Search

Background: Outcomes for patients with high-risk diffuse large B-cell lymphoma (DLBCL) treated with R-CHOP chemotherapy are suboptimal but, to date, no alternative regimen has been shown to improve survival rates. This phase 2 trial aimed to assess the efficacy of a Burkitt-like approach for high-risk DLBCL using the dose-intense R-CODOX-M/R-IVAC regimen., Patients and Methods: Eligible patients were aged 18-65 years with stage II-IV untreated DLBCL and an International Prognostic Index (IPI) score of 3-5. Patients received alternating cycles of CODOX-M (cyclophosphamide, vincristine, doxorubicin and high-dose methotrexate) alternating with IVAC chemotherapy (ifosfamide, etoposide and high-dose cytarabine) plus eight doses of rituximab. Response was assessed by computed tomography after completing all four cycles of chemotherapy. The primary end point was 2-year progression-free survival (PFS)., Results: A total of 111 eligible patients were registered; median age was 50 years, IPI score was 3 (60.4%) or 4/5 (39.6%), 54% had a performance status ≥2 and 9% had central nervous system involvement. A total of 85 patients (76.6%) completed all four cycles of chemotherapy. There were five treatment-related deaths (4.3%), all in patients with performance status of 3 and aged >50 years. Two-year PFS for the whole cohort was 67.9% [90% confidence interval (CI) 59.9-74.6] and 2-year overall survival was 76.0% (90% CI 68.5-82.0). The ability to tolerate and complete treatment was lower in patients with performance status ≥2 who were aged >50 years, where 2-year PFS was 43.5% (90% CI 27.9-58.0)., Conclusions: This trial demonstrates that R-CODOX-M/R-IVAC is a feasible and effective regimen for the treatment of younger and/or fit patients with high-risk DLBCL. These encouraging survival rates demonstrate that this regimen warrants further investigation against standard of care., Trial Registration: ClinicalTrials.gov (NCT00974792) and EudraCT (2005-003479-19)., Competing Interests: Disclosure AKM has received funding for travel and medical advisory board participation from F. Hoffman-La Roche and Amgen, and speakers' fees from F. Hoffman-La Roche. EHP has received research funding from F. Hoffman-La Roche. KMA has received funding for travel expenses and medical advisory board participation from F. Hoffman-La Roche. RPa has received funding for medical advisory board participation from F. Hoffman-La Roche. All other authors declare no relevant conflicts of interest., (Copyright © 2020 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: Individuals with cancer, particularly those who are receiving systemic anticancer treatments, have been postulated to be at increased risk of mortality from COVID-19. This conjecture has considerable effect on the treatment of patients with cancer and data from large, multicentre studies to support this assumption are scarce because of the contingencies of the pandemic. We aimed to describe the clinical and demographic characteristics and COVID-19 outcomes in patients with cancer., Methods: In this prospective observational study, all patients with active cancer and presenting to our network of cancer centres were eligible for enrolment into the UK Coronavirus Cancer Monitoring Project (UKCCMP). The UKCCMP is the first COVID-19 clinical registry that enables near real-time reports to frontline doctors about the effects of COVID-19 on patients with cancer. Eligible patients tested positive for severe acute respiratory syndrome coronavirus 2 on RT-PCR assay from a nose or throat swab. We excluded patients with a radiological or clinical diagnosis of COVID-19, without a positive RT-PCR test. The primary endpoint was all-cause mortality, or discharge from hospital, as assessed by the reporting sites during the patient hospital admission., Findings: From March 18, to April 26, 2020, we analysed 800 patients with a diagnosis of cancer and symptomatic COVID-19. 412 (52%) patients had a mild COVID-19 disease course. 226 (28%) patients died and risk of death was significantly associated with advancing patient age (odds ratio 9·42 [95% CI 6·56-10·02]; p<0·0001), being male (1·67 [1·19-2·34]; p=0·003), and the presence of other comorbidities such as hypertension (1·95 [1·36-2·80]; p<0·001) and cardiovascular disease (2·32 [1·47-3·64]). 281 (35%) patients had received cytotoxic chemotherapy within 4 weeks before testing positive for COVID-19. After adjusting for age, gender, and comorbidities, chemotherapy in the past 4 weeks had no significant effect on mortality from COVID-19 disease, when compared with patients with cancer who had not received recent chemotherapy (1·18 [0·81-1·72]; p=0·380). We found no significant effect on mortality for patients with immunotherapy, hormonal therapy, targeted therapy, radiotherapy use within the past 4 weeks., Interpretation: Mortality from COVID-19 in cancer patients appears to be principally driven by age, gender, and comorbidities. We are not able to identify evidence that cancer patients on cytotoxic chemotherapy or other anticancer treatment are at an increased risk of mortality from COVID-19 disease compared with those not on active treatment., Funding: University of Birmingham, University of Oxford., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Various studies have documented variation in the management of older patients with breast cancer, and some of this variation stems from different approaches to balancing the expected benefit of different treatments, with the ability of patients to tolerate them. Frailty is an emerging concept that can help to make clinical decisions for older patients more consistent, not least by providing a measure of 'biological' ageing. This would reduce reliance on 'chronological' age, which is not a reliable guide for decisions on the appropriate breast cancer care for older patients. This article examines the potential of frailty assessment to inform on breast cancer treatments. Overall, the current evidence highlights various benefits from implementing comprehensive geriatric assessment and screening for frailty in breast cancer patients. This includes a role in supporting the selection of appropriate therapies and improving physical fitness prior to treatment. However, there are challenges in implementing routine frailty assessments in a breast cancer service. Studies have used a diverse array of frailty assessment instruments, which hampers the generalisability of research findings. Consequently, a number of issues need to be addressed to clearly establish the optimal timing of frailty assessment and the role of geriatric medicine specialists in the breast cancer care pathway., (Copyright © 2019. Published by Elsevier Ltd.)

