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Background: Apalutamide plus androgen deprivation therapy (ADT) significantly improved metastasis-free survival (MFS), overall survival (OS), and time to prostate-specific antigen (PSA) progression in the placebo-controlled SPARTAN study of high-risk nonmetastatic castration-resistant prostate cancer (nmCRPC). Objective: To assess the relationships between PSA kinetics, outcomes, and molecular subtypes in SPARTAN. Design, setting, and participants: The authors conducted a post hoc analysis of nmCRPC patients randomized to receive apalutamide (n = 806) or placebo (n = 401) plus ADT and a subset stratified by molecular classifiers. Intervention: Apalutamide 240 mg/d. Outcome measurements and statistical analysis: The association between PSA kinetics and MFS, OS, time to PSA progression, and molecular subtypes was evaluated using the landmark analysis and Kaplan-Meier methods. Results and limitations: By 3 mo, PSA decreased in most apalutamide-treated patients and increased in most placebo-treated patients. After apalutamide, the median time to PSA nadir, confirmed ≥50% PSA reduction, ≥90% PSA reduction, and PSA ≤0.2 ng/ml were 7.4, 1.0, 1.9, and 2.8 mo, respectively. By 6 mo, 90%, 57%, and 32% of apalutamide patients had ≥50% PSA reduction, ≥90% PSA reduction, and PSA ≤0.2 ng/ml, respectively, while only 1.5% of placebo patients experienced ≥50% PSA reduction. PSA reductions were observed within 3 mo and up to 12 mo of apalutamide treatment, and were similar across molecular subtypes. Deep PSA responses (≥90% PSA reduction or PSA ≤0.2 ng/ml) at landmark 6-mo apalutamide treatment were significantly associated with improved time to PSA progression (hazard ratio {HR} [95% confidence interval {CI}] 0.25 [0.18–0.33] or 0.13 [0.08–0.21]), MFS (0.41 [0.29–0.57] or 0.3 [0.19–0.47]), and OS (0.45 [0.35–0.59] or 0.26 [0.18–0.38]; p < 0.001 for all). Conclusions: Apalutamide plus ADT produced rapid, deep, and durable PSA responses by 6-mo treatment regardless of assessed molecular prognostic markers. An early PSA response with apalutamide was associated with clinical benefits, supporting prognostic value of PSA monitoring. Patient summary: In this report, we describe how prostate-specific antigen (PSA) levels relate to outcomes in patients with nonmetastatic castration-resistant prostate cancer treated with apalutamide plus androgen deprivation therapy (ADT). We found that treatment with apalutamide plus ADT resulted in rapid, deep, and durable PSA responses in the majority of patients, including those with high-risk molecular subtypes, which were associated with improved survival.

Background: The phase III SPARTAN study demonstrated that apalutamide significantly improves metastasis-free survival and overall survival vs. placebo in patients with non-metastatic castration-resistant prostate cancer (nmCRPC). However, patients receiving apalutamide experienced falls more frequently vs. those receiving placebo (15.6% vs. 9.0%). Methods: 806 patients with nmCRPC randomized to apalutamide in SPARTAN and treated with apalutamide in addition to ongoing androgen deprivation therapy (ADT) were included in this post-hoc analysis investigating clinical variables associated with a subsequent fall. Time to a fall was assessed with Cox proportional-hazards models adjusted for baseline characteristics and time-varying factors. Statistical inference was based on final multivariable models. Results: Falls were reported for 125/803 (15.6%) patients treated with apalutamide and ADT. Most falls were grade 1 or 2 and did not require hospitalization. Median time from randomization to first fall was 9.2 months (range 0.1–25.3 months). In the final multivariable model of both baseline and after-baseline covariates, baseline patient characteristics (older age, poor Eastern Cooperative Oncology Group performance status, history of neuropathy, and α-blocker use before study treatment) remained significantly associated with fall; after-baseline clinical characteristics significantly associated with time to fall were development of neuropathy, arthralgia, and weight loss before fall. Conclusions: This analysis identified risk factors for fall among nmCRPC patients treated with apalutamide. Clinical management can minimize these identified risks while enhancing patient outcomes. Preventive interventions should be considered when the identified baseline conditions and post-treatment neuropathy, arthralgia, or weight decrease are present, to reduce risk of fall. CA extern

The cancer stem cell hypothesis has gained currency in recent times but concerns remain about its scientific foundations because of significant gaps that exist between research findings and comprehensive knowledge about cancer stem cells (CSCs). In this light, a mathematical model that considers hematopoietic dynamics in the diseased state of the bone marrow and peripheral blood is proposed and used to address findings about CSCs. The ensuing model, resulting from a modification and refinement of a recent model, develops out of the position that mathematical models of CSC development, that are few at this time, are needed to provide insightful underpinnings for biomedical findings about CSCs as the CSC idea gains traction. Accordingly, the mathematical challenges brought on by the model that mirror general challenges in dealing with nonlinear phenomena are discussed and placed in context. The proposed model describes the logical occurrence of discrete time delays, that by themselves present mathematical challenges, in the evolving cell populations under consideration. Under the challenging circumstances, the steady state properties of the model system of delay differential equations are obtained, analyzed, and the resulting mathematical predictions arising therefrom are interpreted and placed within the framework of findings regarding CSCs. Simulations of the model are carried out by considering various parameter scenarios that reflect different experimental situations involving disease evolution in human hosts. Model analyses and simulations suggest that the emergence of the cancer stem cell population alongside other malignant cells engenders higher dimensions of complexity in the evolution of malignancy in the bone marrow and peripheral blood at the expense of healthy hematopoietic development. The model predicts the evolution of an aberrant environment in which the malignant population particularly in the bone marrow shows tendencies of reaching an uncontrollable equilibrium state. Essentially, the model shows that a structural relationship exists between CSCs and non-stem malignant cells that confers on CSCs the role of temporally enhancing and stimulating the expansion of non-stem malignant cells while also benefitting from increases in their own population and these CSCs may be the main protagonists that drive the ultimate evolution of the uncontrollable equilibrium state of such malignant cells and these may have implications for treatment.

BACKGROUND: Epoetin a (EPO) continues to be the initial treatment of choice for most anemic patients with myelodysplastic syndromes (MDS). Over the years, different therapeutic strategies have been adopted to optimize the clinical benefits of EPO in this setting. METHODS: In the current meta-analysis of published literature, erythroid response (ER) rates with EPO as a single agent versus its combination with granulocyte–colony-stimulating factor (G-CSF) or granulocyte-macrophage–colony-stimulating factor (GM-CSF) were compared. RESULTS: The assessment indicated that the ER rates were comparable between the 2 EPO-based therapeutic strategies. Furthermore, EPO monotherapy at a higher dose of 60,000 to 80,000 U weekly produced significantly higher ER rates (64.5%) compared with the standard oncology dose of 30,000 to 40,000 U weekly either as a single agent (49%; P < .001) or in combination with G-CSF/GM-CSF (50.6% P ¼ .007). In addition, when transfusion-dependent patients were assessed separately, both EPO monotherapy and its combination with G-CSF/GM-CSF produced comparable and appreciable levels of transfusion independence (28.8% and 24.8%, respectively). CONCLUSIONS: In the current meta-analysis, higher doses of EPO demonstrated better ER rates compared with EPO at standard doses alone or in combination with G-CSF/GM-CSF. Furthermore, the authors concluded that prospective clinical studies are warranted to evaluate the use of higher doses of EPO in anemic patients with MDS. Cancer 2009;115:706–15. V C 2009 American Cancer Society.

