MUC1 inhibition leads to decrease in PD-L1 levels via upregulation of miRNAs.

The PD-L1/PD-1 pathway is a critical component of the immunosuppressive tumor microenvironment in acute myeloid leukemia (AML), but little is known about its regulation. We investigated the role of the MUC1 oncoprotein in modulating PD-L1 expression in AML. Silencing of MUC1 in AML cell lines suppressed PD-L1 expression without a decrease in PD-L1 mRNA levels, suggesting a post-transcriptional mechanism of regulation. We identified the microRNAs miR-200c and miR-34a as key regulators of PD-L1 expression in AML. Silencing of MUC1 in AML cells led to a marked increase in miR-200c and miR-34a levels, without changes in precursor microRNA, suggesting that MUC1 might regulate microRNA-processing. MUC1 signaling decreased the expression of the microRNA-processing protein DICER, via the suppression of c-Jun activity. NanoString (Seattle, WA, USA) array of MUC1-silenced AML cells demonstrated an increase in the majority of probed microRNAs. In an immunocompetent murine AML model, targeting of MUC1 led to a significant increase in leukemia-specific T cells. In concert, targeting MUC1 signaling in human AML cells resulted in enhanced sensitivity to T-cell-mediated lysis. These findings suggest MUC1 is a critical regulator of PD-L1 expression via its effects on microRNA levels and represents a potential therapeutic target to enhance anti-tumor immunity.

Dynamic analysis of immune and cancer cell interactions at single cell level in microfluidic droplets.

Cell-cell communication mediates immune responses to physiological stimuli at local and systemic levels. Intercellular communication occurs via a direct contact between cells as well as by secretory contact-independent mechanisms. However, there are few existing methods that allow quantitative resolution of contact-dependent and independent cellular processes in a rapid, precisely controlled, and dynamic format. This study utilizes a high-throughput microfluidic droplet array platform to analyze cell-cell interaction and effector functions at single cell level. Controlled encapsulation of distinct heterotypic cell pairs was achieved in a single-step cell loading process. Dynamic analysis of dendritic cell (DC)-T cell interactions demonstrated marked heterogeneity in the type of contact and duration. Non-stimulated DCs and T cells interacted less frequently and more transiently while antigen and chemokine-loaded DCs and T cells depicted highly stable interactions in addition to transient and sequential contact. The effector function of CD8+ T cells was assessed via cytolysis of multiple myeloma cell line. Variable cell conjugation periods and killing time were detected irrespective of the activation of T cells, although activated T cells delivered significantly higher cytotoxicity. T cell alloreactivity against the target cells was partially mediated by secretion of interferon gamma, which was abrogated by the addition of a neutralizing antibody. These results suggest that the droplet array-based microfluidic platform is a powerful technique for dynamic phenotypic screening and potentially applicable for evaluation of novel cell-based immunotherapeutic agents.

Selective in vivo mobilization with granulocyte macrophage colony-stimulating factor (GM-CSF)/granulocyte-CSF as compared to G-CSF alone of dendritic cell progenitors from peripheral blood progenitor cells in patients with advanced breast cancer undergoing autologous transplantation.

Dendritic cells (DCs) are potent antigen-presenting cells that are essential for the initiation of T cell-mediated immunity. DCs develop from myeloid progenitor populations under the influence of granulocyte macrophage colony-stimulating factor (GM-CSF) and pass through an intermediate stage of maturation that is characterized by CD14 expression. Interest has focused on generating human-derived DCs for antigen-specific tumor vaccines to be used as adjuvant immunotherapy in minimal disease settings, such as after autologous transplantation. In the present study, mobilized peripheral blood progenitor cells (PBPCs) were obtained from 18 patients with locally advanced or metastatic breast cancer preparing to undergo autologous stem cell transplantation. PBPCs mobilized in 10 patients with GM-CSF for 1 week, followed by the combination of GM-CSF and G-CSF, were compared with those obtained from patients receiving G-CSF alone with respect to the presence of DC progenitors and the capacity to generate functionally active mature DCs. PBPCs mobilized with GM-CSF/G-CSF were markedly enriched for CD14+ DC progenitor cells as compared with those mobilized with G-CSF alone. Consistent with an immature progenitor population, the CD14+ cells express Ki-67 antigen but not nonspecific esterase. CD14+ cells purified by fluorescence-activated cell sorting from PBPCs mobilized with either regimen and cultured for 1 week in GM-CSF and interleukin-4 generated nearly pure populations of cells with characteristic DC phenotype and function. The addition of GM-CSF to the mobilization regimen resulted in greater yields of functionally active DCs for potential use in posttransplant immunotherapy.