Background: There is little clinical evidence to guide treatment decisions for ductal carcinoma in situ (DCIS) in older women. This study evaluated how the management of DCIS in women aged 70 or more compared with women aged 50-69 in England and Wales., Method: The study identified women aged ≥50 years with new unilateral DCIS diagnosed between 2014 and 2016 from linked cancer registration and routine hospital datasets for England and Wales. Rates of surgery and adjuvant radiotherapy were examined by age, deprivation, fitness measures (comorbidity and frailty), method of presentation and tumour grade using multilevel logistic regression., Results: 12,716 women were diagnosed with unilateral DCIS between 2014 and 2016, of whom 2,754 (22%) were aged ≥70 years and 74% were screen detected. High grade DCIS was common, irrespective of age and method of presentation. Fewer women aged ≥70 had surgery compared to women aged 50-69 (81% vs. 94%), which was only partly explained by poor fitness. Use of radiotherapy following breast conserving surgery was strongly associated with grade, and was received by less than 16% of all patients with low grade tumours. Over 70% of women aged 50-69 with high grade DCIS received radiotherapy, but this fell to 35% among women aged ≥80. Use of radiotherapy was not associated with patient fitness., Conclusion: Treatment decisions for women with DCIS varied by age at diagnosis. Lower rates of surgery and adjuvant radiotherapy in older women were only partly explained by patient fitness. Better evidence is needed to aid treatment selection for older women with DCIS., (Crown Copyright © 2019. Published by Elsevier Ltd. All rights reserved.)

The majority of malignant cells in chronic lymphocytic leukaemia (CLL) circulate in the peripheral blood whereas small lymphocytic lymphoma (SLL) cells reside in tissues. The aim of this study was to detect differences in chemokine receptor expression, DNA single nucleotide polymorphism (SNP) microarray analysis and proteomic profiling to help elucidate why the cells remain in their respective environments. We identified by flow cytometric studies of chemokine receptors and DNA SNP microarray analysis significant differences between cells from CLL and SLL patients. Proteomic analysis revealed two potential markers (m/z 3091 and 8707) to distinguish the two disorders. There was a significantly greater expression of leucocyte trafficking receptor CXCR3 (CD183) and migration and homing receptor CXCR4 (CD184), and significantly lower expression of cell adhesion molecule integrin α 4 chain (CD49d), on CLL cells, compared with SLL cells. Conversely, SNP microarrays revealed greater numbers of copy-neutral loss of heterozygosity chromosomal aberrations, as well as gross chromosomal aberrations, in the SLL group, compared with the CLL group. These findings revealed that there was a significantly greater expression of trafficking, migration and homing receptors and significantly lower expression of adhesion molecules on CLL cells than on SLL cells, and that SLL may be a more progressive disease than CLL, with a more complex genotype., (Copyright © 2017. Published by Elsevier Inc.)

