Observation versus late reintroduction of letrozole as adjuvant endocrine therapy for hormone receptor-positive breast cancer (ANZ0501 LATER): an open-label randomised, controlled trial.

BACKGROUND: Despite the effectiveness of adjuvant endocrine therapy in preventing breast cancer recurrence, breast cancer events continue at a high rate for at least 10 years after completion of therapy. PATIENTS AND METHODS: This randomised open label phase III trial recruited postmenopausal women from 29 Australian and New Zealand sites, with hormone receptor-positive early breast cancer, who had completed ≥4 years of endocrine therapy [aromatase inhibitor (AI), tamoxifen, ovarian suppression, or sequential combination] ≥1 year prior, to oral letrozole 2.5 mg daily for 5 years, or observation. Treatment allocation was by central computerised randomisation, stratified by institution, axillary node status and prior endocrine therapy. The primary outcome was invasive breast cancer events (new invasive primary, local, regional or distant recurrence, or contralateral breast cancer), analysed by intention to treat. The secondary outcomes were disease-free survival (DFS), overall survival, and safety. RESULTS: Between 16 May 2007 and 14 March 2012, 181 patients were randomised to letrozole and 179 to observation (median age 64.3 years). Endocrine therapy was completed at a median of 2.6 years before randomisation, and 47.5% had tumours of >2 cm and/or node positive. At 3.9 years median follow-up (interquartile range 3.1-4.8), 2 patients assigned letrozole (1.1%) and 17 patients assigned observation (9.5%) had experienced an invasive breast cancer event (difference 8.4%, 95% confidence interval 3.8% to 13.0%, log-rank test P = 0.0004). Twenty-four patients (13.4%) in the observation and 14 (7.7%) in the letrozole arm experienced a DFS event (log-rank P = 0.067). Adverse events linked to oestrogen depletion, but not serious adverse events, were more common with letrozole. CONCLUSION: These results should be considered exploratory, but lend weight to emerging data supporting longer duration endocrine therapy for hormone receptor-positive breast cancer, and offer insight into reintroduction of AI therapy. CLINICAL TRIALS NUMBER: Australian New Zealand Clinical Trials Registry (www.anzctr.org.au), ACTRN12607000137493.

EVERSUN: a phase 2 trial of alternating sunitinib and everolimus as first-line therapy for advanced renal cell carcinoma.

BACKGROUND: We hypothesised that alternating inhibitors of the vascular endothelial growth factor receptor (VEGFR) and mammalian target of rapamycin pathways would delay the development of resistance in advanced renal cell carcinoma (aRCC). PATIENTS AND METHODS: A single-arm, two-stage, multicentre, phase 2 trial to determine the activity, feasibility, and safety of 12-week cycles of sunitinib 50 mg daily 4 weeks on / 2 weeks off, alternating with everolimus 10 mg daily for 5 weeks on / 1 week off, until disease progression or prohibitive toxicity in favourable or intermediate-risk aRCC. The primary end point was proportion alive and progression-free at 6 months (PFS6m). The secondary end points were feasibility, tumour response, overall survival (OS), and adverse events (AEs). The correlative objective was to assess biomarkers and correlate with clinical outcome. RESULTS: We recruited 55 eligible participants from September 2010 to August 2012. DEMOGRAPHICS: mean age 61, 71% male, favourable risk 16%, intermediate risk 84%. Cycle 2 commenced within 14 weeks for 80% of participants; 64% received ≥22 weeks of alternating therapy; 78% received ≥22 weeks of any treatment. PFS6m was 29/55 (53%; 95% confidence interval [CI] 40% to 66%). Tumour response rate was 7/55 (13%; 95% CI 4% to 22%, all partial responses). After median follow-up of 20 months, 47 of 55 (86%) had progressed with a median progression-free survival of 8 months (95% CI 5-10), and 30 of 55 (55%) had died with a median OS of 17 months (95% CI 12-undefined). AEs were consistent with those expected for each single agent. No convincing prognostic biomarkers were identified. CONCLUSIONS: The EVERSUN regimen was feasible and safe, but its activity did not meet pre-specified values to warrant further research. This supports the current approach of continuing anti-VEGF therapy until progression or prohibitive toxicity before changing treatment. AUSTRALIAN NEW ZEALAND CLINICAL TRIALS REGISTRY: ACTRN12609000643279.