Dendritic cells: development, function and potential use for cancer immunotherapy.

Dendritic cells (DC) are potent antigen presenting cells that are essential for the initiation of primary immune responses. They richly express MHC, costimulatory and adhesion molecules necessary for the stimulation of naive T cell populations. Dendritic cells are located at sites of antigen capture where they demonstrate phagocytic capacity and subsequently migrate to lymphatic areas for antigen presentation. Their phenotypic and functional characteristics are intimately linked to their stage of maturation. The hematopoietic development of dendritic cells is distinct and may follow several precursor pathways some closely linked to monocytes. Generation of large numbers of cells for potential clinical use has recently been accomplished through the in vitro culturing of progenitors with cytokines. The use of dendritic cell vaccines for cancer immunotherapy has emerged as an exciting new focus of investigation. Various strategies have been adopted to introduce tumor antigens into dendritic cells so that they may be more effectively presented to T cells in the context of costimulation. Animal models demonstrate that dendritic cell tumor vaccines reverse T-cell anergy and result in subsequent tumor rejection. Incorporating the expanding knowledge of dendritic cell biology into vaccine design is essential for the generation of effective immunotherapy for cancer patients.

The interval between courses of high-dose chemotherapy with stem cell rescue: therapeutic hypotheses.

The interval between courses of chemotherapy have classically been kept to a minimum in order to maximize dose intensity. Certain clinical observations suggest that longer intervals, particularly in the high-dose setting, may be more effective. This is based in part on the evidence that resistance is reversible over time and that the interval should be sufficient to allow for such a reversal. Clinical evidence for such reversibility include the following. In metastatic breast cancer, double or double high-dose stem cell rescue (HD-SCR) studies involving a minimal interval, have not, at least as yet, been shown to be superior to single HD-SCR. In Hodgkin's disease, response after relapse correlates directly with duration of time to relapse. In a comparative study of metastatic breast cancer, early HD-SCR preceded by daunorubicin induction was inferior to delayed HD-SCR. The latter was not preceded by induction chemotherapy. In a comparative study of childhood ALL, patients randomized to delayed (4 month interval) intensification had a significantly superior survival as compared to patients randomized to immediate (1 month interval) intensification. Taken together, these clinical observations indicate that resistance is reversible and that optimization of the interval must take this into account. Cytokinetic modeling of those clinical studies also found that delayed HD-SCR could result in a superior effect. Cytokinetic models of minimal residual tumor which were also examined included the Skipper exponential model, the Norton-Simon model which emphasizes the Gompertzian effect, the clonal evolution model, and the Retsky-Demisheli model which derives from a bimodal relapse pattern above. Biological and clinical data have resulted in a clinical protocol in the CALGB wherein patients with metastatic breast cancer are randomly allocated to (1) a single HD-SCR arm; (2) a double HD-SCR with a 5-week interval; and (3) a double HD-SCR arm with a 16 week interval.

Immune reconstitution following high-dose chemotherapy with stem cell rescue in patients with advanced breast cancer.

The present study examines the nature of humoral and cellular immune reconstitution in 28 patients with advanced breast cancer following high-dose chemotherapy with stem cell rescue. Patients underwent testing of T, B, NK and dendritic cell function at serial time points until 1 year post transplant or until the time of disease progression. Abnormalities in T cell phenotype and function were observed following high-dose chemotherapy that persisted for at least 6-12 months. The vast majority of patients experienced an inversion of the CD4/CD8 ratio and demonstrated an anergic response to candida antigen. Mean T cell proliferation in response to PHA and to co-culture with allogeneic monocytes was significantly compromised. In contrast, mean IgG and IgA levels were normal 6 months post transplant and NK cell yields and function were transiently elevated following high-dose chemotherapy. Dendritic cells generated from peripheral blood progenitors displayed a characteristic phenotype and were potent inducers of allogeneic T cell proliferation in the post-transplant period. The study demonstrates that patients undergoing autologous transplantation for breast cancer experience a prolonged period of T cell dysfunction. In contrast, B, NK, and DC recover more rapidly. These findings carry significant implications for the design of post-transplant immunotherapy.