Peripheral T-cell lymphoma (PTCL) represents a relatively rare group of heterogeneous non-Hodgkin lymphomas with a very poor prognosis. Current therapies, based on historical regimens for aggressive B-cell lymphomas, have resulted in insufficient patient outcomes. The majority of patients relapse rapidly, and current 5-year overall survival rates are only 10-30%. It is evident that new approaches to treat patients with PTCL are required. In recent years, prospective studies in PTCL have been initiated, mainly in patients with relapsed/refractory disease. In some of these, selected histologic subtypes have been evaluated in detail. As a consequence, numerous new therapies have been developed and shown activity in PTCL, including: agents targeting the immune system (e.g. brentuximab vedotin, alemtuzumab, lenalidomide); histone deacetylase inhibitors (romidepsin, belinostat); antifolates (pralatrexate); fusion proteins (denileukin diftitox); nucleoside analogs (pentostatin, gemcitabine); and other agents (e.g. alisertib, plitidepsin, bendamustine, bortezomib). A variety of interesting novel combinations is also emerging. It is hoped that these innovative approaches, coupled with a greater understanding of the clinicopathologic features, pathogenesis, molecular biology, and natural history of PTCL will advance the field and improve outcomes in this challenging group of diseases. This review summarizes the currently available clinical evidence on the various approaches to treating relapsed/refractory PTCL, including the role of stem cell transplantation, with an emphasis on potential new drug therapies., (Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Background: The aim of this subgroup analysis of the Mabthera International Trial Group study was to evaluate the impact of chemotherapy and rituximab in primary mediastinal B-cell lymphoma (PMBCL) in comparison to other diffuse large B-cell lymphoma (DLBCL)., Methods: Patients were randomly assigned to six cycles of CHOP-like regimens with or without rituximab., Results: Of 824 patients enrolled, 87 had PMBCL and 627 other types of DLBCL. Rituximab increased the rates of complete remission (unconfirmed) in both PMBCL (from 54% to 80%, P = 0.015) and DLBCL (from 72% to 87%, P < 0.001). In PMBCL, rituximab virtually eliminated progressive disease (PD) (2.5% versus 24%, P < 0.001), whereas without rituximab, PD was more frequent in PMBCL than in DLBCL (24% versus 10%, P = 0.010). With a median observation time of 34 months, 3-year event-free survival (EFS) was improved by rituximab for PMBCL (78% versus 52%, P = 0.012) and for DLBCL (81% versus 61%, P < 0.001). Overall survival benefit was similar for DLBCL (93% versus 85%, P < 0.001) and PMBCL (89% versus 78%, P = 0.158)., Conclusion: In young patients with PMBCL (age-adjusted International Prognostic Index 0-1), rituximab added to six cycles of CHOP-like chemotherapy increases response rate and EFS to the same extent as other DLBCL. The combination of rituximab with CHOP chemotherapy is an effective treatment in PMBCL with good prognosis features.

In vivo alemtuzumab reduces the risk of graft-versus-host disease (GVHD) and nonrelapse mortality after reduced intensity allogeneic transplantation. However, it also delays immune reconstitution, leading to frequent infections and potential loss of graft-versus-tumor responses. Here, we tested the feasibility of alemtuzumab dose deescalation in the context of fludarabine-melphalan conditioning and human leukocyte antigen (HLA)-identical sibling transplantation. Alemtuzumab was given 1-2 days before graft infusion, and dose reduced from 60 mg to 20 mg in 4 sequential cohorts (total n = 106). Pharmacokinetic studies were fitted to a linear, 2-compartment model in which dose reduction led to incomplete saturation of CD52 binding sites and greater antibody clearance. Increased elimination was particularly evident in the 20-mg group in patients who had CD52-expressing tumors at time of transplantation. The 20-mg dose was also associated with greater risk of severe GVHD (acute grade III-IV or chronic extensive) compared with > 20 mg (hazard ratio, 6.7; 95% CI, 2.5-18.3). In contrast, dose reduction to 30 mg on day -1 was associated with equivalent clinical outcomes to higher doses but better lymphocyte recovery at 12 months. In conclusion, alemtuzumab dose reduction to 30 mg is safe in the context of reduced intensity conditioning and HLA-identical sibling transplantation. This trial was registered at http://www.ncrn.org.uk as UKCRN study 1415.

Background: The purpose of this study was to determine whether there was a relationship between disease activity and health functioning, as measured by a range of patient-reported outcome (PRO) measures in patients with follicular lymphoma (FL)., Patients and Methods: A total of 222 patients with FL were recruited from eight sites across the UK and they completed a number of PRO measures. The participants were analyzed across five disease states: 'active disease-newly diagnosed', 'active disease-relapsed', 'partial response', 'complete response' and 'disease free'. The relationship between these disease states and their level of health functioning was assessed as well as the relationship between being 'on' or 'off' chemotherapy and disease state., Results: In terms of health-related quality of life (HRQoL), participants in the relapsed category had the lowest mean physical well-being, emotional well-being, functional well-being and social well-being score. In a regression analysis, the 'active disease-relapsed' group acted as a significant predictor for each PRO variable. In addition, the remission group acted as a significant predictor of high anxiety scores as measured by the Hospital Anxiety and Depression Scale., Conclusion: The results of this study demonstrate that various aspects of patient-reported health outcomes differ according to disease state in patients with FL. For those patients who have relapsed, they are more likely to experience worse HRQoL and other patient-reported health outcomes than patients newly diagnosed, in partial or complete remission or when completely disease free.

