Identifying baseline immune-related biomarkers to predict clinical outcome of immunotherapy.

As cancer strikes, individuals vary not only in terms of factors that contribute to its occurrence and development, but as importantly, in their capacity to respond to treatment. While exciting new therapeutic options that mobilize the immune system against cancer have led to breakthroughs for a variety of malignancies, success is limited to a subset of patients. Pre-existing immunological features of both the host and the tumor may contribute to how patients will eventually fare with immunotherapy. A broad understanding of baseline immunity, both in the periphery and in the tumor microenvironment, is needed in order to fully realize the potential of cancer immunotherapy. Such interrogation of the tumor, blood, and host immune parameters prior to treatment is expected to identify biomarkers predictive of clinical outcome as well as to elucidate why some patients fail to respond to immunotherapy. To approach these opportunities for progress, the Society for Immunotherapy of Cancer (SITC) reconvened the Immune Biomarkers Task Force. Comprised of an international multidisciplinary panel of experts, Working Group 4 sought to make recommendations that focus on the complexity of the tumor microenvironment, with its diversity of immune genes, proteins, cells, and pathways naturally present at baseline and in circulation, and novel tools to aid in such broad analyses.

Cancer classification using the Immunoscore: a worldwide task force.

Prediction of clinical outcome in cancer is usually achieved by histopathological evaluation of tissue samples obtained during surgical resection of the primary tumor. Traditional tumor staging (AJCC/UICC-TNM classification) summarizes data on tumor burden (T), presence of cancer cells in draining and regional lymph nodes (N) and evidence for metastases (M). However, it is now recognized that clinical outcome can significantly vary among patients within the same stage. The current classification provides limited prognostic information, and does not predict response to therapy. Recent literature has alluded to the importance of the host immune system in controlling tumor progression. Thus, evidence supports the notion to include immunological biomarkers, implemented as a tool for the prediction of prognosis and response to therapy. Accumulating data, collected from large cohorts of human cancers, has demonstrated the impact of immune-classification, which has a prognostic value that may add to the significance of the AJCC/UICC TNM-classification. It is therefore imperative to begin to incorporate the 'Immunoscore' into traditional classification, thus providing an essential prognostic and potentially predictive tool. Introduction of this parameter as a biomarker to classify cancers, as part of routine diagnostic and prognostic assessment of tumors, will facilitate clinical decision-making including rational stratification of patient treatment. Equally, the inherent complexity of quantitative immunohistochemistry, in conjunction with protocol variation across laboratories, analysis of different immune cell types, inconsistent region selection criteria, and variable ways to quantify immune infiltration, all underline the urgent requirement to reach assay harmonization. In an effort to promote the Immunoscore in routine clinical settings, an international task force was initiated. This review represents a follow-up of the announcement of this initiative, and of the J Transl Med. editorial from January 2012. Immunophenotyping of tumors may provide crucial novel prognostic information. The results of this international validation may result in the implementation of the Immunoscore as a new component for the classification of cancer, designated TNM-I (TNM-Immune).

Defining the critical hurdles in cancer immunotherapy.

Scientific discoveries that provide strong evidence of antitumor effects in preclinical models often encounter significant delays before being tested in patients with cancer. While some of these delays have a scientific basis, others do not. We need to do better. Innovative strategies need to move into early stage clinical trials as quickly as it is safe, and if successful, these therapies should efficiently obtain regulatory approval and widespread clinical application. In late 2009 and 2010 the Society for Immunotherapy of Cancer (SITC), convened an "Immunotherapy Summit" with representatives from immunotherapy organizations representing Europe, Japan, China and North America to discuss collaborations to improve development and delivery of cancer immunotherapy. One of the concepts raised by SITC and defined as critical by all parties was the need to identify hurdles that impede effective translation of cancer immunotherapy. With consensus on these hurdles, international working groups could be developed to make recommendations vetted by the participating organizations. These recommendations could then be considered by regulatory bodies, governmental and private funding agencies, pharmaceutical companies and academic institutions to facilitate changes necessary to accelerate clinical translation of novel immune-based cancer therapies. The critical hurdles identified by representatives of the collaborating organizations, now organized as the World Immunotherapy Council, are presented and discussed in this report. Some of the identified hurdles impede all investigators; others hinder investigators only in certain regions or institutions or are more relevant to specific types of immunotherapy or first-in-humans studies. Each of these hurdles can significantly delay clinical translation of promising advances in immunotherapy yet if overcome, have the potential to improve outcomes of patients with cancer.