Axitinib versus sorafenib in advanced renal cell carcinoma: subanalyses by prior therapy from a randomised phase III trial.

BACKGROUND: In the AXIS trial, axitinib prolonged progression-free survival (PFS) vs sorafenib in patients with advanced renal cell carcinoma (RCC) previously treated with sunitinib or cytokines. METHODS: In post hoc analyses, patients were grouped by objective response to prior therapy (yes vs no), prior therapy duration (< vs ⩾median), and tumour burden (baseline sum of the longest diameter < vs ⩾median). PFS and overall survival (OS), and safety by type and duration of prior therapy were evaluated. RESULTS: Response to prior therapy did not influence outcome with second-line axitinib or sorafenib. PFS was significantly longer in axitinib-treated patients who received longer prior cytokine treatment and sorafenib-treated patients with smaller tumour burden following sunitinib. Overall survival with the second-line therapy was longer in patients who received longer duration of prior therapy, although not significant in the sunitinib-to-axitinib sequence subgroup; OS was also longer in patients with smaller tumour burden, but not significant in the cytokine-to-axitinib sequence subgroup. Safety profiles differed modestly by type and duration of prior therapy. CONCLUSIONS: AXIS data suggest that longer duration of the first-line therapy generally yields better outcome with the second-line therapy and that lack of response to first-line therapy does not preclude positive clinical outcomes with a second-line vascular endothelial growth factor-targeted agent in patients with advanced RCC.

Neoadjuvant chemotherapy with sequential anthracycline-docetaxel with gemcitabine for large operable or locally advanced breast cancer: ANZ 0502 (NeoGem).

BACKGROUND: Neoadjuvant chemotherapy has a sound rationale for use in women with large operable breast cancer, and achievement of pathological complete response (pCR) is prognostic. Epirubicin and cyclophosphamide followed by docetaxel is a standard chemotherapy regimen for early breast cancer. In metastatic breast cancer the combination of gemcitabine and a taxane has shown promising results. This phase II study investigated the efficacy and safety of incorporating gemcitabine into neoadjuvant therapy. METHODS: Female patients with operable breast cancer that was clinically T2 (≥3 cm) or T3-4, N0-1, M0 were enrolled to receive 24 weeks of neoadjuvant chemotherapy using epirubicin and cyclophosphamide followed by docetaxel and gemcitabine, plus trastuzumab if HER2-positive. The primary endpoint was the pathological complete response (pCR) rate in the breast in separate HER2-negative and HER2-positive cohorts. Secondary endpoints included pCR in both the breast and axillary lymph nodes, clinical and radiological response rates, disease free survival and safety. RESULTS: 81 patients were enrolled: 63 HER2-negative and 18 HER2-positive. 67 (84%) completed all cycles of chemotherapy, and 78 (96%) proceeded to surgery. pCR was achieved by 12 (20%) patients with HER2-negative, and 9 (53%) with HER2-positive disease. At the first interim analysis, addition of prophylactic G-CSF was recommended due to excess neutropenia. The HER2-negative cohort was closed to accrual because it did not meet the pre-specified target for pCR, and the HER2-positive cohort was closed due to slow accrual. At a median follow-up of 24 months, 12 of 81 (15%) patients had experienced a relapse of their breast cancer. CONCLUSION: Neoadjuvant gemcitabine, when added to docetaxel, after epirubicin and cyclophosphamide, did not reach the pre-specified expectations for pCR rate in HER2-negative tumours. Excess neutropenia was observed, requiring growth factor support. Addition of gemcitabine to docetaxel in this schedule cannot be recommended. Australia and New Zealand Clinical Trials Registry (www.anzctr.org.au) registration number ACTRN12606000191594.

Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia.

Congenital neutropenia and cyclic neutropenia are disorders of neutrophil production predisposing patients to recurrent bacterial infections. Recently the locus for autosomal dominant cyclic neutropenia was mapped to chromosome 19p13.3, and this disease is now attributable to mutations of the gene encoding neutrophil elastase (the ELA2 gene). The authors hypothesized that congenital neutropenia is also due to mutations of neutrophil elastase. Patients with congenital neutropenia, cyclic neutropenia, or Shwachman-Diamond syndrome were referred to the Severe Chronic Neutropenia International Registry. Referring physicians provided hematologic and clinical data. Mutational analysis was performed by sequencing polymerase chain reaction (PCR)-amplified genomic DNA for each of the 5 exons of the neutrophil ELA2 gene and 20 bases of the flanking regions. RNA from bone marrow mononuclear cells was used to determine if the affected patients expressed both the normal and the abnormal transcript. Twenty-two of 25 patients with congenital neutropenia had 18 different heterozygous mutations. Four of 4 patients with cyclic neutropenia and 0 of 3 patients with Shwachman-Diamond syndrome had mutations. For 5 patients with congenital neutropenia having mutations predicted to alter RNA splicing or transcript structure, reverse transcriptase-PCR showed expression of both normal and abnormal transcripts. In cyclic neutropenia, the mutations appeared to cluster near the active site of the molecule, whereas the opposite face was predominantly affected by the mutations found in congenital neutropenia. This study indicates that mutations of the gene encoding neutrophil elastase are probably the most common cause for severe congenital neutropenia as well as the cause for sporadic and autosomal dominant cyclic neutropenia.

Myelodysplasia syndrome and acute myeloid leukemia in patients with congenital neutropenia receiving G-CSF therapy.

Granulocyte colony-stimulating factor (G-CSF) has had a major impact on management of "severe chronic neutropenia," a collective term referring to congenital, idiopathic, or cyclic neutropenia. Almost all patients respond to G-CSF with increased neutrophils, reduced infections, and improved survival. Some responders with congenital neutropenia have developed myelodysplastic syndrome and acute myeloblastic leukemia (MDS/AML), which raises the question of the role of G-CSF in pathogenesis. The Severe Chronic Neutropenia International Registry (SCNIR), Seattle, WA, has data on 696 neutropenic patients, including 352 patients with congenital neutropenia, treated with G-CSF from 1987 to present. Treatment and patient demographic data were analyzed. The 352 congenital patients were observed for a mean of 6 years (range, 0.1-11 years) while being treated. Of these patients, 31 developed MDS/AML, for a crude rate of malignant transformation of nearly 9%. None of the 344 patients with idiopathic or cyclic neutropenia developed MDS/AML. Transformation was associated with acquired marrow cytogenetic clonal changes: 18 patients developed a partial or complete loss of chromosome 7, and 9 patients manifested abnormalities of chromosome 21 (usually trisomy 21). For each yearly treatment interval, the annual rate of MDS/AML development was less than 2%. No significant relationships between age at onset of MDS/AML and patient gender, G-CSF dose, or treatment duration were found (P >.15). In addition to the 31 patients who developed MDS/AML, the SCNIR also has data on 9 additional neutropenic patients whose bone marrow studies show cytogenetic clonal changes but the patients are without transformation to MDS/AML. Although our data does not support a cause-and-effect relationship between development of MDS/AML and G-CSF therapy or other patient demographics, we cannot exclude a direct contribution of G-CSF in the pathogenesis of MDS/AML. This issue is unclear because MDS/AML was not seen in cyclic or idiopathic neutropenia. Improved survival of congenital neutropenia patients receiving G-CSF therapy may allow time for the expression of the leukemic predisposition that characterizes the natural history of these disorders. However, other factors related to G-CSF may also be operative in the setting of congenital neutropenia. (Blood. 2000;96:429-436)

Novel endogenous type D retroviral particles expressed at high levels in a SCID mouse thymic lymphoma.