A short course of induction chemotherapy followed by two cycles of high-dose chemotherapy with stem cell rescue for chemotherapy naive metastatic breast cancer: sequential phase I/II studies.

Two cycles of high-dose chemotherapy with stem cell support (HDC) may increase the total dose delivered and dose intensity. A brief induction phase and different non-cross-resistant agents for each HDC cycle were used to avoid drug resistance. Twenty-six women with metastatic BC had induction and stem cell mobilization with two cycles of doxorubicin/G-CSF given every 14 days. Patients with stable disease or better after induction received HD CTCb followed by HD melphalan and dose-escalated paclitaxel. At 475 mg/m(2) of paclitaxel by 24-h infusion, dose-limiting transient peripheral sensory neuropathy was encountered. No toxic deaths occurred. Complete and near complete response after completion of therapy was achieved in 22 (85%) of 26 patients. The median EFS was 38 months. The median OS has not yet been reached. At a median follow-up of 33 (25-43) months, actuarial EFS and OS were 54% (95% confidence interval (CI), 39-69%) and 69% (95% CI, 56-79%), respectively. This double transplant approach lasts only 14 weeks and is feasible, safe, and tolerable. Whilst selection biases may in part contribute to favorable EFS and OS, a randomized comparison of standard therapy vs double transplant in both metastatic and locally advanced breast cancer is warranted.

A short course of induction chemotherapy followed by two cycles of high-dose chemotherapy with stem cell rescue for chemotherapy naive metastatic breast cancer.

A single cycle of high-dose chemotherapy with stem cell support (HDC) in women with responsive metastatic breast cancer (BC) consistently achieves over 50% complete and near complete response (CR/nCR). This significant cytoreduction results in a median event-free survival (EFS) of 8 months, and approximately 20% 3-year and 16% 5-year EFS in selected patients. To improve long-term outcomes, new strategies to treat minimal residual tumor burden are needed. Increased total dose delivered can be achieved with two cycles of HDC. Critical design issues include shortening induction chemotherapy to avoid development of drug resistance and the use of different agents for each HDC cycle. We have determined the maximum tolerated dose (MTD) for paclitaxel combined with high-dose melphalan in the context of a double transplant and explored the impact of a short induction phase. Between June 1994 and August 1996, we enrolled 32 women with metastatic BC on to this phase I double transplant trial. Induction consisted of doxorubicin 30 mg/m2/day days 1-3 given for 2 cycles every 14 days with G-CSF 5 microg/kg s.c. days 4-12. Stem cell collection was performed by leukapheresis in each cycle when the WBC recovered to above 1000/microl. Patients with stable disease or better response to induction were eligible to proceed with HDC. HDC regimen I (TxM) included paclitaxel with dose escalation from 0 to 300 mg/m2 given on day 1 and melphalan 180 mg/m2 in two divided doses given on day 3. HDC regimen II was CTCb (cyclophosphamide 6 g/m2, thiotepa 500 mg/m2, and carboplatin 800 mg/m2 total doses) delivered by 96-h continuous infusion. At the first dose level of 150 mg/m2 paclitaxel by 3 h infusion, four of five patients developed dose-limiting toxicity consisting of diffuse skin erythema and capillary leak syndrome. Only two of these five completed the second transplant. Subsequently, paclitaxel was delivered by 24-h continuous infusion together with 96 h of dexamethasone and histamine receptor blockade. This particular toxicity was not observed again. No toxic deaths occurred and dose-limiting toxicity was not encountered. Three patients were removed from study prior to transplant: one for insurance refusal and two for disease progression. All others completed both cycles of transplant. Complete and near complete response (CR/nCR) after completion of therapy was achieved in 23 (72%) of 32 patients. The median EFS is 26 months. The median overall survival has not yet been reached. At a median follow-up of 58 months, EFS and overall survival are 41% and 53%, respectively. This double transplant approach is feasible, safe, and tolerable. Treatment duration is only 14 weeks and eliminates lengthy induction chemotherapy. The observed event-free and overall survivals are promising and are better than expected following a single transplant. Whilst selection biases may in part contribute to this effect, a much larger phase II double transplant trial is warranted in preparation for a potential randomized comparison of standard therapy vs single vs double transplant.