Recommendations from the iSBTc-SITC/FDA/NCI Workshop on Immunotherapy Biomarkers.

PURPOSE: To facilitate development of innovative immunotherapy approaches, especially for treatment concepts exploiting the potential benefits of personalized therapy, there is a need to develop and validate tools to identify patients who can benefit from immunotherapy. Despite substantial effort, we do not yet know which parameters of antitumor immunity to measure and which assays are optimal for those measurements. EXPERIMENTAL DESIGN: The iSBTc-SITC (International Society for Biological Therapy of Cancer-Society for Immunotherapy of Cancer), FDA (Food and Drug Administration), and NCI (National Cancer Institute) partnered to address these issues for immunotherapy of cancer. Here, we review the major challenges, give examples of approaches and solutions, and present our recommendations. RESULTS AND CONCLUSIONS: Although specific immune parameters and assays are not yet validated, we recommend following standardized (accurate, precise, and reproducible) protocols and use of functional assays for the primary immunologic readouts of a trial; consideration of central laboratories for immune monitoring of large, multi-institutional trials; and standardized testing of several phenotypic and functional potential potency assays specific to any cellular product. When reporting results, the full QA (quality assessment)/QC (quality control) should be conducted and selected examples of truly representative raw data and assay performance characteristics should be included. Finally, to promote broader analysis of multiple aspects of immunity, and gather data on variability, we recommend that in addition to cells and serum, RNA and DNA samples be banked (under standardized conditions) for later testing. We also recommend that sufficient blood be drawn to allow for planned testing of the primary hypothesis being addressed in the trial, and that additional baseline and posttreatment blood is banked for testing novel hypotheses (or generating new hypotheses) that arise in the field.

Molecularly targeted therapies for colorectal cancer: Strategies for implementing translational research in clinical trials.

Few breakthroughs in preclinical research have translated into meaningful benefits, either in clinical terms or quality of life, for patients with advanced colorectal cancer, despite important preclinical discoveries regarding aberrant biological pathways associated with disease development and progression. The many reasons for the slow progress are diverse, ranging from the failure to codevelop biomarkers and targeted therapies, the regulatory burdens imposed on academic investigators, and the failure to collect serial tumor biopsies during clinical trials. This review discusses promising translational research that could help reduce the disparity between preclinical discovery and patient benefit, and advocate the concentration of efforts and resources on the most promising therapeutic targets in colorectal cancer, such as EGFR, VEGF and Fcγ receptor.

A systematic approach to biomarker discovery; preamble to "the iSBTc-FDA taskforce on immunotherapy biomarkers".

The International Society for the Biological Therapy of Cancer (iSBTc) has initiated in collaboration with the United States Food and Drug Administration (FDA) a programmatic look at innovative avenues for the identification of relevant parameters to assist clinical and basic scientists who study the natural course of host/tumor interactions or their response to immune manipulation. The task force has two primary goals: 1) identify best practices of standardized and validated immune monitoring procedures and assays to promote inter-trial comparisons and 2) develop strategies for the identification of novel biomarkers that may enhance our understating of principles governing human cancer immune biology and, consequently, implement their clinical application. Two working groups were created that will report the developed best practices at an NCI/FDA/iSBTc sponsored workshop tied to the annual meeting of the iSBTc to be held in Washington DC in the Fall of 2009. This foreword provides an overview of the task force and invites feedback from readers that might be incorporated in the discussions and in the final document.

How useful are housekeeping genes? Variable expression in melanoma metastases.