A xenograft model of the human disease Langerhans cell histiocytosis (LCH) was investigated with severe combined immunodeficiency (SCID) mice. Transplantation of human LCH biopsy material into SCID mice resulted in the generation of mouse tumors resembling lymphomas. A thymoma cell line (ThyE1M6) was generated from one of these mice and found to display significant levels of Mg2+-dependent reverse transcriptase activity. Electron microscopy revealed particles with type D retroviral morphology budding from ThyE1M6 cells at a high frequency, whereas control cultures were negative. Reverse transcription-PCR of virion RNA with degenerate primers for conserved regions of various mouse, human, and primate retroviruses amplified novel sequences related to primate type D retroviruses, murine intracisternal A particles, Jaagsiekte sheep retrovirus, and murine long interspersed nuclear elements but not other retroviral classes. We demonstrate that these sequences represent a novel group of endogenous retroviruses expressed at low levels in mice but expressed at high levels in the ThyE1M6 cell line. Furthermore, we propose that the activation of endogenous retroviral elements may be associated with a high incidence of thymomas in SCID mice.

Perivascular T cells express the pro-inflammatory chemokine RANTES mRNA in multiple sclerosis lesions.

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the central nervous system (CNS), characterized by accumulation of mononuclear cells. The pathogenesis of MS is complex and probably involves soluble immune mediators, particularly cytokines, and activated memory T cells, that are thought to migrate into the CNS. During lesion formation in MS, cytokines regulate cell functions, such as cell recruitment and migration. Because the chemokine RANTES play a role in both activating and recruiting leucocytes, particularly memory T cells into inflammatory sites, the authors have assessed RANTES mRNA levels at the site of lesions. Expression levels were analysed in brain samples and compared with neurological, infectious and other controls. RANTES was expressed by activated perivascular memory T cells, predominantly located at the edge of active plaques. While RANTES mRNA was detected in all 17 MS brains analysed, it was only found in six of the 14 control patients and generally at a lower expression level. In view of the regulatory and chemotactic properties of RANTES, these results imply that RANTES in MS lesions may play an important role in the activation and/or selective accumulation of memory T cells and, thereby, in the pathogenic events associated with MS.

Leukemia inhibitory factor and interleukin-6 are produced by diseased and regenerating skeletal muscle.

The process of skeletal muscle regeneration following injury or disease involves locally produced growth factors which control cellular proliferation and differentiation. Leukemia inhibitory factor (LIF) and interleukin-6 (IL-6) have previously been shown to promote the proliferation of myoblasts in vitro, and thus may be involved in muscle regeneration. In the present investigation, the in vivo expression of these two myogenic growth factors was examined in regenerating muscle after a crush injury of wild type mice, and in diseased skeletal muscle and diaphragm of the mdxmouse. Using Reverse transcription polymerase chain reaction we have demonstrated that while normal muscle rarely expresses mRNA for these two molecules, there is significant up-regulation following injury, coinciding with the active period of muscle regeneration. This suggests these molecules act as locally produced trauma factors. This observation is reinforced in mdxmouse muscle, which is undergoing a cycle of degeneration and regeneration, and expresses both LIF and IL-6. Using in situ hybridization we have localized mRNA for LIF expression in the mdx diaphragm, suggesting that local production of these molecules by regenerating muscle itself, as well as by other cells in muscle, plays an important role in muscle regeneration.

EWS/FLI-1 fusion transcript detection and MIC2 immunohistochemical staining in the diagnosis of Ewing's sarcoma.