Low levels of 25-hydroxyvitamin D before allogeneic hematopoietic SCT correlate with the development of chronic GVHD.

Vitamin D, a hormone involved in bone and calcium homeostasis, has been shown to have potent immunomodulatory effects. We performed a retrospective cohort analysis to evaluate whether monohydroxyvitamin D levels before allogeneic hematopoietic SCT (HSCT) correlate with the risk of GVHD. Fifty-three patients who underwent myeloablative HSCT were studied. Vitamin D levels were measured in serum samples obtained before HSCT. The median 25-hydroxyvitamin vitamin D level was 21.9 ng/mL (7.8-45.7). The cumulative incidence (CI) of grades II-IV acute GVHD at 100 days was 53.1% in patients with vitamin D<25, versus 33.3% in patients with vitamin D ≥ 25 ng/mL (P=0.13). The CI of chronic GVHD (cGVHD) at 2 years in patients with vitamin D<25 was 63.8%, compared with 23.8% in patients with vitamin D ≥ 25 ng/mL (P=0.009). Similarly, the 2 year CI of extensive cGVHD was significantly greater in patients with vitamin D<25 compared with those with vitamin D ≥ 25 ng/mL (54.5% versus 14.3%, P=0.005). In a multivariable competing risk model, low pre-transplant vitamin D levels remained a significant factor associated with cGVHD (hazard ratio=5.26, P=0.02). Our results demonstrate that vitamin D deficiency before HSCT is associated with an increased risk of cGVHD.

Immunomodulatory effects of vitamin D: implications for GVHD.

GVHD remains a major source of morbidity and mortality after allogeneic BMT. GVHD is mediated by alloreactive T cells derived from the hematopoietic graft that target host tissues. Pre-clinical models have shown that presentation of alloantigens by host DCs results in the activation of donor-derived T cells that mediate GVHD. Strategies that interfere with the Ag-presenting capacity of DCs after allogeneic transplantation may decrease the risk of developing GVHD. Vitamin D is a hormone essential for calcium metabolism that shows immunomodulatory properties. We showed that correction of vitamin D deficiency appeared to mitigate manifestations of GVHD. In pre-clinical studies, we have shown that vitamin D inhibits DC maturation, polarizes T-cell populations toward the expression of Th2 as compared with Th1 cytokines, and blunts allogeneic T-cell proliferation in response to DC stimulation. Exposure to vitamin D resulted in increased expression of IDO, an enzyme responsible for tryptophan metabolism that is upregulated in tolerizing DCs. These data suggest that exposure to vitamin D results in immature DC populations that bias toward tolerizing rather than stimulatory T-cell populations. Vitamin D may therefore have a role in the prevention of GVHD.

Functionally distinct monoclonal antibodies reactive with enzymatically active and binding domains of Pseudomonas aeruginosa toxin A.

Monoclonal antibodies (MAbs) are described which react with two discrete structural domains of Pseudomonas aeruginosa toxin A and which have two distinct functional profiles. The MAbs designated T3-1C7 and T4-1F2 reacted with a 46,000-dalton peptide similar to the putative B or binding fragment of toxin A. These antibodies neutralized the cytotoxic and lethal properties of toxin but had no effect on its ADP-ribosyl transferase activity. T4-1F2 interfered with the binding of toxin A to membrane receptors on mouse fibroblasts (L cells), although the epitope for the antibody appears to be distinct from the actual receptor binding site. The MAb designated T2-1H2 reacted with intact toxin A and with a cloned, enzymatically active carboxy-terminal polypeptide similar to the toxin A fragment. This MAb neutralized the ADP-ribosyl transferase activity of activated holotoxin and of the cloned peptide, but inhibited neither binding of toxin to membrane receptors nor its cytotoxic and lethal actions. The complementary specificity and function of these MAbs confirm the functional specialization of discrete structural domains within the toxin A molecule. Our findings suggest the greater antitoxic potential of antibodies that block binding, compared with those which inhibit the enzymatic activity of toxin A.

Vaccination against infectious disease following hematopoietic stem cell transplantation.