BACKGROUND: There is a certain difference in opinion regarding the optimal choice of housekeeping genes used as normalization factors in gene expression analysis. We have therefore examined the suitability of three housekeeping genes, hypoxanthine phosphoribosyl transferase, beta-glucuronidase and beta2-micro-globulin, for normalization of expression data from melanoma metastases. METHODS: The expression of the three housekeeping genes was quantified by quantitative reverse transcription PCR in snap-frozen sections from 44 melanoma metastases, of which 19 were from patients treated with cisplatinum, dacarbazine and interferon-alpha2b. RESULTS: The expression of each housekeeping gene varied considerably between the different metastases. Histopathological examination of the tissue sections revealed variation in the amount of tumor cells in the tissue, necrosis, varying degrees of lymphocyte infiltration, and lymph node remnants. Based on this examination, 16 biopsies were omitted from further analysis because they had cracked, contained empty or necrotic areas, or were dominated by lymph node tissue. Even in sections with more than 90% tumor cells, a wide variation in the expression of the three housekeeping genes was found. The amount of lymphatic infiltrate in the tumors can have an effect on the expression of housekeeping genes in the metastases, whereas treatment did not seem to influence the expression. CONCLUSIONS: We conclude that the choice of housekeeping genes can have great impact on the normalization of specific genes in melanoma metastases. Furthermore, in the analysis of mRNA expression in tumor tissue, microscopic examination is of great importance to evaluate the integrity and cellular composition of the biopsy.

Preoperative interleukin-6 production by mononuclear blood cells predicts survival after radical surgery for colorectal carcinoma.

BACKGROUND: Colorectal cancer is one of the most common forms of cancer in the Western world. Staging based on histopathology is currently the most accurate predictor of outcome after surgery. Colorectal cancer is curable if treated at an early stage (stage I-III). However, for tumors in stages II and III there is a great need for tests giving more accurate prognostic information defining the patient population in need of closer follow-up and/or adjuvant therapy. Furthermore, tests that provide prognostic information preoperatively could provide a guide both for preoperative oncologic treatment and the surgical procedure. METHODS: Peripheral blood mononuclear cells (PBMCs) were isolated preoperatively, within a week before primary surgery, from 39 patients undergoing surgery for colorectal cancer. The PBMCs were cultured in vitro for 24 hours in the presence of autologous serum and lipopolysaccharide (LPS). Interleukin-6 (IL-6) production was measured with enzyme-linked immunosorbent assay (ELISA). Staging based on histopathology was performed in all patients. Patients were followed for at least 54 months. RESULTS: A production of >5000 pg/mL of IL-6 identified colorectal cancer patients with a poor prognosis. Eight out of 13 patients with >5000 pg/mL IL-6 died from cancer within the follow-up period, whereas no cancer-related deaths were recorded among 21 patients with 5000 pg/mL IL-6 or less. A multivariate Cox regression analysis, stratified for T- and N-stage, identified IL-6 production as an independent prognostic factor. CONCLUSIONS: IL-6 production in vitro by PBMC can predict survival after radical surgery for colorectal cancer.

Effects of cisplatin, alpha-interferon, and 13-cis retinoic acid on the expression of Fas (CD95), intercellular adhesion molecule-1 (ICAM-1), and epidermal growth factor receptor (EGFR) in oral cancer cell lines.

BACKGROUND: Previous studies showed that many chemotherapeutic agents can induce immuno-suppression at therapeutic drug concentrations whereas low drug doses induce immuno-augmentation. METHODS: The effect of low-dose cisplatin, interferon-alpha, and 13-cis retinoic acid on receptors involved in immune-mediated apoptosis (Fas/CD95), cell growth (epidermal growth factor receptor) and lymphocyte adhesion (intercellular adhesion molecule-1) was investigated in two oral cancer cell lines (UT-SCC-20A and UT-SCC-24A). Different methods for cell preparation were studied: mechanical and enzymatic detachment, and culture on chamber slides. Receptor expression was investigated using immunohistochemical staining. The amount of soluble and cell-bound Fas was determined with the ELISA technique, and the functional relevance of Fas expression, apoptosis induction, was analyzed. RESULTS: Cisplatin enhanced cytoplasm and membrane staining for Fas in both cell lines. After cisplatin treatment, the amount of soluble Fas was increased in UT-SCC-20A cultures, but no effect was observed in the UT-SCC-24A cell line. Apoptosis, measured as enhanced caspase-3 activity, was induced by an agonistic Fas antibody (CH11) after cisplatin treatment in UT-SCC-24A cells. CONCLUSIONS: Low-dose cisplatin treatment enhanced Fas expression in both cell lines and increased susceptibility to apoptosis in one of them.