Hematopoietic stem cell/hematopoietic progenitor cell (HSC/HPC) homing to specific microenvironmental niches involves interactions between multiple receptor ligand pairs. Although CXCL12/CXCR4 plays a central role in these events, CXCR4 regulators that provide the specificity for such cells to lodge and be retained in particular niches are poorly defined. Here, we provide evidence that the sialomucin endolyn (CD164), an adhesion receptor that regulates the adhesion of CD34+ cells to bone marrow stroma and the recruitment of CD34+CD38(lo/-) cells into cycle, associates with CXCR4. The class II 103B2 monoclonal antibody, which binds the CD164 N-linked glycan-dependent epitope or CD164 knockdown by RNA interference, significantly inhibits the migration of CD133+ HPCs toward CXCL12 in vitro. On presentation of CXCL12 on fibronectin, CD164 associates with CXCR4, an interaction that temporally follows the association of CXCR4 with the integrins VLA-4 and VLA-5. This coincides with PKC-zeta and Akt signaling through the CXCR4 receptor, which was disrupted on the loss of CD164 though MAPK signaling was unaffected. We therefore demonstrate a novel association among 3 distinct families of cell-surface receptors that regulate cell migratory responses and identify a new role for CD164. We propose that this lends specificity to the homing and lodgment of these cells within the bone marrow niche.

Background: In patients with multiply relapsed Hodgkin's lymphoma allogeneic stem-cell transplantation has been limited by prohibitive non-relapse-related mortality rates and by a lack of definitive evidence for a therapeutic graft-versus-tumour effect. Therefore, we aimed to assess the graft-versus-tumour effect of reduced-intensity allogeneic transplantation., Methods: We undertook reduced-intensity transplantation in 49 patients with multiply relapsed Hodgkin's lymphoma, 44 (90%) of whom had progression of disease after previous autologous transplantation (median age 32 years [range 18-51], number of previous treatment courses was five [range 3-8], and time from diagnosis 4.8 years [range 0.6-4.8]). 31 patients had HLA matched donors who were related and 18 had donors who were unrelated. Median follow-up was 967 days (range 102-2232). The primary endpoints were engraftment, toxic effects, non-relapse-related mortality, incidence of graft-versus-host disease (GVHD), and the toxic effects of adjuvant donor-lymphocyte infusion., Findings: All patients engrafted. Eight of 49 (16%) had grade II-IV acute GVHD and seven (14%) had chronic GVHD before donor-lymphocyte infusion. 16 (33%) patients received donor-lymphocyte infusion from 3 months after transplantation for residual disease or progression. Six (38%) of the 16 developed grade II-IV acute GVHD and five developed chronic GVHD. Nine (56%) showed disease responses after infusion (eight complete, one partial). Non-relapse-related mortality was 16.3% at 730 days (7.2% for patients who had related donors vs 34.1% for those with unrelated donors, p=0.0206). Projected 4 year overall and progression-free survival were 55.7% and 39.0%, respectively (62.0% and 41.5% for related donors)., Interpretation: These data show the potential for durable responses in patients who have previously had substantial treatment for Hodgkin's lymphoma. The low non-relapse-related mortality suggests the procedure could be undertaken earlier in the course of the disease.

Background: We have investigated a novel nonmyeloablative conditioning regimen in 44 patients with hematological malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant., Methods: Recipient conditioning consisted of CAMPATH-1H 20 mg/day on Days -8 to -4, fludarabine 30 mg/m(2) on Days -7 to -3 and melphalan 140 mg/m(2) on Day -2. Thirty-six recipients received unmanipulated G-CSF mobilized PBSC from HLA identical siblings and eight received unmanipulated BM from MUD. GvHD prophylaxis was with CYA alone for 38 patients and CYA plus MTX for six sibling recipients., Results: Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite PCR indicate that 18 of 31 patients studied were full donor chimeras, while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range, 3-29 months) 33 patients remain alive in CR, or with no evidence of disease progression. Seven patients relapsed or progressed post-transplant and four of them subsequently died. Four patients died from regimen-related complications. There were no cases of Grades III-IV acute GvHD. Only two patients developed Grade II acute GvHD and only one had chronic GvHD. The estimated probability of non-relapse mortality at 1 year was 11%.Results: Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity and low incidence of GvHD.