Ewing's sarcoma (ES) and other primitive peripheral neuroectodermal tumors (pPNETs) can present a significant diagnostic problem, as they may morphologically resemble other small round cell tumors (SRCTs) of childhood. However, ES/pPNET is known to carry a characteristic t(11;22)(q24;q12), the detection of which may aid diagnosis. The recent identification of the EWS and FLI-1 genes flanking the translocation break point has enabled reverse transcriptase-polymerase chain reaction (RT-PCR) to be used to detect the putative chimeric transcription factor mRNA produced by the fusion gene. We have assessed the RT-PCR method of detection by examining 40 cases of ES for the presence of EWS/FLI-1 transcripts. Twenty-six (76%) of the 34 cases with intact mRNA yielded fusion transcripts. Four different transcript sizes were detected and two tumors contained two transcripts of different size. No transcripts were detected in a control group of non-ES/pPNET SRCTs. Eight cases with intact mRNA were transcript negative. The MIC2 cell surface antigen, which is reported to be present in over 95% of ES/pPNETs, was present in 32 of 33 tumors (97%), including all 24 EWS/FLI-1 transcript-positive cases examined. Hence MIC2 is a useful screen for ES, with RT-PCR detection of t(11;22) being the optimal method for confirming the diagnosis.

Cytogenetic and DNA analysis of two neuroectodermal tumors without a simple t(11;22).

Cytogenetic analysis was conducted on tumor biopsy material from two pediatric, small, round, blue-cell tumors whose histology failed to give a clearcut diagnosis. The first case showed a complex composite karyotype within which there were two normal chromosomes 11 and one abnormal chromosome 22 present. The composite karyotype in the second case was similarly complex but this time included an abnormal chromosome 11 but no corresponding abnormal chromosome 22. Analysis of tumor mRNA from both cases using a Reverse Transcriptase PCR test with primers derived from a Ewing's sarcoma t(11;22)(q24;q12) breakpoint sequence showed both to have abnormal, chimeric transcribed messengers, each of different lengths. Further analysis of case 2 using chromosome painting and centromeric probing confirmed the abnormal chromosome 11 to be a der(11)t(11;22)(q24;q12) and also revealed two additional minor clones containing a der(22), which may be the karyotypic locations of the t(11;22) fusion sequences. Taken into consideration with clinical and histologic information, the results of these investigations indicated that both were neuroectodermal tumors (Ewing sarcomas of the chest wall/Askin tumors). The comparative values of both cytogenetic and molecular analysis in the diagnosis of neuroectodermal tumors and the detection of covert chromosome rearrangements are discussed.

Soluble antigen can cause enhanced apoptosis of germinal-centre B cells.

Germinal centres are dynamic microenvironments of B-lymphocyte differentiation, which develop in secondary lymphoid tissues during immune responses. Within germinal centres, activated B lymphocytes proliferate and point mutations are rapidly introduced into the genes encoding their immunoglobulin receptors. As a result, new specificities of B cells are created, including those with a heightened capacity to bind the immunizing antigen. Immunoglobulin gene mutation can also lead to reactivity to self antigens. It has been suggested that any newly formed self-reactive B cells are eliminated within the germinal centre in order to avoid autoimmunity. Here we present evidence that antigen-specific, high-affinity, germinal-centre B cells are rapidly killed by apoptosis in situ when they encounter soluble antigen. The effect seems to act directly on the B cells, rather than through helper T cells. Furthermore, the apoptosis is unique to germinal-centre cells, and is only incompletely impeded by constitutive expression of the proto-oncogene bcl-2. This phenomenon may reflect clonal deletion of self-reactive B cells within germinal centres.

bcl-2 transgene expression can protect neurons against developmental and induced cell death.

The bcl-2 protooncogene, which protects various cell types from apoptotic cell death, is expressed in the developing and adult nervous system. To explore its role in regulation of neuronal cell death, we generated transgenic mice expressing Bcl-2 under the control of the neuron-specific enolase promoter, which forced expression uniquely in neurons. Sensory neurons isolated from dorsal root ganglia of newborn mice normally require nerve growth factor for their survival in culture, but those from the bcl-2 transgenic mice showed enhanced survival in its absence. Furthermore, apoptotic death of motor neurons after axotomy of the sciatic nerve was inhibited in these mice. The number of neurons in two neuronal populations from the central and peripheral nervous system was increased by 30%, indicating that Bcl-2 expression can protect neurons from cell death during development. The generation of these transgenic mice suggests that Bcl-2 may play an important role in survival of neurons both during development and throughout adult life.