Production of IL-1Ra by human mononuclear blood cells in vitro: influence of serum factors.

In vitro cell culture models that measure cytokine production can be of great value when analyzing regulatory mechanisms underlying various pathological conditions. However, testing the function of peripheral blood cells has to take into consideration that serum factors are likely to be of importance in maintaining their function. Interleukin-1 receptor antagonist (IL-1Ra) is a cytokine of key importance in immune regulation and is believed to be involved in numerous pathological processes, such as autoimmunity and cancer. We investigated the influence of normal, human serum on spontaneous production of IL-1Ra by human peripheral blood mononuclear cells (PBMC) in vitro. IL-1Ra production in vitro spanned over a wide range of concentrations, which could be attributed to a combined effect of both cellular parameters and properties of the serum used. The production of IL-1Ra in vitro could be correlated to the level of immobilized IgG, especially IgG1 and IgG3, which is adsorbed from the serum and bound to the tissue culture wells during culture. However, the amount of serum IgG adsorbed to the tissue culture wells could not necessarily be predicted based on the serum concentration of IgG.

Effects of cytokines on matrix metalloproteinase expression in oral squamous cell carcinoma in vitro.

CONCLUSIONS: The intra-tumoral cytokines IL-6, hepatocyte growth factor (HGF) and tumour necrosis factor-a (TNF-a) stimulate oral cancer cells to enhanced secretion of matrix metalloproteinase (MMP)-1 and -9. These results contribute to an understanding of the extracellular events necessary for tumour progression. OBJECTIVE: MMPs play an important role in enhanced intra-tumoral proteolytic activity, promoting angiogenesis and invasion by acting on extracellular matrix substances. Cytokines secreted by tumour-infiltrating immune cells, fibroblasts and tumour cells can modulate MMP expression and secretion by cancer cells. The objective of this study was to investigate the effects of IL-6, soluble IL-6 receptor (sIL-6R), HGF, TNF-a and IL-8 on MMP-1, -2 and -9 expression by two oral squamous cell carcinoma cell lines (UT-SCC-20A and -24A). MATERIAL AND METHODS: ELISA was used to analyse secretion of total MMP protein and gelatin zymography was used for activity analysis. RESULTS: IL-6 had a moderate stimulatory effect on MMP-1 secretion in both cell lines, whereas sIL-6R had no effect. When these cytokines were added together, a dose-dependent, synergistic stimulatory effect was observed. HGF also upregulated MMP-1 secretion, especially in one cell line (UT-SCC-24A), and a synergistic effect was observed when HGF was added to IL-6 in both cell lines. MMP-9 secretion by UT-SCC-24A was increased when stimulated with HGF and IL-6 combined with sIL-6R, whereas no effect was found in the other cell line. TNF-a stimulated MMP-9 secretion in both cell lines, but only stimulated MMP-1 secretion in one (UT-SCC-24A). The zymographic results were consistent with the ELISA results, indicating an upregulation of active enzyme when a stimulatory effect on protein expression was detected.

Phase I investigation of recombinant anti-human vascular endothelial growth factor antibody in patients with advanced cancer.

We assessed the tolerability, safety, pharmacokinetics and dose-limiting toxicity (DLT) of the recombinant humanized IgG4 anti-vascular endothelial growth factor (VEGF) monoclonal antibody, HuMV833, in patients with advanced cancer. Cohorts of patients with progressive solid tumours received escalating doses of HuMV833 as a 1-h intravenous (I.V.) infusion on days 1, 15, 22, and 29. Twenty patients (median Eastern Cooperative Oncology Group (ECOG) score 1) were accrued. HuMV833 infusions were well tolerated and there were no grade III or IV toxicities definitely related to the antibody. Grade I or II toxicities probably related to the antibody included fatigue, dyspnoea and rash. There were two episodes of asymptomatic hypocalcaemia, one at grade III and one grade IV, which were recorded in early follow-up. There were eight grade I episodes of asymptomatic elevation of activated partial thromboplastin time (APTT) and two grade III events; one in a patient receiving 1 mg/kg and the other receiving extended doses of 10 mg/kg. Pharmacokinetic analysis revealed a non-linear kinetic and an elimination half-life of between 8.2 (0.3 mg/kg) and 18.7 (10 mg/kg) days. One patient with ovarian cancer experienced a partial response (PR) of 9 months duration and eight had disease stabilisation (SD) including one patient with colorectal carcinoma whose disease was stable for 14 months. In 13 of the 14 samples taken from 12 patients, the plasma concentration of hepatocyte growth factor (HGF) was reduced 24 h after drug administration. HuMV833 is safe and lacked DLT at doses up to 10 mg/kg on this schedule. Multiple doses were well tolerated, despite occasional asymptomatic elevations in APTT. By combining pharmacokinetic, pharmacodynamic and toxicity data, we can identify doses of 1 and 3mg/kg for further investigation. HuMV833 appears to possess some clinical activity.