A novel nonmyeloablative conditioning regimen was investigated in 44 patients with hematologic malignancies. The median patient age was 41 years. Many of the patients had high-risk features, including 19 patients with a previous failed transplant. Recipient conditioning consisted of CAMPATH-1H, 20 mg/day on days -8 to -4; fludarabine, 30 mg/m(2) on days -7 to -3; and melphalan, 140 mg/m(2) on day -2. Thirty-six recipients received unmanipulated granculocyte colony-stimulating factor-mobilized peripheral blood stem cells from HLA-identical siblings, and 8 received unmanipulated marrow from matched unrelated donors. GVHD prophylaxis was with cyclosporine A alone for 38 patients and cyclosporine A plus methotrexate for 6 sibling recipients. Forty-two of the 43 evaluable patients had sustained engraftment. Results of chimerism analysis using microsatellite polymerase chain reaction indicate that 18 of 31 patients studied were full-donor chimeras while the other patients were mixed chimeras in one or more lineages. At a median follow-up of 9 months (range 3 to 29 months), 33 patients remain alive in complete remission or with no evidence of disease progression. Seven patients relapsed or progressed post-transplantation, and 4 of them subsequently died. Four patients died of regimen-related complications. There were no cases of grades III-IV acute GVHD. Only 2 patients developed grade II acute GVHD, and only 1 had chronic GVHD. The estimated probability of nonrelapse mortality was 11%. Although longer follow-up is needed to establish the long-term remission rates, this study demonstrates that this nonmyeloablative preparative regimen is associated with durable engraftment, minimal toxicity, and low incidence of GVHD.

Objective: To compare the characteristics and growth patterns of human peritoneal mesothelial cells (HPMCs) from advanced epithelial ovarian cancer (EOC) patients with those from non-EOC patients., Methods: Peritoneal and omental biopsies were obtained from treatment-naïve patients. Formalin-fixed biopsies and cytologic touch preparations were studied immunochemically. HPMCs were isolated from tissue biopsies or malignant ascites and cultured with or without growth factors. Cell growth was determined by the MTT assay. Cultured HPMCs were further characterised by flow cytometry analysis (FACS)., Results: Peritoneal biopsies showed a continuous flat mesothelial cell layer in non-EOC patients, whereas in advanced EOC, the mesothelium was a discontinuous layer of rounded cells. In all peritoneal biopsies, the mesothelium expressed cytokeratin 8/18, vimentin, and the mesothelioma cell antigen but not E-cadherin. In touch preparations, expression of the putative fibroblast antigen ranged from negative to weakly positive. HPMC from non-EOC cases grew slowly in vitro except when exposed to L-cysteine (L-cys 30 micrograms/mL) during the initial 24 hours of culture. Conversely, cells from EOC sources grew more rapidly, especially when exposed to both epidermal growth factor (EGF 10 ng/mL) and hydrocortisone (HC 400 ng/mL). HPMC coexpressed cytokeratin 8/18 and vimentin in vitro, but the expression of the putative fibroblast antigen increased during primary culture, whereas that of the mesothelioma cell antigen decreased in successive passages. Furthermore, in FACS, cultured HPMC did not express CD14, CD16, or CD34., Conclusion: In peritoneal biopsies from non-EOC and EOC patients, HPMCs showed different morphology but similar immunostaining characteristics, whereas cultured cells from different sources were similar in both morphology and phenotype. L-cysteine enhanced the growth of non-EOC but not of EOC-derived HPMCs, which had a maximal response to EGF and HC. The growth advantage of HPMCs from EOC in vitro suggests these cells are in a primed or activated state.