Patients undergoing hematopoietic stem cell transplantation (HSCT) experience a prolonged period of dysfunctional immunity associated with an increased risk of bacterial and viral infections. Effective approaches toward vaccinating patients against common pathogens are being explored but are limited by poor levels of responsiveness. Relevant studies examining the nature of reconstitution of cellular and humoral immunity and its impact on vaccination strategies against infectious pathogens are reviewed. Following transplantation, deficiencies in cellular immunity are characterized by the inversion of CD4/CD8 ratios, a decreased proliferative response to mitogens, and the development of anergy to recall antigens as measured by delayed-type hypersensitivity testing. The impact on humoral immunity consists of decreased levels of circulating immunoglobulin, impaired immunoglobulin class switching, and a loss of complexity in immunoglobulin gene rearrangement patterns. In this setting, a loss of protective immunity has been demonstrated against viral and bacterial pathogens previously targeted by childhood vaccination. Infections due to encapsulated bacterial organisms such as Streptococcus pneumoniae and Haemophilus influenzae type B remain prevalent even in the late posttransplantation period. The efficacy of vaccination following HSCT is influenced by the time elapsed since transplantation, the nature of the hematopoietic graft, the use of serial immunization, and the presence of graft-versus-host disease. Strategies to enhance vaccine efficacy include pretransplantation immunization of the stem cell donor and the use of cytokine adjuvants.

Activation of antitumor cytotoxic T lymphocytes by fusions of human dendritic cells and breast carcinoma cells.

We have reported that fusions of murine dendritic cells (DCs) and murine carcinoma cells reverse unresponsiveness to tumor-associated antigens and induce the rejection of established metastases. In the present study, fusions were generated with primary human breast carcinoma cells and autologous DCs. Fusion cells coexpressed tumor-associated antigens and DC-derived costimulatory molecules. The fusion cells also retained the functional potency of DCs and stimulated autologous T cell proliferation. Significantly, the results show that autologous T cells are primed by the fusion cells to induce MHC class I-dependent lysis of autologous breast tumor cells. These findings demonstrate that fusions of human breast cancer cells and DCs activate T cell responses against autologous tumors.

Neutropenic enterocolitis as a complication of high dose chemotherapy with stem cell rescue in patients with solid tumors: a case series with a review of the literature.

BACKGROUND: This case series with an accompanying review of the literature describes neutropenic enterocolitis as a complication of high dose chemotherapy with stem cell rescue in patients with solid tumors. METHODS: Neutropenic enterocolitis is documented in two patients undergoing autologous stem cell transplantation. A review of MEDLINE from 1970 to the present was performed to delineate the prior related disease settings in which this condition has been described. The nature of the clinical syndrome and controversies regarding management are discussed. RESULTS: The authors report two patients who each received autologous stem cell transplantation for the treatment of a solid tumor. Both patients presented with progressive abdominal pain, signs of peritoneal irritation on examination, and pneumatosis intestinalis. In the first case, the syndrome developed rapidly over several hours, requiring emergent surgical intervention during the period of postchemotherapy nadir. In the second case, the patient developed a slowly progressive clinical picture that was managed successfully with medical therapy alone. A review of the literature suggests that neutropenic enterocolitis arises due to drug-induced bowel wall mucosal injury followed by superinfection with colonic and opportunistic organisms. Surgical intervention potentially is beneficial in patients who develop an acute syndrome that is unresponsive to antibiotic and supportive therapy. Those patients who can be stabilized with medical management often will recover once reengraftment occurs. CONCLUSIONS: Neutropenic enterocolitis is described in two patients undergoing autologous stem cell transplantation for solid tumors. The management of this life-threatening complication is controversial and governed by the nature of the clinical presentation.

Dendritic cells and macrophages can mature independently from a human bone marrow-derived, post-colony-forming unit intermediate.