CD28 expression in sentinel node biopsies from breast cancer patients in comparison with CD3-zeta chain expression.

BACKGROUND: Immunosuppression is documented in several malignant diseases, including breast cancer. Subsequently, future therapeutic concepts might include immunological approaches. However, detailed knowledge about tumor immunogenicity and host immunoreactivity, and how to assess these adequately, is still limited. We studied CD28 and CD3-zeta expression in sentinel node biopsies (SNB) from breast cancer patients to analyze tumor-related changes in T cell activity. METHOD: 25 women underwent surgery for primary breast cancer, including SNB. Frozen sections from 21 sentinel nodes could be analyzed with a double-staining technique. CD28 expression was studied in CD4+ and CD8+ T-lymphocyte subsets and compared with CD3-zeta expression in three specified nodal regions. RESULTS: The degree of CD28 expression varied between the different lymph node areas. The lowest degree of CD28 expression was observed in CD4+ T-lymphocytes in the paracortex and germinal centers. Here, a good agreement with CD3-zeta expression was found. A higher CD28 expression was noted in CD4+ T-cells in the primary follicles, where concordance with CD3-zeta expression was weaker. The CD8+ T-lymphocyte subset displayed generally a higher degree of CD28 expression than the CD4+ subset. CONCLUSION: Sentinel lymph nodes from breast cancer patients displayed local immunosuppression of varying extent. In the areas with the lowest degree of CD28 expression an accordingly low CD3-zeta expression was found. The SNB might prove an important diagnostic tool for the evaluation of interactions between tumor and the host immune system, helping to select patients who might benefit from adjuvant immunotherapy.

Differences in adsorption of serum proteins and production of IL-1ra by human monocytes incubated in different tissue culture microtiter plates.

In vitro cell culture models can be of great value in order to further analyze the regulatory mechanisms underlying the inappropriate function of the immune system in diseases such as autoimmunity and cancer. Cell culture conditions have to be well controlled in a way that they mirror the in vivo situation. The objective of this study was to compare tissue culture microtiter plates from different manufacturers with respect to their ability to support monokine production by human monocytes cultured in human serum. Tissue culture ware, made of polystyrene, undergoes treatment by the manufacturers to make the surface more suitable for culture of adherent cell populations. It is possible that quality differences in this treatment can lead to variations in protein binding properties and thereby influence the adherence and functional properties of monocytes. We measured spontaneous interleukin-1 receptor antagonist (IL-1ra) production by peripheral blood monocytes, cultured in human serum, in five different microtiter plates made for adherent cell culture. Culture in plates from two of the five manufacturers resulted in significantly lower amounts of secreted IL-1ra. IL-1ra release by human monocytes can be induced by adherent IgG cross-linking membrane receptors for the Fc part of IgG (FcgammaR). We found that reduced IL-1ra production coincided with a reduced capacity for binding of serum IgG in one case. Furthermore, this brand of microtiter plate also displayed the lowest level of adsorption of human albumin. We conclude that the protein adsorption properties of the plastic tissue culture ware have to be taken into consideration when assessing monokine production by human monocytes in vitro.

Heparan sulphate epitope-expression is associated with the inflammatory response in metastatic malignant melanoma.