Low levels of telomerase activity have recently been detected in human primitive hematopoietic cells, however, blood cells exhibit telomere shortening on cell proliferation. This challenging observation led us to study telomerase regulation and telomere length in human hematopoietic progenitor cells from fetal liver (FL), cord blood (CB), peripheral blood (PB), and bone marrow (BM). We found telomerase activity in CD34+/CD38+ cells exceeding levels in CD34+/CD38-, CD34- , and mononuclear cells (P < .05). Baseline telomerase activity was highest in BM (n = 5) CD34+ cells, followed by PB (n = 20), CB (n = 11), and FL (n = 1). Within 48 hours to 72 hours of in vitro culture of CD34+ cells in the presence of cytokines (KL, interleukin-3 [IL-3], IL-6, erythropoietin, granulocyte colony-stimulating factor), telomerase activity was upregulated, peaked after 1 week of culture, and decreased to baseline levels or below detection after 3 to 4 weeks. Stimulation of CD34+ cells with single cytokines resulted in no or minor telomerase upregulation, whereas cytokine combinations resulted in a significant telomerase increase (P < .001). There was a correlation between telomerase activity, cell cycle status by BrdU incorporation, and induction of phosphorylated retinoblastoma protein, CDC2, CDK2, cyclin D1, and cyclin A, but not cyclin E and B1 after 72 hours with multiple (but not single) cytokines. In nonexpanding CD34+ cells, telomerase was low or undetectable. Secondary CD34+ cells showed a reduced ability to upregulate telomerase activity. Antiproliferative cytokines such as transforming growth factor-beta1 and high concentrations of all-trans-retinoic acid in cytokine-supported CD34+ cultures downmodulated telomerase activity. Average telomere lengths were 10.4 kbp, 7.4 kbp, and 7.6 kbp in CB, PB, and BM CD34+ cells, respectively. In ex vivo expansion cultures, an average telomeric DNA loss of 1 to 2 kbp over 4 weeks was observed. However, the rate of base pair loss per population doubling was significantly lower during the first 2 weeks, when telomerase was upregulated, than during weeks 3 and 4 of culture. In summary, telomerase is upregulated in response to cytokine-induced proliferation and cell cycle activation in primitive hematopoietic cells. Telomerase is downregulated between weeks 3 and 4 of ex vivo expansion culture linked with decreased proliferation and greater expansion of more mature cell subsets. Our data suggest that telomerase activity in hematopoietic cells reduces, but does not prevent, telomere shortening on proliferation.

Endothelial cells are a major component of the bone marrow (BM) microenvironment that regulate the trafficking and homing of hematopoietic progenitor and stem cells. In this paper, we provide evidence that BM endothelial cells (BMECs) also support multilineage hematopoiesis by elaboration of soluble cytokines. Hematopoietic progenitor cells incubated in direct contact with BMEC monolayers, or physically separated by microporous membrane, expanded five-fold to sevenfold at 7 days, in the absence of exogenous cytokines. Flow cytometric analysis of proliferating progenitor cells grown in the presence of BMEC monolayers showed that by day 14 of coculture, 70% to 80% of hematopoietic cells were myeloid, expressing CD15 or CD14, and 14% to 19% were megakaryocytic, expressing GPIIb/IIIa or GPIb. CD34+ cells derived from umbilical cord blood, cultured in the upper chamber of transwell culture plates, as well as the cells grown in direct contact with BMEC monolayers, generated progenitors for up to 70 days. Unstimulated BMEC monolayers constitutively produce interleukin-6, Kit-ligand, granulocyte colony-stimulating factor, and granulocyte macrophage colony-stimulating factor. These data suggest that BMEC regulate proliferation of hematopoietic progenitor cells and long-term culture initiating cells by elaboration of lineage-specific cytokines.

Five years ago the haemopoietic growth factors were introduced to clinical practice with the aim of reducing the depth and duration of chemotherapy induced neutropenia. Now, they have a wider remit, with important roles in supporting dose intensive treatments and mobilising BPC. Similarly, BPC themselves have until now been predominantly used in autologous transplantation following myeloablative treatments. In the next five years we can expect to see BPC from novel sources manipulated to feature in many new roles, including allogeneic transplantation, multicyclic dose-intensive chemotherapy and gene therapy.

Autologous blood progenitor cell (BPC) support is used in conjunction with high-dose chemotherapy regimens and also permits the use of increasing dose-intensity schedules of conventional cytotoxic chemotherapy. However, our understanding of the optimal BPC support to be used in these different conditions is incomplete. These difficulties result from the use of different BPC collection procedures in different centres, to different patient populations and considerable variability between patients. For such treatment to be cost effective, greater knowledge is required about the best way of achieving mobilisation, collection, storage and engraftment of BPC. The use of G-CSF in combination with chemotherapy can yield sufficient BPC at a single apheresis to achieve haematopoietic reconstitution after high-dose therapy. This was partly achieved by accurate prediction of the optimum timing of cell collection, which coincided with the early exponential increase in white blood cells (WBC) after the chemotherapy induced nadir. BPC in apheresis product or whole blood from patients primed with chemotherapy and filgrastim (G-CSF) were found to be viable for 48 hours when stored at 4 degrees C. Patients receiving multicyclic ifosfamide, carboplatin and etoposide (ICE) chemotherapy received BPC in apheresis product or whole blood, which had been stored at 4 degrees C, on day 3 of each treatment cycle. Such an approach allowed a 200% increase in dose intensity without any increase in toxicity or the need for supportive care. Optimal use of BPC support may increase the effectiveness of chemotherapy for the common solid tumours.