Viable bone marrow stromal cells are required for the in vitro survival of B-cell precursor acute lymphoblastic leukemic cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. A number of studies have examined the binding of BCP ALL cells to BM stromal cells and extracellular matrix components. To date there have been no studies examining the effect of such binding on the growth and survival of BCP ALL cells. In this study, by measuring the growth parameters of these cells with use of a lipophilic fluorescent probe, PKH 26 GL, we demonstrate the positive effect of viable BM stromal cells on BCP ALL cell survival in 10 patient samples. At the same time, by comparing these cultures with cultures of the same patient samples in the presence of glutaraldehyde-fixed stromal cells, deoxycholic acid-derived stromal cell matrices, purified laminin, collagen or fibronectin, the role of various stromal cell-derived contact components in BCP ALL survival was tested. It was shown that the survival of BCP ALL cells in vitro was dependent upon viable BM stromal cells present in co-culture as the various contact components did not show any functional effect on BCP ALL cell survival.

Flow cytometric analysis of intercellular adhesion between B-cell precursor acute lymphoblastic leukemic cells and bone marrow stromal cells.

The growth of B-cell precursor acute lymphoblastic leukemic (BCP ALL) cells in vitro is dependent on interactions with bone marrow (BM) stromal cells. We have recently demonstrated that the rate of cell division of BCP ALL cells increases when cultured in direct contact with BM stromal cells. In this study we describe a new method for examining the direct binding of BM stromal cells and BCP ALL cells at a cellular level. For this binding assay, BCP ALL cells from six patient samples were first stained with the lipophilic fluorescent probe PKH 26 GL and mixed with BM stromal cells in suspension. In all cases, aggregates between BCP ALL and BM stromal cells were identified by flow cytometry and isolated. Using this assay we have examined some of the mechanisms involved in this binding process. The pattern of aggregate formation at various leukemic/stromal cell ratios showed that the aggregate formation increased by increasing the number of either cell type and that the binding could not be saturated. This suggests that the interaction between these cells is an equilibrium reaction. Functional studies showed that the majority of BCP ALL-BM stromal cell binding is dependent on the presence of divalent cations and requires active cellular metabolism. Finally, by use of inhibitory monoclonal antibodies (moAbs) directed against cell adhesion molecules including anti-CD29, VCAM and CD18, we have demonstrated that the involvement of these molecules in the direct cellular interactions could be detected by this method. However, the maximum inhibition observed was 36% which suggests either that the avidity is low or that other adhesion molecules are involved. The data show that the use of flow cytometric analysis of aggregate formation (rather than cell binding to intact cell layers) allows the study of cell interactions at the individual cell level which can reveal additional cellular adhesion mechanisms.

Kiz-1, a protein with LIM zinc finger and kinase domains, is expressed mainly in neurons.

