Francesco Lisa

The relevance of the CD4+ CD26- subset in the identification of circulating Sézary cells.

Background  The lack of specific markers for the phenotyping of circulating neoplastic T cells in Sézary syndrome (SS) patients makes it difficult both to ascertain the presence of clonal cells and to quantify the tumour burden in the peripheral blood. In previous reports we showed that the lack of CD26 (dipeptidyl‐aminopeptidase IV) is a characteristic feature of circulating Sézary cells (SC).

Prognostic factors in Sézary syndrome: A multivariate analysis of clinical, haematological and immunological features

BACKGROUND Sézary syndrome (SS) prognostic factors are not well defined because of the rarity of this disease. The specific goal of this prospective study was to assess by multivariate analysis the predictive value with respect to survival of a series of clinical, haematological and immunological parameters taken at SS diagnosis. PATIENTS AND METHODS A cohort of 62 SS patients diagnosed and followed since 1975 was examined, and 51 were included in the multivariate analysis model. RESULTS The median survival time was 31 months (range: 1 month-15.7+ years), and the five-year survival rate 33.5%. The following variables were found by univariate analysis to be associated with a poor prognosis at the time of SS diagnosis: previous history of mycosis fungoides (P = 0.013), high number of circulating leukocytes (P = 0.001), Sézary cells (SC) (P < 0.001) and CD4+ cells (P < 0.001), presence of large circulating SC (P < 0.001), above normal range LDH serum levels (P = 0.015), presence of PAS-positive inclusions in the cytoplasm of circulating SC (P < 0.001), high CD4/CD8 ratio (P = 0.004) and a CD7 negative circulating SC phenotype (P < 0.001). Among them, the stepwise multivariate analysis selected as adverse independent prognostic factors: PAS-positive cytoplasmic inclusions (P = 0.001), CD7 negative phenotype (P = 0.018) and presence of large circulating SC (P = 0.045). CONCLUSIONS Two low-/high-risk groups have been singled out on the basis of the risk index. Patients with no or one adverse prognostic feature(s) (risk index < or = 1; n = 31) share a slow disease course and a relatively favorable prognosis (five-year survival: 58%); on the other hand, patients with 2 or 3 adverse prognostic feature (risk index > 1; n = 20) are characterized by an aggressive disease course not modifiable by traditional therapies (five-year survival: 5%).

Collagenase digestion and mechanical disaggregation as a method to extract and immunophenotype tumour lymphocytes in cutaneous T-cell lymphomas.

Various enzymatic or mechanical methods have been proposed in the past to dissociate cells from different solid tissues. An automated mechanical disaggregation device (Medimachine™) has recently been proposed. Unfortunately, most of these techniques are associated with a high cellular damage and a low cell recovery and are difficult to apply to skin biopsies. In this paper, we propose a combined enzymatic and mechanical method based on Medimachine™, useful for the isolation of skin infiltrating T‐lymphocytes from small cutaneous biopsies. As this method is easy and allows for a more correct qualitative and quantitative cytofluorimetric analysis of the lymphocyte subsets, it may be useful in the immunophenotyping of cutaneous T‐cell lymphomas.

Thymostimulin therapy in melanoma patients: Correlation of immunologic effects with clinical course

Thirty-two nonmetastatic melanoma patients with low T-lymphocyte values were treated with a thymic extract, thymostimulin (TS) (8 patients), DTIC (8 patients), or surgery alone (16 patients). In the 8 patients receiving TS, active E-rosette (T-Ea) and total E-rosette (T-Et) counts rose to normal levels and there was a significant rise in IgM and IgD receptors. Six out of eight patients treated with TS showed no evidence of metastases after 34 months, while 7/8 patients on DTIC and 13/16 patients on surgery alone developed metastases. Twenty metastatic patients with low T-lymphocyte values received either DTIC plus TS or DTIC alone. Total lymphocyte, T-Ea, and T-Et counts did not increase in either group nor was there a significant difference between the group on DTIC plus TS and the group on DTIC alone. The survival rate of patients on DTIC plus TS did not differ significantly from that on DTIC alone.

The prognostic value of T-lymphocyte levels in malignant melanoma. A five-year follow-up.

