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.

Dose-intensive cyclophosphamide with etoposide and vincristine for pediatric solid tumors: a phase I/II pilot study by the Australia and New Zealand Childhood Cancer Study Group.

PURPOSE: This pilot study of the Australia and New Zealand Childhood Cancer Study Group investigated the effectiveness and toxicity of a regimen incorporating vincristine (VCR), etoposide, and divided-dose, escalating cyclophosphamide (CPA) (VETOPEC) in 23 patients aged 1 to 20 years with solid tumors. PATIENTS AND METHODS: Seventeen patients (group A) had recurrent or refractory tumors after prior multiagent therapy, and six patients (group B) with adverse prognostic indicators were treated at initial presentation. Treatment cycles were 21 to 28 days and consisted of vincristine (0.05 mg/kg) on days 1 and 14, with etoposide (2.5 mg/kg/d) plus escalating CPA on days 1, 2, and 3. The CPA dosage was escalated from 30 mg/kg/d in cycle no. 1 by 5 mg/kg/d in each cycle to a maximum of 55 mg/kg/d in cycle no. 6. RESULTS: Of 20 patients assessable for tumor response, 19 (95%) responded after two to six cycles of VETOPEC: seven complete responses (CRs); eight very good partial responses (VGPRs); and four partial responses (PRs). In group A, 13 of 14 (93%) assessable patients responded (five CRs, four VGPRs, four PRs), and in group B, five stage IV and one stage III patient achieved two CRs and four VGPRs. The principal toxicity was myelosuppression. Grade IV neutropenia occurred after 98% of cycles, and the incidence of grade IV thrombocytopenia increased from 37% after cycle no. 1 to 91% after cycle no. 6 (P = .002). A total of 115 cycles delivered were followed by 62 febrile admissions (54%), and showed a significant rise with increasing cycles (P = .001). One patient died of septicemia. CONCLUSION: This combination and scheduling produced a high response rate in patients with recurrent, refractory, or advanced solid tumors of childhood. Further studies of this regimen and of strategies to reduce hematologic toxicity are warranted.

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.

SCL gene in human tumors.

The SCL gene encodes a member of the helix-loop-helix (HLH) family of transcription factors and is reportedly involved in up to 25% of T-cell acute lymphoblastic leukemia (T-ALL). We have surveyed over 120 primary human tumors including melanomas, myeloid, and lymphoid leukemias, and other solid tumors without evidence of rearrangements involving SCL. These results are further supported by low level expression of SCL in these tumors (as assessed by a polymerase chain-reaction-based method). We conclude that rearrangement/translocation with subsequent activation of SCL occurs infrequently in myeloid leukemias and melanomas.

Fractionation of normal and beta-thalassemic human hemopoietic progenitor cells by immunomagnetic beads.

Depletion of adherent cells, followed by simultaneous immunomagnetic bead depletion of Leu 4+, Leu 7+, Leu 11+, Leu M1+, Leu M3+, B1+, WEM-G11+, and Glycophorin A+ cells from normal bone marrow mononuclear cells, consistently led to recoveries of erythroid and nonerythroid colony-forming cells of greater than 100% and enrichment of 13- to 99-fold. Similarly, the recovery of mixed erythroid colony-forming cells was greater than 100%, with enrichments of 7.5- to 262-fold. This simple procedure, when used on normal bone marrow and beta-thalassemic peripheral blood mononuclear cells, as well as providing significant enrichment, suggests that colony assays on unfractionated human mononuclear cells specifically underestimate the number of BFU-E, Mix-CFC, and nonerythroid-CFC present.

Characterization of stroma-adherent colony-forming cells: a clonogenic assay for early hemopoietic cells?

Hemopoietic cells that adhered to preformed selected marrow stromal cell layers were characterized on the basis of progenitor cell production capacity, colony-formation kinetics and forward light scatter (FLS) properties. It was shown that early hemopoietic cells attached to the stromal layers within 2 hours of incubation, and were responsible for the initial production of the more differentiated granulocyte-macrophage colony-forming cells (day 14 GM-CFC) in long-term cultures (LTC). In a clonogenic assay system, hemopoietic cells that adhere to stromal layers can be detected by the formation of small colonies of blast-like cells and are designated as stroma-adherent CFC. Cell fractionation on the basis of FLS and counting colonies on days 5, 14 and 21 revealed that there was a succession of colony formation, indicating that the stroma-adherent CFC consisted of subpopulations with different lag-phases before initiation of proliferation. Day 5, day 14 and day 21 stroma-adherent CFC were shown to have a high, intermediate and low FLS, respectively. The cells that produced GM-CFC by day 21 showed FLS properties similar to those of day 21 stroma-adherent CFC, suggesting a correlation between day 21 stroma-adherent CFC and CFC-producing cells in LTC. The CFC present on day 21 required the synergistic action of GM-CSF+IL-3 + stem cell factor (SCF) for optimal proliferation. The prolonged lag-phase, the low FLS and the multifactor-responsive progeny are properties similar to those reported for other early cells, and it is proposed that day 21 stroma-adherent CFC represent an early hemopoietic cell type whereas day 5 stroma-adherent CFC represent a more mature stage of differentiation.

Studies by radioiodination of normal adult, fetal and leukemic cell membranes.