The olfactory epithelium is the only neuronal tissue capable of generating new neurons during adult life and hence must express genes responsible for this phenomenon. Therefore, we have used mRNA from immortalized olfactory epithelial cells to search for novel protein tyrosine kinases by polymerase chain reaction, using as primers conserved sequences from the catalytic domain of known kinase genes. A full-length complementary DNA clone corresponding to one such polymerase chain reaction product was isolated and sequenced. This complementary DNA, designated Kiz-1, encodes a protein containing two prominent domains; the NH2-terminal region contains a cysteine/histidine-rich moiety previously identified as a zinc-finger domain in proteins of the LIM family, while the COOH-terminus contains a kinase domain. Kiz-1 is expressed mainly in the brain of adult mice but also in a range of cultured cell lines, regardless of their tissue of origin. Immunohistochemical studies on adult mouse brain demonstrated that Kiz-1 is expressed exclusively in neurons, not in astrocytes or oligodendrocytes. In the developing embryo, however, Kiz-1 is expressed in all tissues. In COS cells transfected with Kiz-1 complementary DNA and in the immortalized olfactory epithelial cells, Kiz-1 was found mainly in the cytoplasm, but in neurons of the adult brain, it resided also in the nucleus. Two Kiz-1 mRNA species are expressed in cell lines as well as in the murine and human brain. One transcript lacks a region of 60 nucleotides, which lies within the catalytic domain of the kinase and is encoded by a separate exon. Our results suggest that Kiz-1 may play distinct roles in dividing cells and in differentiated neurons.

The role of cytokines in the pathogenesis of Langerhans cell histiocytosis.

Langerhans cell histiocytosis (LCH) is characterised by an accumulation of cells ('LCH cells') with the same phenotypic features as normal Langerhans cells found in skin and other organs. The pathogenesis of LCH is unknown but there is increasing evidence to implicate the involvement of lymphokines and proinflammatory cytokines in the tissue damage seen in this disorder. Apart from histiocytes, the lesions contain giant cells, macrophages, neutrophils, eosinophils, lymphocytes, plasma cells and occasional mast cells that are the hallmark of an inflammatory process. The role of cytokines in the recruitment of haemopoietic cells within inflammatory lesions has only recently been recognised. In this article, we review the possible role of cytokines in the pathogenesis of LCH, and provide an overview of the methods currently used to detect and quantitate them. An appreciation of the type, distribution and amount of different cytokines released within lesions can provide clues to the possible aetiology of LCH. Using immunoassays, in situ hybridisation and RT-PCR, increased amounts of IL-1, IL-3, IL-4, IL-8, GM-CSF, TNF alpha, TGF beta and LIF have been demonstrated in LCH lesions. Lymphocytes constitutively produce GM-CSF and IL-3 and, to a lesser degree, IL-1, IL-4 and LIF whilst histiocytes produce TNF alpha, IL-1 beta and GM-CSF.

Human bone marrow stromal cell contact and soluble factors have different effects on the survival and proliferation of paediatric B-lineage acute lymphoblastic leukaemic blasts.

Recent studies have confirmed that in vitro viability and proliferation of precursor B-cell leukaemia (ALL) cells are linked to the presence of bone marrow derived stromal cells. To investigate whether this effect is mediated by direct contact or through the action of soluble factors, using a method we have recently described, the growth parameters of ALL bone marrow blast cells from eight newly diagnosed patients were determined with the lipophilic fluorescent probe PKH 26 GL. The viability of ALL cells and the rate of cell division in cultures containing either medium alone; stromal cell conditioned medium; stromal cell layers allowing direct contact, or in 0.4 microns microporous membrane cultures suspended above stromal cell layers were examined. In all eight samples an improved maintenance of ALL cells in a viable state in cultures containing bone marrow stromal cells was observed. The survival of leukaemic cells was equivalent in 0.4 microns microporous membrane cultures suspended above stromal cell layers and in cultures of leukaemic cells in direct contact with stromal cell layers. It was thus demonstrated that this effect was mediated by the action of soluble factor(s) present in these cultures. However, the improved maintenance of ALL cells in a viable state was observed in only one of the eight cases when ALL cells were cultured in stromal cell conditioned medium alone. The highest rate of cell division of leukaemic cells was observed in ALL cells in direct contact with bone marrow stromal cells. The activities of stromal cell derived soluble factors could not be reproduced by recombinant forms of likely candidate factors including IL-1 beta, IL-4, IL-6, IL-7, SCF, TNF alpha, TGF beta, LIF, NGF or a mixture of these factors when examined in cultures of the same patient samples. This study implicates the existence of a novel bone marrow derived factor(s) that improves survival of ALL cells in vitro.