly higher number of acute leukemias in the myeloid group, 21/62 (33.9%), than in the erythroid group, 7/55 (12.7%). Of the 117 cases, 64 had marrow culture studies performed using implanted diffusion chambers (DC) in an irradiated rat host. There was a highly statistically significant correlation between the development of leukemia and abnormal growth in DC during the RDA stage.

A series of monoclonal antibodies reactive with normal myeloid cells at different stages of differentiation (anti-MY4, -MY7, -MY8, -Mo1, -Ia) were used to characterize the leukemic cells of 70 patients with acute myeloblastic leukemia (AML). Sixty-two of the leukemias expressed a phenotype corresponding to a recognizable immature normal myeloid cell. These 62 cases could be divided into 4 phenotype groups, corresponding approximately to the normal CFU-C (group I, 21%), myeloblast (group II, 26%), promyelocyte (group III, 8%), and promonocyte (group IV, 45%). Morphological subtyping of these leukemias tended to agree with the immunologic phenotype, particularly with more "differentiated" morphological subtypes, such as acute monocytic leukemia or acute promyelocytic leukemia. However, each phenotype group contained more than one morphological type of AML, indicating that the level of differentiation of the surface membrane of AML cells may not always be concordant with morphology. The phenotype groups were also analyzed with respect to cytochemical staining patterns, age, the presence of Auer rods, and complete remission rates. Statistically significant differences among the groups were noted in the distribution of myeloperoxidase staining, nonspecific esterase staining, and Auer rods. The complete remission rates varied from 60% (groups III and IV) to 88% (group II). These results suggest that surface marker analysis in AML may be used as a highly reproducible classification system that will provide additional information about the leukemic cells in conjunction with morphological analysis.

Circulating peripheral blood tumor cells in four cases of chronic lymphoproliferative disease were immunologically characterized. By the use of T-cell-specific heteroantisera and indirect immunofluorescence, all were shown to involve proliferation of malignant T cells. Three cases demonstrated morphologic and clinical features consistent with chronic lymphocytic leukemia (CLL), and one case presented as a lymphosarcoma cell leukemia. Antisera specific for normal human T-cell subsets defined the malignant T cells in each case as arising from the TH2--subset. This subset normally constitutes approximately 80% of human peripheral blood T cells. Terminal deoxynucleotidyl transferase (TdT) was not detected in any of the T-cell CLL cases, thus supporting the notion that T-cell CLL represents a malignancy of a mature phenotype. The one patient with lymphosarcoma whose tumor cells were TdT-positive subsequently developed T-cell acute lymphoblastic leukemia (ALL). Moreover, la-like antigen (p23,30) was detected on two of these tumor cell populations. In addition, it was shown that not all tumor cells were E-rosette-positive, since only cells from 3 of 4 patients were capable of forming spontaneous rosettes. These findings demonstrate that heteroantisera can provide an additional important tool for dissecting the heterogeneity of T-cell leukemias and for relating them to more differentiated normal T cells.

A new human megakaryocytic cell line (Dami) has been established from the blood of a patient with megakaryoblastic leukemia. The Dami cells grow primarily in suspension with a doubling time of 24 to 30 hours. By light and electron microscopy, the Dami cells range in size from 12 to 120 micron in diameter and have lobulated nuclei characteristic of megakaryocytes. At least 89% of the cells react with monoclonal antibodies against platelet glycoproteins (GP) Ib and IIB/IIIa, and glycophorin. The cells do not react with antibodies against lymphoid, monocyte, granulocyte, or macrophage antigens. Thirteen percent of the cells become polyploid, spontaneously achieving greater than 4N DNA ploidy levels. In response to phorbol myristate acetate (PMA), the proportion of cells with ploidy levels greater than 4N increased threefold and could be separated into discrete ploidy groups. PMA also increased the expression of GPIb, the GPIIb/GPIIIa complex,l and von Willebrand factor. Cytogenetic analysis revealed a human male hyperdiploid karyotype with a modal chromosome number of 54 to 64 and several consistent clonal chromosomal abnormalities. These included a partial deletion of chromosome 5 and a translocation involving chromosome 3. In contrast to other megakaryocytic cell lines in which only a small portion of the cells express the megakaryocytic phenotype, nearly all of the Dami cells express platelet glycoproteins. Thus, the Dami cells provide a superior model in which to study human megakaryocyte biochemistry and differentiation.

Thirty-four patients with acute promyelocytic leukemia (APL) (median age 37 years, range 20 to 69 years) received induction treatment between 1974 and 1985 with cytosine arabinoside (ara-C) and an anthracycline. Bone marrow hypercellularity was present at the time of diagnosis in all patients, although the median peripheral leukocyte count was 2,600/microL. A second course of induction therapy consisting of further ara-C and anthracycline was initiated 15 days after the start of treatment if bone marrow hypocellularity could not be documented. Karyotypic analysis of bone marrow blasts was performed on 15 of 34 patients; 11 of 15 had abnormalities in chromosomes 15 and/or 17. Twenty-nine of 34 (85%) patients had laboratory evidence of disseminated intravascular coagulopathy. Of the 29 patients surviving 14 days, 24 (83%) received a second course of induction therapy. Complete remission was achieved in 25 of 34 (74%) patients, with four of 25 (16%) requiring one course of induction chemotherapy and 21 of 25 (84%) receiving two courses. Bone marrow specimens obtained 15 days after the start of therapy from the 25 patients who eventually attained complete remission showed the continued presence of dysplastic promyelocytes in 21 cases; three specimens were technically inadequate and only one was truly devoid of promyelocytes. Seventeen of 25 (68%) patients still had persistence of abnormal bone marrow promyelocytes seven or more days after the second course of therapy. Patients in complete remission received various forms of postremission therapy. Ten of the 25 (40%) completely responding patients remain alive in continuous complete remission. Neither the absence of bone marrow hypocellularity nor the persistence of dysplastic promyelocytes during induction exerted any influence on the probability for survival. These findings confirm and extend prior reports that complete remission in APL, in contrast to other subtypes of acute nonlymphocytic leukemia (ANLL), can frequently be achieved without bone marrow aplasia. Whether this observation signifies that complete remission in APL is due to leukemic cell differentiation or selective cytotoxicity is unknown. The absence of therapy-induced bone marrow hypoplasia in APL is not an absolute indication of induction failure or a poor ultimate prognosis.