Heparan sulphate (HS) represents a heterogeneous class of molecules on cell membranes and extracellular matrices. These molecules are involved in a variety of biological processes, including immune responses, through their binding and functional modulation of proteins. Recently a panel of HS-epitope-specific, human single chain antibodies have been generated by phage display, facilitating analysis of the structural heterogeneity of HS in relation to pathological conditions. In a pilot study a heterogeneous staining pattern in melanoma metastases was observed with one of the clones (EW4G1). Using a double-staining technique, the expression of this epitope was studied in 12 metastatic melanoma lesions in relation to the presence of a CD3(+) cell infiltrate. Different staining patterns with EW4G1 were observed in the different lesions. The different staining patterns were associated with the presence and pattern of inflammation with CD3(+) cells. A pronounced staining pattern of blood vessels with EW4G1 was associated with a more or less brisk presence of CD3(+) cells, while a pronounced staining of tumour cells or tumour cell matrix or absence of staining with EW4G1 was associated with absence of CD3(+) cells. These results suggest a dualistic role for HS in the recruitment and intratumoural migration of CD3(+) cells, depending on the location of expression of its epitope recognized by EW4G1. Further characterization of the structural diversity of HS and its function in T-cell recruitment and migration is therefore warranted, since detailed understanding of this relation may provide new targets for therapeutic intervention, such that better homing and migration of T cells (in)to tumours might be achieved in immunologically based treatment strategies.

On the effect of biochemotherapy in metastatic malignant melanoma: an immunopathological evaluation.

Although immunotherapy and biochemotherapy have shown promise, producing a subset of durable responses, for the majority of patients with metastatic melanoma the prognosis is still poor. Therefore there is a great need for predictive tests to identify patients with a high probability of responding. Furthermore, there is also a need for a better understanding of the mechanisms of action during treatment in order to be able to monitor the relevant antitumour reactivity during treatment and to optimize the efficacy of future immunotherapy and biochemotherapy. In the present study histopathological regression criteria were used to study the efficacy of biochemotherapy. Thirty-two patients with metastatic malignant melanoma (18 with regional disease and 14 with systemic disease) were treated with biochemotherapy (cisplatin 30 mg/m2 intravenously on days 1-3, dacarbazine 250 mg/m2 intravenously on days 1-3 and interferon-alpha2b 10 million IU subcutaneously 3 days a week, every 28 days). Pre-treatment fine needle aspirates were obtained from metastases to analyse the number of tumour-infiltrating CD4+ lymphocytes. Therapeutic efficacy was evaluated in metastases resected after treatment using histopathological criteria of tumour regression. Comparisons were also made with metastases from 17 untreated patients, all with regional disease. Regressive changes of 25% or more (of the section area) were found in two of the 17 untreated patients with regional disease compared with 13 of the 18 patients with regional disease and 10 of the 14 patients with systemic disease after biochemotherapy. Fifty per cent of the patients with regional disease showed a high degree of regressive changes (75-100% of the section area) after biochemotherapy. These results demonstrate the occurrence of an antitumour reactivity in the majority of patients. Patients with extensive regressive changes in 75-100% of the analysed biopsies were also found to have a longer overall survival (P = 0.019). In patients with regional disease there was a close correlation between a larger number of CD4+ lymphocytes pre-treatment and a higher degree of regressive changes post-treatment (P < 0.05). Thus, immunohistochemical analysis of tumour biopsies shortly after treatment seems to be a good surrogate endpoint. This technique also allows detailed analysis of antitumour reactivity and escape mechanisms.

Bcl-2 expression in metastatic malignant melanoma. Importance for the therapeutic efficacy of biochemotherapy.