International audience; Stemming from current emerging paradigms related to the cancer stem cell hypothesis, an existing mathematical model is expanded and used to study cell interaction dynamics in the bone marrow and peripheral blood. The proposed mathematical model is described by a system of nonlinear differential equations with delay, to quantify the dynamics in abnormal hematopoiesis. The steady states of the model are analytically and numerically obtained. Some conditions for the local asymptotic stability of such states are investigated. Model analyses suggest that malignancy may be irreversible once it evolves from a nonmalignant state into a malignant one and no intervention takes place. This leads to the proposition that a great deal of emphasis be placed on cancer prevention. Nevertheless, should malignancy arise, treatment programs for its containment or curtailment may have to include a maximum and extensive level of effort to protect normal cells from eventual destruction. Further model analyses and simulations predict that in the untreated disease state, there is an evolution towards a situation in which malignant cells dominate the entire bone marrow - peripheral blood system. Arguments are then advanced regarding requirements for quantitatively understanding cancer stem cell behavior. Among the suggested requirements are, mathematical frameworks for describing the dynamics of cancer initiation and progression, the response to treatment, the evolution of resistance, and malignancy prevention dynamics within the bone marrow - peripheral blood architecture.

The association of cytogenetic abnormalities with cancer is well established. However, due to the historic lack of specific insight into the functional role of these anomalies, they have mostly served as diagnostic and/or prognostic indicators. Recent developments in chronic myelogenous leukemia and breast cancer have raised hopes for specific cytogenetic alterations to serve as therapeutic targets. This article reviews the aid provided by molecular diagnostics in these exciting developments in the cancer arena.

The bone marrow of patients with myelodysplastic syndromes (MDS) shows excessive intramedullary apoptosis, particularly in S-phase cells. In the light of previous reports that showed a link between experimental overexpression of the E2F1 transcription factor and apoptosis in the S phase, we compared the status of E2F1 protein in bone marrow mononuclear cells of MDS patients with that of healthy donors. Nearly 67% of MDS marrow samples showed higher expression of E2F1 transcription factor than in healthy donors. The retinoblastoma gene product, Rb, is a major negative regulator of E2F1 activity; however, Rb protein levels were found to be normal in MDS marrow samples. Amplification of genomic DNA by the polymerase chain reaction (PCR) showed no E2F1 gene amplification or mutation in the Rb-binding region of E2F1 in MDS patients, nor was transcriptional up-regulation noted when E2F1 messenger RNA (mRNA) levels were estimated with real-time reverse transcriptase-PCR. Furthermore, the overexpression of E2F1 was paralleled by its increased transcriptional activity, as reflected by the increased mRNA levels for one of its target genes, dihydrofolate reductase. Importantly, in a subset of the studied MDS patients for whom a simultaneous measurement of apoptosis in S-phase cells was possible, the E2F1 protein levels showed a significant positive correlation with this phenomenon. Previously, increased E2F1 activity in human disease had been found primarily as a consequence of Rb derailment. Hence, the observation in MDS of increased E2F1 activity in the presence of normal Rb levels is novel and unique, and E2F1 activity in association with apoptosis in S-phase cells may thus have significant therapeutic implications.

Labeling index (LI), apoptosis, levels of 2 pro-apoptotic cytokines tumor necrosis factor-alpha (TNF-alpha) and transforming growth factor-beta(TGF-beta), and the number of monocyte/macrophage cells that are the likely source of the cytokines were simultaneously measured in plastic-embedded bone marrow (BM) biopsy sections of 145 patients with myelodysplastic syndromes (MDS). TNF-alpha was correlated with TGF-beta (P = .001) and with monocyte/macrophage cells (P = .003). Patients with excess blasts in their marrows had a higher TGF-beta level (P = .01) and monocyte/macrophage number (P = .05). In a linear regression model,TGF-beta emerged as the most significant biological difference between patients who have excess of blasts and those who do not (P = .01). We conclude that in addition to TNF-alpha, TGF-beta also plays a significant role in the initiation and pathogenesis of MDS, and that a more precise definition of its role will likely identify better preventive and therapeutic strategies.

Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c-oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age-matched controls. Cytochrome c-oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential 'hot-spots'. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age-matched controls in all cell fractions (P < 0.05), with bone marrow high-density fraction (BMHDF) showing a higher mutation rate than other fractions (P < 0.05). MDS marrows showed higher levels of apoptosis than normal controls (P < 0.05), and apoptosis in BMHDF was directly related to cytochrome c-oxidase I gene mutations (P < 0.05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron-laden mitochondria. These results suggest a role for mt-DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.

Transmission electron microscopic (TEM) studies have not been reported in bone marrow (BM) biopsies of patients with myelodysplastic syndromes (MDS) owing to failure to overcome the technical impediment of maintaining ultrastructural detail in decalcified tissue. Using a modified technique to physically separate pieces of bone from marrow tissue under a dissecting microscope, and embedding the material directly for TEM, ultrastructural studies were performed in 15 MDS patients and four normal BM biopsies. Biopsy tissue was also used to initiate long-term in vitro cultures and 12-week plates were sacrificed for TEM analysis. Features noted in freshly obtained decorticated tissue included an excessive apoptosis in both haematopoietic and stromal cells, ringed sideroblasts with iron-laden mitochondria and highly active, enormously increased phagocytosis. In addition, type IV nuclear inclusion body variants (NIB-v) and confronting cylindrical cisternae (CCC) were readily identified in up to 40% of stromal cells in vivo, providing an important footprint of a possible infectious agent in the pathology of MDS. Cultured stromal cells did not show excessive apoptosis and only 2-4% fibroblasts showed the presence of NIB-v or CCC, underscoring the artificial nature of ex vivo systems. We conclude that ultrastructure studies using decorticated tissue can be a powerful tool to investigate the biology and aetiology of a variety of haematopoietic disorders as it enables the direct examination of BM biopsies with their intimate stromal parenchymal cell associations preserved intact.

Abiraterone acetate, a prodrug of the CYP17A1 inhibitor abiraterone that blocks androgen biosynthesis, is approved for treatment of patients with metastatic castration-resistant prostate cancer (mCRPC) in combination with prednisone or prednisolone 5 mg twice daily. This review evaluates the basis for the effects of prednisone on mineralocorticoid-related adverse events that arise because of CYP17A1 inhibition with abiraterone. Coadministration with the recommended dose of glucocorticoid compensates for abiraterone-induced reductions in serum cortisol and blocks the compensatory increase in adrenocorticotropic hormone seen with abiraterone. Consequently, 5 mg prednisone twice daily serves as a glucocorticoid replacement therapy when coadministered with abiraterone acetate, analogous to use of glucocorticoid replacement therapy for certain endocrine disorders. We searched PubMed to identify safety concerns regarding glucocorticoid use, placing a focus on longitudinal studies in autoimmune and inflammatory diseases and cancer. In general, glucocorticoid-related adverse events, including bone loss, immunosuppression, hyperglycemia, mood and cognitive alterations, and myopathy, appear dose related and tend to occur at doses and/or treatment durations greater than the low dose of glucocorticoid approved in combination with abiraterone acetate for the treatment of mCRPC. Although glucocorticoids are often used to manage tumor-related symptoms or to prevent treatment-related toxicity, available evidence suggests that prednisone and dexamethasone might also offer modest therapeutic benefit in mCRPC. Given recent improvements in survival achieved for mCRPC with novel agents in combination with prednisone, the risks of these recommended glucocorticoid doses must be balanced with the benefits shown for these regimens.