Limiting-dilution analysis of long-term culture-initiating cells (LTCIC) is a quantitative method of estimating hematopoietic stem cell activity in clinical samples. We compared the numbers of LTCIC in bone marrow (BM), umbilical cord blood, and blood progenitor cells (obtained from patients with solid tumors at leukapheresis after mobilization with induction chemotherapy and filgrastim administration), using a two-stage long-term culture system and a limiting-dilution technique, scoring cobblestone areas of greater than 15 hematopoietic cells weekly for up to 8 weeks. Samples were obtained from 30 normal BMs, 20 human umbilical cords, and 32 leukapheresis products. Direct comparison of LTCIC in the three sources showed that the median proportions of cells generating hematopoietic foci from unfractionated mononuclear cells at 5 and 8 weeks, respectively, were 1:13,314 and 1:33,949 for BM, 1:12,506 and 1:34,546 for umbilical cord blood, and 1:10,302 and 1:12,891 for leukapheresis product. The estimated proportions of LTCIC from unfractionated mononuclear cells and CD34+ cells were similar in experiments with leukapheresis product. Leukapheresis product was superior to umbilical cord blood and cord blood to BM at 5 and 8 weeks of culture (P = .01). In two-stage long-term cultures, more colonies per flask and CD34+ cells were found in assays of leukapheresis product than in BM or umbilical cord blood cultures (P = .0005). Results obtained by this simplified limiting-dilution analysis correlated well with standard long-term cultures and can be used as a measure of the stem cell population. These data suggest that the incidence of putative stem cells in leukapheresis product and umbilical cord blood are at least comparable with that of BM.

Here we review the evidence that cytotoxic dose intensity is an important determinant of outcome in high- and intermediate-grade non-Hodgkin's lymphoma. The results of retrospective analyses and prospective studies support this proposition but confirmatory studies are required. We discuss the role of haemopoietic growth factors and mobilized peripheral blood progenitor cells to increase dose intensity. Studies using these modalities will enable the importance of dose intensity to be tested.

We report a reliable approach for sequencing lymphoma-specific CDRIII regions. CDRIII regions present in DNA prepared from routinely fixed and paraffin-embedded diagnostic lymph node material were amplified by the use of consensus VH and JH primers via PCR. PCR products were subcloned directly, without purification or modification of PCR fragments. Only small amounts of miniprep plasmid DNA of recombinant clones were required for cycle sequencing, resulting in autoradiograms of high quality. The easy and reproducible method which we describe has enabled us to determine the lymphoma-specific CDRIII region in 7/11 high-grade non-Hodgkin's lymphomas as well as in 3/3 cases of ALL and 1/1 case of a centroblastic/centrocytic lymphoma. The obtained sequence data can serve to generate lymphoma-specific oligonucleotides, which then can be used as PCR primers or hybridization probes for the detection of minimal residual disease in individual patients.

Myeloablative treatment and peripheral blood progenitor cell (PBPC) transplantation are increasingly used for lymphomas and leukemias. We have sought to optimize conditions for priming, collection, and engraftment of the leukapheresis product. Fifty-four consecutive adult patients were eligible, 31 with high-grade non-Hodgkin's lymphoma of poor prognosis, 12 with Hodgkin's disease in chemosensitive relapse, and 11 with poor prognosis acute lymphoblastic leukemia. Filgrastim was administered after routine chemotherapy with VAPEC-B or HiCCOM to mobilize PBPC. A rapidly increasing white blood cell count was used to predict the time of peak PBPC release and plan leukapheresis. Forty-five patients underwent leukapheresis. A median of 14 L of blood was processed at a single apheresis. A median of 2.4 x 10(8)/kg mononuclear cells (MNCs), 1.04 x 10(6)/kg granulocyte-macrophage colony-forming cells (GM-CFCs), and 10.6 x 10(6)/kg CD34+ cells were obtained. Slightly fewer MNCs were obtained from the heavily pretreated Hodgkin's disease group. There were no other significant differences in the size or composition of the leukapheresis harvest in the three patient groups. Forty patients underwent high-dose therapy and PBPC transplantation. Filgrastim was administered by daily subcutaneous injection until the absolute neutrophil count was > or = 1 x 10(9)/L for 2 consecutive days. Rapid and sustained hematopoietic engraftment occurred in all patients. The median time to achieve a neutrophil count > or = 0.5 x 10(9)/L was 9 days (range, 8 to 16 days); to achieve a platelet count > or = 20 x 10(9)/L was 10 days (range, 6 to 88 days); and to achieve a platelet count > or = 50 x 10(9)/L was 15.5 days (range, 10 to 100 days). Neutrophil recovery was faster than that of a historical control group treated with autologous bone marrow transplantation and filgrastim, but platelet recovery times were halved in the PBPC group. There was no secondary engraftment failure. Requirements for blood and platelet transfusions, antibiotic use, and parenteral nutrition were similar in the three patient groups. The median number of days in the hospital was 13 (range, 10 to 55) in the PBPC patients, compared with 19 (range, 14 to 51) in the historical controls. Leukapheresis yields (MNC, GM-CFC, and CD34+ cell numbers) were not useful for predicting the times to engraftment. We have shown that sufficient PBPC for transplantation can be obtained at a single leukapheresis after mobilization with routine chemotherapy and filgrastim in patients with non-Hodgkin's lymphoma, Hodgkin's disease, and acute lymphoblastic leukemia, even those heavily pretreated.(ABSTRACT TRUNCATED AT 400 WORDS)