CD34+ precursors in normal human bone marrow (BM) generate large numbers of dendritic cells alongside macrophages and granulocytic precursors when cultured for 12 to 14 days in c-kit ligand, granulocyte-macrophage colony-stimulating factor (GM-CSF), and tumor necrosis factor-alpha (TNF-alpha). This study reports an intermediate cell type that develops by day 6, and has the potential to differentiate into either macrophages or dendritic cells. When the d6 progeny are depleted of mature macrophages and residual CD34+ precursors, a discrete CD14+ HLA-DR+ population persists in addition to immunostimulatory CD14- HLA-DR() dendritic cells. Half of the CD14+ HLA-DR+ population is in cell cycle (Ki-67+), but colony-forming units (CFUs) are no longer detectable. The calls are c-fms+, but lack myeloperoxidase and nonspecific esterase. They also possess substantial phagocytic and allostimulatory activity. These post-CFU, CD14+ HLA-DR+ intermediates develop into typical macrophages when recultured in the absence of exogenous cytokines. M-CSF supports up to approximately 2.5-fold expansion of macrophage progeny. In contrast, the combination of GM-CSF and TNF-alpha supports quantitative differentiation into dendritic cells, lacking c-fms, CD14, and other macrophage properties, and expressing HLA-DR, CD1a, CD83, CD80, CD86, and potent allostimulatory activity. Therefore, normal CD34+ BM precursors can generate a post-CFU bipotential intermediate in the presence of c-kit ligand, GM-CSF, and TNF-alpha. This intermediate cell type will develop along the dendritic cell pathway when macrophages are removed and GM-CSF and TNF-alpha are provided. Alternatively, it can differentiate along a macrophage pathway when recultured with or without M-CSF.

Fusions of human ovarian carcinoma cells with autologous or allogeneic dendritic cells induce antitumor immunity.

Human ovarian carcinomas express the CA-125, HER2/neu, and MUC1 tumor-associated Ags as potential targets for the induction of active specific immunotherapy. In the present studies, human ovarian cancer cells were fused to human dendritic cells (DC) as an alternative strategy to induce immunity against known and unidentified tumor Ags. Fusions of ovarian cancer cells to autologous DC resulted in the formation of heterokaryons that express the CA-125 Ag and DC-derived costimulatory and adhesion molecules. Similar findings were obtained with ovarian cancer cells fused to allogeneic DC. The fusion cells were functional in stimulating the proliferation of autologous T cells. The results also demonstrate that fusions of ovarian cancer cells to autologous or allogeneic DC induce cytolytic T cell activity and lysis of autologous tumor cells by a MHC class I-restricted mechanism. These findings demonstrate that fusions of ovarian carcinoma cells and DC activate T cell responses against autologous tumor and that the fusions are functional when generated with either autologous or allogeneic DC.

A major fraction of human bone marrow lymphocytes are Th2-like CD1d-reactive T cells that can suppress mixed lymphocyte responses.

Murine bone marrow (BM) NK T cells can suppress graft-vs-host disease, transplant rejection, and MLRs. Human BM contains T cells with similar potential. Human BM was enriched for NK T cells, approximately 50% of which recognized the nonpolymorphic CD1d molecule. In contrast to the well-characterized blood-derived CD1d-reactive invariant NK T cells, the majority of human BM CD1d-reactive T cells used diverse TCR. Healthy donor invariant NK T cells rapidly produce large amounts of IL-4 and IFN-gamma and can influence Th1/Th2 decision-making. Healthy donor BM CD1d-reactive T cells were Th2-biased and suppressed MLR and, unlike the former, responded preferentially to CD1d(+) lymphoid cells. These results identify a novel population of human T cells which may contribute to B cell development and/or maintain Th2 bias against autoimmune T cell responses against new B cell Ag receptors. Distinct CD1d-reactive T cell populations have the potential to suppress graft-vs-host disease and stimulate antitumor responses.

Rituximab and ifosfamide, mitoxantrone, etoposide (RIME) with Neupogen support for B-cell non-Hodgkin's lymphoma prior to high-dose chemotherapy with autologous haematopoietic transplant.

A phase I/II study was performed to analyse the ability of ifosfamide-based chemotherapy with rituximab to produce a turmour-free graft as well as the safety of retuximab prior to stem cell harvest and post high-dose chemotherapy. Twenty-two patients with B-cell non-Hodgkin's lymphoma were enrolled either having aggressive large-cell disease in relapse or at high/high-intermediate risk of relapse, or refractory lymphoma or mantle cell lymphoma, or indolent lymphoma. Chemotherapy consisted of ifosfamide 2 g/m2, days 1-3 with mesna, etoposide 100 mg/m2, days 1-3, and mitoxantrone 8 mg/m2 day 1, with figrastim. Rituximab was given at 375 mg/m2 for 4 doses. An encouraging overall response rate of 90%, including 11 CRs was achieved. CD34+ cells were successfully mobilized in 18 or 19 patients analysed so far with a median number of 3.4 x 10(6) cells/kg. The combination of ifosfamide-based chemotherapy with rituximab significantly reduced the number of contaminating B-cells in the stem cell product and so far there has only been a single relapse post high-dose chemotherapy with autologous haematopoietic transplant. The RIME regimen was generally well tolerated with minimal non-haematological toxicity and most of the treatment was done completely on an outpatient basis. Haematological toxicity was manageable with filgrastim, there were some infectious complications.