We used bone marrow biopsies instead of mononuclear cells to maintain long-term cultures from 103 patients belonging to all five sub-categories of myelodysplastic syndromes (MDS), as well as 12 normal controls. By week 4, 30-50% confluency was reached and could be maintained for up to 12 weeks with 100% confluency. The four prominent cells were fibroblasts, macrophages, endothelial cells and adipocytes. Immunohistochemical and electron microscopic studies provided lineage confirmation. Normal hematopoiesis was well supported by MDS stroma. Neither the FAB nor cytogenetics was co-related with the potency of growth. MDS stroma appears to be both morphologically and functionally normal.

The bone marrow (BM) in myelodysplastic syndromes (MDS) undergoes pathobiological changes that mimic an inflammatory process, and hence, an infectious etiology was suspected in these disorders. In the present report, we examined the bone marrow mononuclear cells (BMMNC) of 19 MDS patients and seven normal donors for the expression of one latency-related (Latency membrane protein 1 (LMP-1) and immediate early protein (IEP)) and one activation-related (BZLF and DNA-Pol) m-RNA each for two herpes viruses, Epstein-Barr virus (EBV) and cytomegalovirus (CMV), respectively. Reverse transcriptase polymerase chain reaction was used for this purpose. The latency-related transcripts (EBV-LMP-1 and CMV-IEP) were present in all the MDS and normal specimens. Intriguingly, 10/19 MDS specimens ( approximately 53%) and 2/7 normal donors ( approximately 28%) were positive for active EBV-BZLF (P=0.0067), while 2/19 MDS specimens ( approximately 11%) with 1/7 normal ( approximately 14%) showed active CMV-DNA-Pol (P=0.1588). Later, from another set of MDS patients (n=7) and normal donors (n=4), BM stromal cultures were established, which, at a 75% confluency, were overlaid with cord blood mononuclear cells (CBMNC). IEP was detectable in the CBMNC before and after co-incubation with MDS, as well as normal stroma. So, it was also present both in MDS and normal stromal cells. The other three were absent both in MDS and normal stromal layers. In CBMNC though, active EBV-BZLF and CMV-DNA-Pol m-RNA were detectable in one of seven MDS co-cultures each, albeit from different patients. None of the normal co-cultures showed active virus, either in stroma or CBMNC. Thus, the present report demonstrates, for the first time, the presence of active herpes viruses in the BMMNC of MDS patients and reveals the ability of the MDS stroma to support the viral activation.

A higher percentage of apoptotic cells (apoptotic index or AI) is consistently found in bone marrow (BM) biopsies compared to BM aspirates of patients with myelodysplastic syndrome (MDS). Most studies have only investigated the low-density fraction (LDF) mononuclear cells from BM aspirates following density separation for AI determination. In the present study, both LDF and high-density fraction (HDF) cells for AI were examined by electron microscopy (EM) in 10 MDS patients and 4 healthy donors. Matched BM biopsies were subjected to AI detection by in situ end labeling (ISEL) of fragmented DNA. The results indicate that in LDF and HDF cells, AI is consistently higher in MDS patients (8.5% vs 1.5%, respectively; P = .039) compared to healthy donors (27% vs 4%, respectively; P = .004). The BM biopsy AI was also higher in MDS patients than in healthy donors (3+ vs 0+, respectively; P = .036). In addition, in MDS patients, more apoptotic cells were found in HDF cells than in LDF cells (27% vs 8.5%, respectively;P = .0001). All stages of maturation, ranging from blasts to terminally mature cells belonging to all 3 lineages, were represented in the dying cells in both compartments. Using EM, typical Pelger-Huett–type cells appeared to be apoptotic granulocytes. Both LDF and HDF cells should be examined for an accurate estimation of apoptotic cells because AI would be underestimated if only the LDF cells were studied. Ultrastructural studies consistently show a higher AI in BM biopsies compared to BM aspirates despite the correction factor of HDF cells provided by AI. This may represent the actual extant state, which could conceivably be due to a higher concentration of proapoptotic signals in the biopsies.

Myelodysplastic syndromes (MDS) have previously been reported to show competitively high rates of apoptosis and proliferation in the bone marrow (BM). Using a double-labelling technique in the present study, we demonstrated that a significantly high number of S-phase cells were simultaneously apoptotic (signal antonymy; SA) in MDS (mean ± s.e.m. 53·5 ± 6·7%, n = 24, P

Apoptosis has been documented in chondrocytes both in the growth plates of young, healthy cartilages and in osteoarthritic cartilages; little, however, is known about apoptosis in chondrocytes of normal adult articular cartilage. For the current study, apoptosis in adult chondrocytes was evaluated by labeling DNA fragments using the ISEL in situ end labeling of 3'-recessed strand breaks) or TUNEL (5'-recessed or blunt-ended strand breaks with terminal deoxynucleotidyl transferase-mediated nick end labeling) techniques in primary cultures of chondrocytes in monolayer. Apoptosis was induced in the chondrocytes by either Tumor Necrosis Factor alpha (TNF alpha), Interleukin 1-beta (IL-1 beta), or anti-Fas antibody but only after 48 hours in culture. At 4 and 24 hours, there was no detectable DNA fragmentation. With TNF alpha, IL1 beta, and anti-Fas antibody, chondrocytes show evidence of at least two types of DNA strand breaks within the same cell (as assessed by simultaneous labeling with ISEL and TUNEL). Therefore, some pathways leading to apoptosis in chondrocytes appear to involve more than one type of endonuclease activity. When the chondrocytes were cultured as explants with the articular matrix intact (ex vivo), neither IL-1 beta, TNF alpha, the anti-Fas antibody, nor fibronectin fragments were able to induce apoptosis in the chondrocytes. In normal human adult cartilage that was untreated and uncultured (in situ), DNA fragmentation was undetectable; however, a significant number of chondrocytes in osteoarthritic cartilage did contain strand breaks. These data suggest that apoptosis occurs in chondrocytes in which the matrix has been disrupted experimentally or destroyed by the osteoarthritic disease process. The results of these studies suggest that the ECM may be an essential survival factor for chondrocytes.