For the majority of patients with metastatic malignant melanoma the prognosis is poor. Immunotherapy and biochemotherapy have shown promise with a subset of durable responses, but there is still a great need for a better understanding of the mechanisms of action during treatment to optimize future treatment schedules. In the present study Bcl-2 expression was studied in biopsies from ten patients with metastatic malignant melanoma (five with regional disease and five with systemic disease) treated with biochemotherapy (cisplatinum 30 mg/m2 days 1-3, DTIC 250 mg/m2 days 1-3 i.v. and Interferon-alpha2b 10 MIU s.c. 3 days a week, on a 28-day cycle). The expression of Bcl-2 by the tumour cells was separately recorded in areas of histopathological regressive changes and in areas of unaffected tumour growth. Comparisons were made with biopsies from 14 untreated patients. In 10 of 10 treated patients a high expression of Bcl-2 by the tumour cells was found in areas of unaffected tumour growth. In contrast, only in 5 of 13 untreated patients was a high expression of Bcl-2 by the tumour cells found in these areas ( P=0.008). A significant difference was also found in the expression of Bcl-2 by the tumour cells between areas of unaffected tumour growth and areas of histopathological regressive changes ( P=0.03). The significantly higher expression of Bcl-2 by the tumour cells in areas of unaffected tumour growth in treated patients compared to untreated patients indicates that clones with a high expression of Bcl-2 may be present after therapy, preventing apoptosis and eventually in many patients resulting in progressive disease. Supporting this concept, a difference was also found between the expression of Bcl-2 in areas of unaffected tumour growth, i.e. in areas of treatment failure, and the expression in areas of histopathological regressive changes. Thus immunohistochemical analysis of tumour biopsies shortly after therapy seems to be a good surrogate endpoint that allows a detailed analysis of Bcl-2 expression. The high expression of Bcl-2 shown in unaffected tumour areas after therapy suggests the need for additional treatment, e.g. Bcl-2 antisense therapy.

Biochemotherapy of metastatic malignant melanoma. On down-regulation of CD28.

Immunotherapy and combination treatments such as biochemotherapy have shown promise, with higher response rates and a subset of durable responses; however, as the majority of responses are still of short duration, they do not provide any survival benefit. There is therefore a great need to better understand the mechanisms whereby tumours escape immune surveillance. The present study examines the expression of CD28 in patients with untreated and treated melanoma metastases. Twenty-eight patients with metastatic malignant melanoma were treated by biochemotherapy (cisplatinum 30 mg/m(2) days 1-3, DTIC 250 mg/m(2) days 1-3 i.v., and IFN-alpha2b 10 million IU s.c. three days a week for 28 days treatment cycle). Tumours were resected post-biochemotherapy and analysed for the expression of CD28 in CD4(+) and CD8(+) lymphocytes in areas where histopathological regressive changes had occurred, and close to tumour cells in areas of unaffected tumour growth using a double-staining technique. A high percentage of the lymphocytes in areas with regressive changes were found to be CD4(+)CD28(-). In contrast, the vast majority of CD4(+) lymphocytes migrating close to the tumour cells were found to be CD28(+) (P<0.001). A similar difference in the expression of CD28 was also found for the CD8(+) subset (P=0.004). A difference in down-regulation of the expression of CD28 was found between CD4(+) and CD8(+) lymphocytes both in the areas of regressive changes and in the unaffected tumour areas. The present study demonstrates that extensive down-regulation of the co-stimulatory factor CD28 is found in metastases following biochemotherapy. These results indicate that parameters of importance for the immune function have already undergone modification after one or two treatment cycles and that this down-regulation occurs in particular in areas with regressive tumour changes.

Down-regulation of the CD3-zeta chain in sentinel node biopsies from breast cancer patients.

BACKGROUND: In several neoplastic diseases, immunosuppression has been shown to correlate with disease stage, progression, and outcome. As the prognosis for metastatic breast cancer is still pessimistic, additional strategies are being sought to improve survival. Local immunosuppression in sentinel node biopsies from 24 evaluable breast cancer patients was studied as a possible way of selecting patients for immunotherapy. METHOD: Sentinel node biopsy was performed in 24 out of 25 women operated on for primary breast cancer (one was not evaluable). Specimens were snap-frozen and double-stained for the zeta-chain of the T-cell receptor. The degree of down-regulation of the zeta-chain was evaluated in three different lymph-node areas: primary follicles, secondary follicles, and paracortex. RESULTS: Down-regulation of varying degrees was noted in all 24 sentinel node biopsies. A high degree of down-regulation (more than 50% of T-cells not expressing zeta-chain) was seen in the primary follicles in six patients (25%), in the secondary follicles in 13 patients (72%), and in the paracortex in 19 patients (79%). CONCLUSION: Local down-regulation of an immune function parameter was seen in sentinel node biopsies from breast cancer patients. In addition to possible prognostic implications, the sentinel node might be an appropriate location for detecting early-stage immunological down-regulation, which might open a possibility of selecting patients who could benefit from immunotherapy.