The impact of induction duration and the number of high-dose cycles on the long-term survival of women with metastatic breast cancer treated with high-dose chemotherapy with stem cell rescue: an analysis of sequential phase I/II trials from the Dana-Farber/Beth Israel STAMP program.

Although high-dose chemotherapy (HDC) with stem cell rescue for the treatment of women with metastatic breast cancer (MBC) is currently a controversial strategy, we report the long-term outcomes of women undergoing high-dose therapy for MBC over the past 12 years while participating in a sequence of research studies transitioning between a single to a double intensification approach. Univariate and multivariate analyses provide a framework to understand the prognostic factors important for event-free and overall survival. Between May 1988 and April 1998, we enrolled 188 women with MBC into 3 trials of previously reported sequential transplantation strategies. Trial I (long induction/single transplantation) accepted 62 women in partial or complete response to an unspecified induction therapy and treated them with high-dose CTCb (cyclophosphamide, thiotepa, and carboplatin) supported by marrow or peripheral blood progenitor cells (PBPC). Trial II (long induction/double transplantation) accepted 68 women in partial or complete response to an unspecified induction therapy, and mobilized stem cells with 2 cycles of AF (doxorubicin and 5-fluorouracil) with granulocyte colony-stimulating factor (G-CSF). These women then received 1 cycle of high-dose single-agent melphalan followed 3 to 5 weeks later by CTCb, each with marrow or PBPC support. Trial III (short induction/double transplantation) enrolled 58 women prior to chemotherapy treatment for metastatic disease. Induction/mobilization consisted of 2 cycles given 14 days apart of doxorubicin and G-CSF. In contrast to trials I and II, patients with stable disease or better response to induction were eligible to proceed ahead with 2 cycles of HDC, 1 being CTCb and the other being dose escalated paclitaxel together with high-dose melphalan (TxM). These 2 HDC regimens were administered 5 weeks apart. TxM was given first in 32 patients and CTCb was given first in 26 patients. The median follow-up periods for trials I, II, and III were 98, 62, and 39 months from the initiation of induction chemotherapy and 92, 55, and 36 months from last high-dose therapy, respectively. The patient characteristics upon entry into these trials were similar. Important differences were that only those patients achieving a partial response or better to induction therapy were enrolled and analyzed for trials I and II, but all patients were analyzed on an intent-to-treat basis for trial III, including those who did not receive intensification. The median event-free survival (EFS) times from induction chemotherapy were 13, 19, and 27 months for trials I, II, and III, respectively (III versus I + II, P = .0004; III versus I, P = .0005; III versus II, P = .005; II versus I, P = .25). The median overall survival (OS) times from induction chemotherapy were 30, 29, and 57 months for trials I, II, and III, respectively (III versus I + II, P = .002; III versus I, P = .003; III versus II, P = .009; II versus I, P = .47). By multivariate Cox regression, participation in the short induction/double transplantation trial III and having no prior adjuvant chemotherapy remained favorable prognostic factors for both EFS and OS. The presence of visceral disease shortened EFS, and hormone sensitivity was of borderline significance. No substantive differences in the characteristics of the patient populations between the 3 trials appeared to interact with outcomes. In conclusion, we found that single transplantation in responding patients after long induction achieves a small cohort of long-term survivors, similar to the results reported by other transplantation centers. Adding a cycle of single-agent high-dose melphalan in this context delayed median time to relapse but did not affect long-term EFS or OS. The double transplantation approach using CTCb and TxM early in the course of treatment was associated with the best EFS and overall survival and was safe, feasible, and tolerable. Treatment duration was only 14 weeks, and this treatment option eliminated lengthy induction chemotherapy. Although selection biases may have in part contributed to this effect, a randomized comparison of standard therapy versus short induction/double transplantation is warranted.