Myelodysplastic syndromes (MDS) are a group of hematopoietic disorders characterized by the concomitant presence of peripheral cytopenias and normocellular to hypercellular BM. This paradox has been proposed to be due to the presence of excessive proliferation matched by excessive intramedullary apoptosis of hematopoietic cells. When cultured in vitro MDS BM mononuclear cells (BMMC) undergo apoptosis within 4 h. We measured caspase-1-like and caspase-3-like activity in 22 MDS and 4 normal BM immediately following cell separation or after 4 h culture. When cultured in vitro, MDS BMMC demonstrated an increased apoptotic index within 4 h as measured by in situ end-labeling of fragmented DNA that was matched by a concurrent increase in caspase-3-like specific activity, and the two were significantly correlated. During the 4 h culture, a sequential activation of caspase-1-like and caspase-3-like activities was detected. Caspase-1-like specific activity was detected early and transiently at approximately 15 min, followed by a gradual increase in caspase-3-like-specific activity peaking at 2 h. When the broad-spectrum caspase inhibitor, Z-VAD.FMK, was included in the MDS BM aspirate 4 h culture, apoptosis was attenuated. We conclude that sequential activation of caspase-1-like and caspase-3-like activities may form the central biochemical pathway of apoptosis in BMMC from some MDS patients, and prevention of this process by caspase inhibitors may be of significant therapeutic value for these patients, in whom supportive care continues to be the mainstay of therapy.

Increased intramedullary apoptotic death of hematopoietic cells is thought to contribute to the ineffective hematopoiesis in myelodysplastic syndromes (MDS). Furthermore, high amounts of tumor necrosis factor alpha (TNF alpha) have previously been correlated with apoptosis in MDS marrows. The present studies were undertaken to examine the status of two key downstream effectors of TNF alpha signaling, i.e. Caspase 1 and Caspase 3 enzymes, using a fluorometric assay in the bone marrow aspirate mononuclear cells (BMMNC) in relation to apoptotic DNA fragmentation detected by in situ end-labeling (ISEL) of DNA and with localization of TNF alpha in the corresponding biopsies from 14 MDS patients. Both Caspase 1 and 3 were detectable in freshly harvested BMMNC, albeit median Caspase 3 levels (47.5 units/mg protein) being almost 10 times higher than Caspase 1 (4.0 units/mg protein). Upon short-term culture for 4 h in a serum-supplemented medium in vitro a significant increase was seen in Caspase 3 activity (58.8 +/- 13.9 at 0 h vs. 177.8 +/- 55.2 units/mg protein at 4 h, n = 14, P = 0.017) and in percent cells labeled by ISEL (apoptotic index or AI%: 0.76% +/- 0.25% vs. 3.99% +/- 1.1%, n = 14, P = 0.004, respectively). Caspase 1 activity increased after 15 min in culture. Interestingly, TNF alpha levels measured by immunohistochemistry correlated with the net increase in Caspase 3 activity after 4 h (p = 0.517, n = 13, P = 0.07) and the starting levels of Caspase 1 at 0 h correlated with the Caspase 3 levels attained at 4 h (p = 0.593, n = 13, P = 0.033). Additionally when TNF alpha-positive bone marrows (8/14) were compared with the negative marrows (6/14) the Caspase 3 levels were significantly higher in the TNF alpha-positive marrows (189.6 +/- 66.2 vs. 25.0 +/- 14.6 units/mg protein, respectively, P = 0.043). The increase in AI%, though not statistically significant, was also higher in the TNF alpha-positive marrows. Finally in HL60 cells the effects of different Caspase inhibitors and pentoxifylline (PTX) (interferes with lipid signaling of cytokines) on TNF alpha-induced apoptosis were evaluated. TNF alpha treatment significantly increased AI% (P < 0.003) as compared to the untreated controls. A co-treatment with three Caspase inhibitors, zVAD.FMK (inhibitor of Caspases 1 and 3, 10 microM/l), Ac.YVAD.FMK (Caspase 1 inhibitor, 1 microM/l), Ac.DEVD.FMK (Caspase 3 inhibitor, 10 microM/l) as well as PTX (250 microM/l) significantly curtailed the AI% induced by TNF alpha. The present studies thus identify the downstream effectors of TNF alpha-inducible apoptosis in MDS and so also the suppressors of TNF alpha apoptotic signaling. These results may have significant clinical implications in the therapy of MDS in the future.

Rates of proliferation and apoptosis as well as expression of tumor necrosis factor alpha (TNF-alpha), transforming growth factor beta (TGF-beta) and the number of macrophages were measured in bone marrow (BM) biopsies of 33 patients who presented with hypocellular (cellularity30%) myelodysplastic syndromes (MDS). Results showed that 2/3 of the patients had high apoptosis, high cytokine levels and large number of macrophages in their biopsies while 1/3 did not. Apoptosis and TNF-alpha levels were directly related (r = 0.583, P = 0.003, n = 24) as was apoptosis and the degree of anemia (P = 0.033, n = 18). A subgroup of patients with abnormalities of chromosomes 5 or 7 had higher platelets (P = 0.026) and higher apoptosis (P = 0.038) when compared with the rest of the group. Eight patients had no evidence of apoptosis and almost no detectable TNF-alpha in their biopsies. We conclude that within the hypocellular variant of MDS, there may be two distinct sub-groups of patients, one who present with high cytokine-mediated intramedullary apoptosis and the other who may be better characterized as having a stem-cell failure defect since they showed no evidence of apoptosis.

We previously reported excessive apoptosis and high levels of tumor necrosis factor-alpha (TNF-alpha) in the bone marrows of patients with myelodysplastic syndromes (MDS), using histochemical techniques. The present studies provide further circumstantial evidence for the involvement of TNF-alpha in apoptotic death of the marrow cells in MDS. Using our newly developed in situ double-labeling technique that sequentially employs DNA polymerase (DNA Pol) followed by terminal deoxynucleotidyl transferase (TdT) to label cells undergoing apoptosis, we have characterized DNA fragmentation patterns during spontaneous apoptosis in MDS bone marrow and in HL60 cells treated with TNF-alpha or etoposide (VP16). Clear DNA laddering detected by gel electrophoresis in MDS samples confirmed the unique length of apoptotic DNA fragments (180-200 bp). Surprisingly, however, phenotypically heterogeneous population of MDS cells as well as the homogenous population of HL60 cells showed three distinct labeling patterns after double labeling--only DNA-Pol reaction, only TdT reaction, and a combined DNA Pol + TdT reaction, albeit in different cohorts of cells. Each labeling pattern was found at all morphological stages of apoptosis. MDS mononuclear cells, during spontaneous apoptosis in 4 hr cultures, showed highest increase in double-labeled cells (DNA Pol + TdT reaction). Interestingly, this was paralleled by TNF-alpha-induced apoptosis in HL60 cells. In contrast, VP16 treatment of HL60 cells led to increased apoptosis in cells showing only TdT reaction. The double-labeling technique was applied to normal bone marrow and peripheral blood mononuclear cells after treatment with known endonucleases that specifically cause 3' recessed (BamHI), 5' recessed (PstI), or blunt ended (DraI) double-stranded DNA breaks. It was found that the DNA-Pol reaction in MDS and HL60 cells corresponds to 3' recessed DNA fragments, the TdT reaction to 5' recessed and/or blunt ended fragments, and a combined "DNA Pol + TdT reaction" corresponds to a copresence of 3' recessed with 5' recessed and/or blunt ended fragments. Clearly, therefore, apoptotic DNA fragments, in spite of a unique length, may have differently staggered ends that could be cell (or tissue) specific and be selectively triggered by different inducers of apoptosis. The presence of TNF-alpha-inducible apoptotic DNA fragmentation pattern in MDS supports its involvement in these disorders and suggests that anti-TNF-alpha (or anticytokine) therapy may be of special benefit to MDS patients, where no definitive treatment is yet available.