Primitive hematopoietic cells released into the peripheral blood (PB) were studied in 50 patients with high-grade non-Hodgkin's lymphoma enrolled in a phase III trial of intensive weekly chemotherapy (VAPEC-B) alone or with granulocyte colony-stimulating factor (G-CSF). Mononuclear cells numbers were monitored and their in vitro growth potential assessed in clonogenic progenitor cell assays and in long-term culture. Total colony-forming cells (granulocyte-macrophage [GM], burst-forming unit, erythroid [BFU-E], Mix-CFC) were increased 40-fold (median) over baseline with chemotherapy alone and 106-fold with chemotherapy and G-CSF after the final dose. CD34+ cells were increased to a median of 4%, equivalent to that in normal bone marrow (BM) controls. Circulating colony-forming cell levels were maximal when the recovering total white blood cell (WBC) count reached 5 to 10 x 10(9)/L. The timing of the maximum was reproducible in individual patients. Therefore the WBC count can be used as a guide to the timing of leukapheresis. PB cells from normal controls' and patients' prechemotherapy were unable to sustain hemopoiesis in two-stage long-term cultures. In contrast, PB cells collected from patients primed with chemotherapy alone or chemotherapy with G-CSF at the time of predicted maximal colony-forming cell release were able to generate and sustain hematopoiesis in long-term cultures at a level comparable or superior to normal BM. These findings indicate that the use of G-CSF after routine outpatient chemotherapy stimulates maximal release of primitive hemopoietic cells into the circulation, including colony-forming cells and long-term culture-initiating cells. Their numbers are comparable with those in normal BM and are such that a single leukapheresis will usually yield enough cells for hemopoietic reconstitution after myeloablative chemotherapy.

The effect of granulocyte colony-stimulating factor (G-CSF) on neutropenia, infection, and cytotoxic chemotherapy administration was studied in a randomized trial in patients receiving intensive weekly chemotherapy for non-Hodgkin's lymphoma (NHL). Eighty patients (aged 16 to 71 years) with high-grade NHL (Kiel) of any stage were randomized to receive VAPEC-B chemotherapy alone (39 patients) or with G-CSF administered as a daily subcutaneous dose of 230 micrograms/m2 (41 patients). Prophylactic ketoconazole and cotrimoxazole were administered to all patients throughout treatment. The protocol specified identical dose modification and antibiotic treatment criteria bor both groups. Neutropenia (absolute neutrophil count [ANC] less than 1.0 x 10(9)/L) occurred in 15 of 41 (37%) of the G-CSF-treated patients and in 33 of 39 (85%) of the controls, giving a relative risk for control patients of 2.31 (95% confidence interval [CI], [1.51, 3.54]; P = .00001). Fever (greater than or equal to 37.5 degrees C) with neutropenia (ANC less than 1.0 x 10(9)/L) occurred in 9 of 41 (22%) of the G-CSF group and in 17 of 39 (44%) of the controls (relative risk for control, 2.26; 95% CI [1.01, 5.06]; P = .04). There were fewer treatment delays, with shorter duration (P = .01) in patients receiving G-CSF. Chemotherapy doses were reduced in 4 of 41 (10%) of the G-CSF patients and 13 of 39 (33%) of the controls (P = .01). The dose intensity of cytotoxic chemotherapy was significantly increased in patients receiving G-CSF (median of 95% in G-CSF group compared with 83% in control patients). Three vascular deaths occurred in the G-CSF group. Delays in the control group most commonly resulted from neutropenia (19 patients, compared with 2 patients in the G-CSF-treated group, P = .000007). Severe mucositis was the major dose-limiting toxicity in G-CSF-treated patients, but did not occur more frequently than in controls (15 patients in each group). Overall, patients randomized to receive G-CSF achieved a greater dose intensity than control patients, but this did not result in significant differences in drug toxicity (other than neutropenia), intravenous antibiotic usage, or hospitalization between the two groups.