A comparison was made between cord blood lymphocytes, normal adult lymphocytes and leukemic cells after membrane iodination with lactoperoxidase. A double-labeling technique using lactoperoxidase iodination with 125I and 131I followed by analysis on polyacrylamide gel electrophoresis revealed a number of membrane differences between leukemic, normal and fetal cells. There was a reduction in the 70,000 molecular weight component in cord blood cells compared to adult lymphocytes, and an increase in membrane peptides with molecular weights of 35,000, 20,000, 9,000 and 4,000. Although smaller molecular weight peptides were also present in chronic lymphatic leukemia as well as acute myeloid leukemia, these were shown to be distinct from fetal type membrane components.

Clonal proliferation in vitro of individual murine and human hemopoietic cells after fluorescence-activated cell sorting.

A modification of the fluorescence-activated cell sorter (FACS) was used to rapidly and reliably study the clonal proliferation of single hemopoietic cells. Murine FDC-P1 and human cord blood progenitor cells were examined for their ability to proliferate from single cells in 96-well microtiter plates containing agar medium and appropriate stimuli. FACS-sorted FDC-P1 single cells formed colonies in 345 out of 558 wells (62%), which compared favorably with control cultures (53%) and micromanipulated single cells (55%). Similarly, the frequency and type of day-14 colonies arising from cord blood progenitor cells when sorted as single cells by the FACS compared favorably with those grown from micromanipulated single cells or in control cultures.

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.

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.

Measurement of the growth parameters of precursor B-acute lymphoblastic leukaemic cells in co-culture with bone marrow stromal cells; detection of two cd10 positive populations with different proliferative capacities and survival.

A new assay system using the fluorescent probe PKH 26 GL was employed to investigate the regulation of precursor B-cell acute lymphoblastic leukaemic (ALL) cell growth. PKH 26 GL is a lipophilic fluorescent probe which becomes incorporated into the plasma membrane upon the staining of cells. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. Bone marrow derived ALL cells from seven newly diagnosed cases were stained with PKH 26 GL, and cultured for 14 days in control cultures without stimulus, or in cultures with preformed human bone marrow stromal cell layers. Viable leukaemic cells from these cultures were identified on the basis of forward light scatter, 90 degrees light scatter, propidium iodide exclusion, PKH 26 GL staining and CD10 expression by flow cytometry at the beginning of the culture period and on days 2, 6, 10 and 14. The growth parameters of these leukaemic cells were determined by analysis of their pattern of PKH 26 GL fluorescence. A higher rate of proliferation and survival of cells was observed in cultures with stromal cells compared with control cultures, without stromal cells. In the presence of stromal cells, survival and proliferation continued throughout the culture period; in contrast in five of seven control cultures no viable cells could be detected after 6-10 days. Interestingly, two populations of leukaemic cells were distinguished on the basis of their different rates of proliferation, when co-cultured with stromal cells. The results indicate that this technique provides a means for studying and quantitating leukaemic cell growth within a complex stroma-dependent system.

A novel approach to the measurement of different in vitro leukaemic cell growth parameters: the use of PKH GL fluorescent probes.

The application of the fluorescent cell membrane probes PKH2 and PKH 26 GL in the measurement of leukaemic cell growth was examined on four cell lines K562, NALM-6, ACV (a pre-B cell line) and HL-60 using flow cytometry. As the amount of probe per cell reduces at each cell division, the fluorescence can be used to measure cell proliferation. By measuring the mean fluorescence intensity of the cells at the beginning of culture and at various time points, and by combining this information with a viable cell count, it was possible to determine: (1) the number of viable cells; (2) their rate of proliferation; (3) their number of cell divisions; and (4) the maintenance of cells in a viable state over a period of time. It was demonstrated that these parameters could be reliably established using the red fluorescent probe PKH26 GL. In contrast, the green fluorescent probe PKH2 GL showed dye transfer resulting in an underestimation of the number of cell divisions and an overestimation of the maintenance of cells in a viable state. The potential advantages of the use of PKH26 GL over conventional assays for the measurement of leukaemic cell growth parameters are discussed.

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.

Osmotic fragility of leukaemic and normal lymphocytes.

The osmotic fragility of cord blood lymphocytes (CBLs), normal adult lymphocytes (NALs) and leukaemic cells was studied using hypotonic saline. Leukaemic cells were more resistant to hypotonic treatment than cord blood or adult lymphocytes. At low hypotonic saline concentrations (less than 0.2% NaCl), almost all of the NALs and CBLs were lysed after thirty minutes at 4 degrees C, whilst a large proportion of chronic lymphatic leukaemia cells remained intact. This phenomenon could conceivably be used to separate tumour cells from normal lymphoid cells.

Activated killer cell lymphoma: an erythrophagocytic syndrome simulating histiocytic medullary histiocytosis.

We report a detailed analysis of a lymphoma-induced erythrophagocytic syndrome mimicking histiocytic medullary reticulosis. Phenotypic analysis of cell surface molecules demonstrated a T cell-like phenotype. However, more extensive analysis showed that this phenotype was not typical of any element of the normal T cell lineage. The markers were consistent with a subset of natural killer cells, the lymphokine-activated killer (LAK) cell. The lymphoma cells, like normal LAK cells, were shown to be capable of non-specific cytotoxicity. Moreover, consistent with the reported regulatory effects of certain non-specific killer cells on hemopoiesis, the lymphoma cells were able to suppress in-vitro hemopoiesis, especially maturation of erythroid precursors, although a soluble factor could not be directly demonstrated. Both of these activities were blocked by a monoclonal antibody (9.IC3) which inhibits NK cell function. These findings imply that this tumour is a neoplastic counterpart of the cell identifiable in vitro as an LAK cell.

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.

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.