Rates of proliferation, apoptosis and cytokine expression were measured in bone marrow (BM) biopsies of 164 myelodysplastic syndrome (MDS) patients. There were 107 males and 57 females. Median age was 69 years and 101 had refractory anemia (RA), 17 RA with ringed sideroblasts (RARS), 38 with RA and excess blasts (RAEB) and 8 with RAEB in transformation (RAEB-t). Apoptosis measured by in-situ end labeling (ISEL) was directly related to the number of macrophages (p = 0.028, n = 83). Mean tumor necrosis factor alpha (TNF-alpha) and ISEL positivity were higher in RAEB + RAEB-t patients (p = 0.0554 and p = 0.06 respectively) while hemoglobin was higher for RA + RARS group (p = 0.0472). Patients with high apoptosis had lower white blood cell counts (p = 0.0009), lower percentage of blasts (p = 0.0009) and higher number of macrophages (p = 0.0086). We conclude that measurements of apoptosis, proliferation and cytokine expression provide important biological information which helps to distinguish RA + RARS patients from RAEB + RAEB-t patients, and may be of additive prognostic significance.

Par-4 overexpression has been shown by Boehrer et al. to augment the apoptotic death of Jurkat T cells induced by TNF related Apoptosis Inducing Ligand (TRAIL). Previously, the same authors have demonstrated a parallel augmentation of chemotherapeutic drug-induced apoptosis in the same cell line overexpressing Par-4. Through these and other studies, Par-4 appears to emerge as a general intracellular facilitator of both extrinsic and intrinsic pathways of apoptosis. The ability of Par-4 to modulate relevant inhibitors of apoptosis and to facilitate activation of alternative initiator or executioner caspases in contingent situations may support its role as a common facilitator/enhancer of apoptotic cell death. Further understanding of Par-4 biology will have significant clinical implications especially in overcoming drug resistance phenomenon in cancer.

Extensive apoptosis or programmed cell death (PCD) of both hematopoietic (erythroid, myeloid, megakaryocytic) and stromal cells in myelodysplastic syndromes (MDS) cancels the high birth-rate resulting in ineffective hematopoiesis and has been demonstrated as the probable basis for peripheral cytopenias in MDS by our group. It is proposed that factors present in the microenvironment are inducing apoptosis in all the cells whether stromal or parenchymal. To investigate this hypothesis further, bone marrow biopsies from 46 MDS patients and eight normal individuals were examined for the presence of three cytokines, tumor necrosis factor-alpha (TNF-alpha), transforming growth factor-beta (TGF-beta) and granulocyte macrophage-colony stimulating factor (GM-CSF) and one cellular component, macrophages, by the use of monoclonal antibodies immunohistochemically. Results showed the presence of TNF-alpha and TGF-beta in 41/46 and 40/46 cases of MDS respectively, while only 15 cases showed the presence of GM-CSF. Further a significant direct relationship was found between the degree of TNF-alpha and the incidence of PCD (p= 0.0015). Patients who showed high PCD also had an elevated TNF-alpha level. Thus, the expression of high amounts of TNF-alpha and TGF-beta and low amounts of the viability factor GM-CSF may be responsible for the high incidence of PCD leading to ineffective hematopoiesis in MDS. Future studies will be directed at attempting to reverse the lesion in MDS by using anti-TNF-alpha drugs such as pentoxifylline.

Our previous studies using in situ end labeling (ISEL) of fragmented DNA revealed extensive apoptotic cell death in the bone marrows (BM) of patients with myelodysplastic syndromes (MDS) involving both stromal and hematopoietic cells. In the present report we show greater synthesis of interleukin-1 beta (IL-1 beta) in 4 hour cultures of density separated BM aspirate mononuclear (BMAM) cells from MDS patients as compared to the cultures of normal BM from healthy donors or lymphoma patients (1.7 +/- 0.37 pg/10(5) cells, n = 29 v 0.42 +/- 0.24 pg/10(5) cells, n = 11, respectively, P = .049). Further, these amounts of IL-1 beta in MDS showed a significant correlation with the extent of apoptosis detected by ISEL in corresponding plastic embedded BM biopsies (r = .480, n = 30, P = .007). In contrast normal BMs did not show any correlation between the two (r = .091, n = 12, P = .779). No significant correlation was found between the amounts of IL-1 beta and % S-phase cells (labeling index; LI%) in MDS determined in BM biopsies using immunohistochemistry following in vivo infusions of iodo- and/or bromodeoxyuridine. Neither anti-IL-1 beta antibody nor IL-1 receptor antagonist blocked the apoptotic death of BMAM cells in 4 hour cultures (n = 5) determined by ISEL (apoptotic index; AI%), although the latter led to a dose-dependent accumulation of active IL-1 beta in the culture supernatants. On the other hand, a specific tetrapetide-aldehyde inhibitor of ICE significantly retarded the apoptotic death of BMAM cells at 1 mumol/L in 5/6 MDS cases studied (AI% = 2.99 +/- 0.30 in controls v 1.58 +/- 0.40 with ICE-inhibitor, P = .05) and also reduced the levels of active IL-1 beta synthesized (5.59 +/- 2.63 v 2.24 +/- 0.93 pg/10(6) cells, respectively). In normal cells, neither IL-1 blockers nor the ICE inhibitor showed any effect on the marginal increase in apoptosis observed in 4 hour cultures. Our data thus suggest a possible involvement of an ICE-like protease in the intramedullary apoptotic cell death in the BMs of MDS patients.

Cell-cycle kinetics were measured in situ after infusions of iododeoxyuridine and/or bormodeoxyuridine in 50 patients with myelodysplastic syndromes (MDS) and the median labeling index in bone marrow (BM) biopsy samples was 28.6%. Unfortunately, 26 of 50 patients showed that > or = 75% of hematopoietic cells of all three lineages were undergoing programmed cell death (PCD) in their biopsy samples as shown by the in situ end labeling (ISEL) technique. Ten patients had 1/3 and eight had 2/3 ISEL+ cells. Stromal cells were frequently ISEL+ and often S-phase cells were also found to be simultaneously ISEL+. Nucleosomal DNA fragments as a ladder in agarose gel were present in BM aspirates of four patients who showed high ISEL and were absent in two who had no ISEL staining in biopsy samples, but only when DNA was extracted after a 4-hour in vitro incubation in complete medium. Therefore, laddering data confirmed the ISEL findings that the majority of hematopoietic cells in MDS are in early stages of PCD. We conclude that extensive intramedullary cell death may explain the paradox of pancytopenia despite hypercellular marrows in MDS patients. Investigating approaches that protect against PCD in some MDS subsets would be of interest.

Jakob Nielsen1 described us as information foragers; that is, we decide how to consume information in a way that optimizes our benefits relative to the costs. For example, if a wolf is planning to hunt in a forest that is populated by large and small rabbits; which ones will the wolf go after? We would initially say: the “large rabbits” because they provide more meat. But if the large rabbits are faster and are harder to catch, the benefit decreases. It is much better to eat lots of small tasty bites if tiny bunnies are easier to nab (Jakob Nielsen, see footnote). The real question is therefore not which prey provides the most food, but how we get the most food with the least effort (Jakob Nielsen, see footnote). It is the same for us, informavores. A long article will contain more information, but it will take longer to read. Readers may abandon the article and read shorter, easier pieces elsewhere. In such a consideration, therefore, our new publication, the Leukemia Research Reports is committing to support an open access rapid online publication of case reports, brief communications, valuable perspectives and hypotheses. We sincerely hope that the hematology–oncology community will welcome and embrace this new forum.

We describe a novel double-labeling method to simultaneously investigate proliferation and apoptosis from plastic-embedded biopsy specimens (PEBs). Infusions of bromo- and/or iododeoxyuridine (BrdU/IudR) were given to 10 patients, five with acute myeloid leukemia (AML) and five with myelodysplastic syndromes (MDS), and S-phase cells were measured in PEBs using a monoclonal anti-IudR/BrdU antibody. Apoptosis was measured by in situ end-labeling (ISEL) of DNA. The results demonstrate that both AML and MDS are highly proliferative disorders but that there is almost no apoptosis in the former, whereas extensive apoptosis was observed in the latter. Double labeling revealed that large numbers of S-phase cells in MDS were simultaneously undergoing apoptosis. We conclude that the high cell death in MDS cancels the high cell birth, resulting in a functionally aplastic marrow and thus accounting for the observed ineffective hematopoiesis. On the other hand, AML is rapidly fatal, probably owing to high cell birth with no or minimal cell death. Therapeutic strategies to prevent intramedullary programmed cell death of hematopoietic precursors should be evaluated in MDS, and efficacy of chemotherapy in AML can be assessed by measuring the induction of apoptosis in post-treatment biopsy specimens.

Huet first observed an abnormal hyposegmented neutrophil granulocyte in 1928, and a year later Pelger reported an additional case. Such a cell later became known as Pelger-Huet anomaly. In 1932 Huet pointed out its hereditary and systemic character.[1][1] The Pelger-Huet anomaly is a familial

Dexamethasone combined with abiraterone acetate might result in improved survival in the treatment of castration-resistant prostate cancer compared with the Food and Drug Administration-approved prednisone combination, but the hypothesis must be confirmed with with clinical studies comparing the two combinations.

Refractory anaemia associated with excessive intramedullary erythroid apoptosis and dysplasia is a major feature of myelodysplastic syndromes (MDS). Recombinant human erythropoietin (specifically, epoetin alfa [EPO]) has been used in the therapy of MDS for many years. Initially, the erythroid response rates were modest, as EPO was used in all subgroups of MDS patients without discretion. However, with increased sophistication in patient selection and response evaluation criteria, there has been a significant improvement in the response rates to EPO therapy. This review discusses the evolution of therapeutic strategies incorporating EPO for the treatment of MDS.

The 21st century is witnessing emergence of a new era in medicine called “Genomic Disease Management.” Chromosomes, which house the essential components of this new faculty of medicine, have gained scientific attention since the late 19th century. However, it took nearly 100 yr before the understanding of these vital molecules could be taken to a patient’s bedside. The association of chromosomal abnormalities with congenital malformation disorders and cancer has been suspected since the late 1950s (1–4). In particular, demonstration by these studies of trisomy of one of the smallest chromosomes in Down’s syndrome (chromosome 21, as shown later), monosomy X in Turner’s syndrome, and the chromosomal analysis of leukemias laid foundation for the coming years. It was the development of a variety of staining methods assigning uniqueness to individual chromosomes that really marked the beginning of genomic analysis (for review, see ref. 5). As shown in Table 1, these techniques are collectively known as chromosome banding. A realization that using certain stains like Giemsa and Quinacrine mustards, the AT-rich and GC-rich regions of chromosomes could be distinguished into a unique banding pattern for every chromosome, indeed, invited clinical interest into this fascinating field (6–7). Using a metaphase cell preparation, it became possible to clearly identify chromosomal abnormalities in clinical specimens. The banding techniques underscored the fact that the association of chromosomal abnormalities with disease might not be random or an epiphenomenon. A demonstration of two consistent chromosomal translocations—t(9;22) related to Philadelphia chromosome in chronic myeloid leukemia (CML) and t(15;17) found in acute promyelocytic leukemia (APL)—provided direct testimony for a specificity of chromosomal anomalies in hematological malignancies (4,8,9). Ever since, chromosomal banding became the gold standard for both clinical and basic science studies in cytogenetics. Many authors view the progress of cytogenetics in three phases: (1) the Prebanding era (until 1970), (2) the banding era (1970–1980), and (3) the present-day constantly evolving molecular cytogenetics era (post-1980) (10,11). The roots of this astoundingly progressive molecular phase in the last two decades actually could be seen as early as 1969, when the concept of in situ oligonucleotide hybridization (ISH) was introduced by several groups simultaneously (12–14). Albeit, the use of radioisotopic labeling and the autoradiographic visualization method of ISH restricted its general application. Between 1986 and 1988, two major advances illuminated the field of cytogenetics: (1) the development of nonradioactive fluorescent oligonucleotide probe labeling technique and (2) the construction of human-chromosomespecific libraries (15–18). During this period, the concept of “interphase cytogenetics” was born, which offered a tremendous technical ease in the ISH protocol and reduced the assay time considerably (17). The fluorescence in situ hybridization (FISH) has since come a long way, metamorphosing from a concurrent detection of several chromosomal abnormalities to the identification of previously uncharacterized abnormalities with multiplex-FISH (M-FISH) or spectral karyotyping (SKY™), further to genomic screening with FISH-based metaphase-comparative genomic hybridization and most recently to the solid-phase genomic DNA arrays (for review, see refs. 11 and 19). The present chapter describes the principles of ISH and FISH and their potential clinical applications and, finally, introduces the seeds of future developments in FISH technology.

Myelodysplastic syndromes (MDS) are considered to be stem cell disorders. High incidence of intramedullary apoptosis has been associated with the peripheral cytopenia and refractory anemia in these disorders. The investigations on the cell of origin in the bone marrow have invariably been hampered by a poor yield of CD34+ cells from these marrows. Interestingly, even though limited in number, these studies raised more questions and dilemmas than providing answers. While the enigma surrounding the clonality of these marrows continues, the controversies regarding incidence of apoptosis, proliferation, and potential of clonogenic expansion may be closer to a settlement. The present review proposes a model depicting interplay between extraneous apoptogenic factors and intracellular apoptosis-susceptibility determinants that contributes significantly toward the progression of MDS and how a shift in dynamics of this interplay may provide grounds to accumulate additional mutations with a probable block in differentiation eventually leading to a leukemic transformation.

The field of cytogenetics has already entered the molecular era and a rapid expansion of its contribution is seen in genomic disease management. Among the evolving advanced molecular techniques, with an impeccable balance of high specificity, sensitivity and assay rapidity, fluorescence in situ hybridization has made its home in routine clinical laboratory. Today, its clinical application is vivid in every phase of disease management of a number of malignancies. The rapid growth in the knowledge of specific associations between distinct chromosomal abnormalities and different types of cancers will necessitate simultaneous detection of multiple abnormalities using multicolor/multiplex fluorescence in situ hybridization tests more often in the near future. Also, as the human genome sequence is ascertained, genome-wide screening with microarray technology will gain eminence in the clinical scenario, yield better solutions and bring the concept of personalized medicine in cancer closer to reality than ever before.

Fas-Fas-ligand, the most well investigated apoptotic signaling system, plays a pivotal role in several human diseases. Its signaling through multiple pathways; multiple, complex regulatory points; and recently described tendency to undergo ligand-independent self-assembly warrant a comprehensive understanding of its physiologically well controlled function. In this review, we attempt to develop such a perspective on the regulation of Fas signaling. The multistep Fas regulation model presented here should be helpful in devising effective interventions for conditions resulting from the dysregulation of Fas.

Mitochondria (mt) play an important role in both apoptosis and haem synthesis. The present study was conducted to determine DNA mutations in mitochondrial encoded cytochrome c-oxidase I and II genes. Bone marrow (BM) biopsy and aspirate, peripheral blood (PB) and buccal smear samples were collected from 20 myelodysplastic syndrome (MDS) patients and 10 age-matched controls. Cytochrome c-oxidase I (CO I) and II (CO II) genes were amplified using polymerase chain reaction and sequenced. CO I mutations were found in 13/20 MDS patients and the CO II gene in 2/10 normal and 12/20 MDS samples, irrespective of MDS subtype. Mutations were substitutional, deletional and insertional. CO I mutations were most common at nucleotide positions 7264 (25%) and 7289 (15%), and CO II mutations were most common at nucleotide positions 7595 (40%) and 7594 (30%), suggesting the presence of potential 'hot-spots'. Mutations were not found in buccal smears of MDS patients and were significantly higher in MDS samples compared with age-matched controls in all cell fractions (P0.05), with bone marrow high-density fraction (BMHDF) showing a higher mutation rate than other fractions (P0.05). MDS marrows showed higher levels of apoptosis than normal controls (P0.05), and apoptosis in BMHDF was directly related to cytochrome c-oxidase I gene mutations (P0.05). Electron microscopy revealed apoptosis affecting all haematopoietic lineages with highly abnormal, iron-laden mitochondria. These results suggest a role for mt-DNA mutations in the excessive apoptosis and resulting cytopenias of MDS patients.

As described in many chapters in this volume it is evident that excessive intramedullary apoptosis constitutes a salient feature of the pathobiology of myelodysplastic syndromes (MDS). Since the first indication of increased apoptosis upon examining the bone marrow (BM) histology in 1990 by Clark and Lampert, followed by histochemical demonstration by our group in 1994–95 ([Mundle et al., 1994a] and [b]; [Raza et al., 1995a] and [b]), a number of studies using a variety of techniques in independent demographic populations around the globe have repeatedly confirmed this finding ([Yoshida & Mufti, 1999]). In spite of such an endorsement, it must be acknowledged that as pointed out by [Dar et al. (1999)], exceptions to this rule showing undetectable apoptosis are also commonly found in MDS. In cases with high apoptosis, the most striking feature is the engagement of all types of hematopoietic and stoma] cells in the apoptotic death process. As a result the presence of an apoptosis-inducing microenvironmental factor(s) with a wide range of target cells was suspected. Different studies have revealed exceedingly high levels of proinflammatory factors like tumor necrosis factor alpha (TNFa), interferon gamma (IFN y), interleukin (IL) 1 and 6, etc. ([Mundle et al., 1996]; [Shetty et al., 1996]; [Kitagawa et al., 1997]; [Gersuk et al., 1998]; [Deeg et al., 2000]). Subsequent studies highlighted TNFa. as probably the most apical trigger of apoptosis in MDS ([Mundle et al., 1999 a] and [b]).

Serum lipid profiles were obtained in 108 patients with myelodysplastic syndrome (MDS) and compared to 28 healthy volunteers. Serum cholesterol and low-density and high-density lipoproteins (LDL and HDL) were found to be significantly lower in MDS patients than in normals (p = 0.0001, 0.0038 and 0.037, respectively). This difference was significant for all MDS categories. Serum cholesterol and HDL were negatively related to biopsy cellularity (p = 0.001 and 0.0001, respectively), and serum triglycerides were negatively related to labeling index (p = 0.0003). No differences were noted in the lipid profiles of MDS patients with normal versus abnormal karyotypes. However, low-risk MDS patients with abnormal karyotypes had significantly lower triglyceride levels compared with the high-risk patients (p = 0.027), as did low-risk patients with normal cytogenetics (p = 0.015). Serum HDL levels were significantly higher for the low-risk group with normal cytogenetics as well (p = 0.003). We conclude that serum cholesterol, LDL, and HDL are significantly reduced in MDS patients, probably indicating excessive intracellular lipid biosynthesis in the expanding clone. These relatively simple measurements could serve as important prognostic markers and reliable indicators of disease activity in individual patients. Prospective studies to determine their utility as independent variables that guide the need for active therapeutic intervention are warranted.

Spontaneous intramedullary apoptosis was measured in bone marrow (BM) biopsies of 175 patients with myelodysplastic syndromes (MDS) using in situ end-labeling (ISEL) of fragmented DNA. Two groups of high (n = 71) versus low (n = 43) levels of apoptosis were identified while 61 patients were ISEL-negative. Semiquantitative assessment of 3 cytokines, the number of macrophages and in vivo labeling indices (LI) were also determined from consecutive sections of the biopsy. Patients with high apoptosis levels tended to have a high LI (p = 0.013), more macrophages in their BM biopsies (p = 0.006) and higher tumor necrosis factor alpha (TNF-α) levels (not significant) compared to patients with no apoptosis. In addition, low risk MDS patients had significantly lower rates of apoptosis (p = 0.047) and lower levels of TNF-α (p = 0.055) compared to high-risk MDS patients. We conclude that the genesis of cytopenias in MDS is of multifactorial origin and that cytokine-associated apoptosis clearly identifies a distinct biological subgroup of patients who may benefit selectively by use of anti-cytokine therapies.

The effects of the administration of a 3-day course of 13-cis retinoic acid in combination with interferon a [RA/IFN] on the leukemia cells was measured in vivo in 43 patients with chronic myelogenous leukemia. The administration of RA/IFN was associated with a significant fall in the white blood cell count of patients with chronic-phase disease and with a fall in the percentage S-phase cells in CML patients regardless of the stage of their leukemia. In two thirds of the patients studied the administration of RA/IFN was also associated with an increase in marrow apoptosis. The cytokine combination also suppressed bcl-2 and myc expression in a minority of patients and such expression appears to be associated with response to a treatment regimen which includes RA/IFN. These studies are the first to directly assess the effects of the combination of RA/IFN on chronic myelogenous leukemia cells in vivo in patients. These effects, if seen in other malignant diseases, could account for the therapeutic benefit which has been associated with the administration of this combination of biological agents to patients with malignant disease.

Forty-three patients with myelodysplastic syndromes (MDS) were treated with a combination of pentoxifylline and ciprofloxacin (PC) with the addition of dexamethasone (PCD) in 18 patients who failed to respond to PC. There were 15 females and 28 males, and the median age was 67 years. A total of 18 patients either showed a hematopoietic improvement, a partial or complete cytogenetic response or a combination of both for an overall response rate of 42%. Seven PC only patients responded, four showing hematologic improvement, two cytogenetic responses and one patient showing a combined response. This 16% response rate to PC was increased to 61% by the addition of dexamethasone with 11/18 patients showing a response. Four of the 7 patients who responded initially to PC were given dexamethasone after at least 12 weeks of PC therapy, and only 1 showed a further improvement in response. Thus, we conclude that the combination of PCD provides an encouraging novel approach to treating MDS. The mechanism of action is probably related to the suppression of a veriety of cytokines which in turn attenuate the excessive intramedullary apoptotic death of hematopoetic cells in MDS, an observation which has been speculated to be the basis of the paradox of variable cytopenias despite cellular marrows in MDS. Larger numbers of patients need to be treated and followed for longer periods to determine the true efficacy of this therapy, especially the nature and duration of the cytogenetic responses.

Myelodysplastic syndromes (MDS) are highly proliferative bone marrow (BM) disorders where the primary lesion presumably affects a CD34+ early progenitor or stem cell. We investigated the proliferative characteristics of CD34+ cells of 33 untreated MDS patients (19 RA, 5 RARS, 7 RAEB, 2 RAEBt) and five patients with acute myeloid leukemia after MDS (sAML). All patients received a 1-h infusion of the thymidine analogue iodoor bromodeoxyuridine intravenously before a BM aspirate and biopsy was taken. A double-labeling immunohistochemistry technique by monoclonal anti-CD34 (QBend/10) and anti-IUdR/BrdU antibodies was developed and performed. By this technique we recognised CD34+ and CD34- cells actively engaged in DNA synthesis or not. As MDS evolves a significant increase occurred in the percentage of CD34+ cells of all myeloid cells (mean value: RA/RARS 1.67%; RAEB(t) 8.68%; sAML 23.83%) as well as in the percentage of proliferating CD34+ cells of all myeloid cells (RA/RARS 0.19%; RAEB(t) 0.43%; and sAML 3.30%). This was associated with a decreasing trend in the overall myeloid labeling index (LI: RA/RARS 25.8%, RAEB(t) 24.6% and sAML 21.5%). This decrease in overall myeloid LI is due to an exponential increase in the proportion of CD34+ cells of the proliferating compartment during MDS evolution (RA/RARS 0.35%, RAEB(t) 1.44% and sAML 11.98% of all S-phase cells). These CD34+ cells appeared to proliferate more slowly than their more mature CD34 negative counterparts, since we found a progressive increment in the mean total cell cycling time (Tc) of all myeloid cells during MDS progression (RA/RARS 39.8, RAEB(t) 45.2 and sAML 65.8 h). This study showed that during MDS evolution to sAML the CD34+ compartment develops a growth advantage leading to apparent expansion.

Chronic myelogenous leukemia (CML) has a progressive course but little is known about the biologic characteristics of disease progression. This study was designed to assess the changes in cell proliferative characteristics, apoptosis, the expression of the bcl-2 and c-myc genes between the time of initial diagnosis and entrance into the blastic phase of the disease. We observed that the rate of cell proliferation decreased and the cell death rate did not significantly change as the disease accelerated. The level of bcl-2 expression was significantly higher in accelerated/blastic phase cells than in the chronic phase cells in the population as a whole, however, the bcl-2 expression level did not change in blast cell subpopulation. c-myc Expression was significantly higher in the blast cell subpopulation of accelerated/blastic phase than in that of earlier phases of the disease. In conclusion, the characteristics of CML cells, namely proliferation rate, c-myc and bcl-2 change during the course of the disease. It is possible that the change in c-myc expression plays a causative role in evolution of the blastic phase from the chronic phase.

The study was carried out on 22 patients with non-Hodgkin's lymphoma (NHL) who had received sequential infusions of two thymidine analogues iododeoxyuridine (IUdR) and bromodeoxyuridine (BrdU). Cell cycle kinetic studies seemed to differentiate distinctly between low grade lymphoma (n = 8, LI = 2.6%) compared to that of intermediate grade (n = 9, LI = 13%, p = 0.0001) and high grade NHL (n = 5, LI = 16.3%, p = 0.0062). While the majority of 14 intermediate and high grade lymphomas had a high labeling index there were 3/14 patients with a LI of 5.5%, 5.5% and 4.1% respectively. A decrease in the rate of programmed cell death (PCD) or apoptosis due to the overexpression of bcl-2 has been implicated as the possible pathogenesis for follicular lymphoma. We determined the presence of bcl-2 protein immunohistochemically and apoptosis by in situ end labeling of DNA which detects cells in early stages of PCD not recognized morphologically. Nine NHL patients demonstrated PCD ranging from 1%-40%, while it was undetectable in 13/22 patients. Of these 13 cases, 6 showed the presence of bcl-2 expression. To understand the relationship of the microenvironment to the lymphoma cells, the presence of transforming growth factor beta (TGF-beta) was determined immunohistochemically. TGF-beta was present in all the cases where bcl-2 was present, except one. This study highlights some of the key biological features of NHL cells and their microenvironment.

Despite hypercellular bone marrows (BM), peripheral cytopenias are the rule in patients with myelodysplastic syndromes (MDS). This study examined the roles played by cell birth and cell death rates in generating this paradox. Cell kinetics from BM biopsies of 35 MDS patients were measured using intravenous infusions of either iododeoxyuridine or bromodeoxyuridine, or both. Degree of apoptosis or programmed cell death (PCD) was estimated using in situ end-labeling of DNA directly from BM biopsies, which were simultaneously double-labeled for proliferation/PCD. MDS were found to be highly proliferative disorders with large numbers of myeloid, erythroid, and megakaryocytic cells synthesizing DNA. Median cycling time (Tc) of myeloblasts was more rapid than that of patients with acute myeloid leukemia (44.1 hr vs. 56.0 hr). Interestingly, most marrow cells of all three lineages in 32 of 34 evaluable cases were undergoing PCD. In 19 of 32 patients, greater than 75% cells were apoptotic. Surprisingly, large numbers of S-phase cells were found to be simultaneously undergoing PCD, as were stromal cells of the BM microenvironment. We conclude that the extensive apoptosis in hematopoietic cells effectively cancels the high birth rate resulting in ineffective hematopoiesis and accounting for deficient bone marrow function. ©1995 Wiley-Liss, Inc.

In vivo administration of HSPI (10.7 kDa FSH suppressing peptide of prostate) to intact adult male rats was found to suppress the basal levels of incorporation of 3H-thymidine into DNA of ventral and dorsolateral lobes of the prostate, in a dose-dependent manner. Interestingly, microscopic examination of the prostate histology of HSPI treated rats revealed a significantly increased incidence of apoptotic bodies which are indicative of cell death. In another experiment, HSPI was also found to suppress the active DNA synthesis in testosterone-induced regrowth of prostate in castrated rats. Thus HSPI can suppress the basal and stimulated DNA synthesis and also induce apoptotic cell death in rat prostate.

Prostatic inhibin purified from human seminal plasma (10.7 kDa, 94 amino acids) is very well known for its endocrine action on pituitary to suppress synthesis and secretion of FSH. In the present report we have revealed its antiproliferative action on two fibroblast cell lines, NRK-49F (ED50 = 2.5 ng/ml) and Balb/c 3T3 (ED50 = 24.5 ng/ml) which may mark its emergence as a negative growth regulator.