Serial immunologic tests (active E‐rosettes = T‐Et; total E‐rosettes = T‐Et; total lymphocytes and null cells) were performed every 3 months for 5 years on 113 melanoma patients. A significant reduction in absolute T‐Ea, T‐Et, null cells, and total lymphocytes was noted in the patients who died, by comparison with those who are still alive. The latter presented a significant reduction in absolute T‐Et only, plus a significant increase in null cells when compared with normals. The 38 patients without metastases, at the end of the study, presented a reduction in T‐Et and an increase in null cells compared with the normals, while the 75 patients with metastases presented a reduction in T‐Et, null cells and total lymphocytes when compared with the patients without metastases and a reduction in T‐Ea, T‐Et, and total lymphocytes when compared with the normals. Null cells show a linear decrease in patients who died and a linear increase in those who survived. A total of 80.2% of patients with a fall in T‐Et displayed metastases usually within 2 to 10 months (mean, 6.8). Patients with normal T‐Ea, T‐Et, and total lymphocyte values showed a significant prolonged survival when compared to those with lower values. In addition, survival seemed to be always a function of immunologic test values, irrespective of the tumor site.

Macrophage-mediated immunostimulation modulates therapeutic efficacy of interleukin-2 based chemoimmunotherapy in advanced metastatic melanoma patients.

&NA; The biological mechanisms of chemoimmunotherapy efficacy in vivo have not been fully clarified; furthermore, few data are available to predict its efficacy on the basis of clinical and immunological pretreatment factors. In this paper, pre‐ and post‐treatment serum levels of cytokines (interleukin [IL]‐6, IL‐10, IL‐12 and neopterin) and soluble IL‐2 receptors (sIL‐2R), as well as circulating levels of T‐cell and NK subpopulations, were analysed according to clinical outcome in 66 advanced metastatic melanoma (MM) patients treated with subcutaneous IL‐2 in association with interferon‐&agr;, cisplatin and tamoxifen. Our purpose was to correlate the immune modifications during treatment with the clinical response and to define pretreatment factors with predictive value for clinical outcome. The overall response rate was 35%, with a median overall survival of 11.3 months. During treatment, responding patients showed a common marked increase in IL‐12 (mainly released by activated macrophages), sIL‐2R and neopterin serum levels, associated with high levels of total lymphocytes and circulating natural killer lymphocytes; progressing patients were characterized by an increase in IL‐6 serum levels (directly related to the increase in tumour burden). Multivariate analysis showed that high pretreatment IL‐12 levels (P = 0.05) and, to a lesser extent, lactate dehydrogenase levels in the normal range (≤ 450 U/I; P = 0.061) are independent favourable prognostic factors for survival. Our results show that macrophage activation in an immunostimulating way either before or during treatment is associated with a better clinical response and improved survival in advanced MM patients treated with IL‐2‐based chemoimmunotherapy.

Soluble lnterleukin-2 Receptor, CD4 and CD8 Levels in Melanoma A Longitudinal Study

Serum levels of soluble(s) interleukin-2 receptor (IL-2R), sCD4 and SCD8 were analysed in 227 melanoma patients, using a sandwich enzyme immunoassay. Different stages of the disease were considered, and a longitudinal study with a 2-year follow-up was performed. Mean values of sIL-2R were significantly higher than in normal controls in all stages and correlated with the disease progression. sCD8 increases with stage progression were less striking, while sCD4 values were always in the normal range. We conclude that sIL-2R measurement is a useful clinical parameter in monitoring disease evolution in melanoma.

Functional properties in Sézary cells with an unusual phenotype

The immunological and functional characteristics of Sézary cells with an unusual phenotype are reported. The clinical, histologic, and hematologic picture was typical for Sézary syndrome. Studies with monoclonal antibodies showed that 80% Sézary cells had an CD3+, CD4+, CD5+, CD7-, CD8-, Leu-7+, Leu-8-, Leu-11-, OKM1- phenotype. By two-color immunofluorescence assay 80% FACS-sorted Leu-7+ cells coexpressed CD4 antigen and did not express the myeloid antigen OKM1, CD8, and antigens characteristic of immature T cells. The cells had no NK activity but did display a high helper activity. Unseparated and FACS-sorted Leu-7+ and Leu-7- Sézary cells did not respond to mitogens but were able to grow in the presence of exogenous IL-2. FACS sorted Leu-7- cells, cultured for 7 days in the presence of 20% IL-2, acquired the receptors for Leu-7. IL-2 and IFN-gamma production was studied in unseparated Leu-7+ and Leu-7- FACS-sorted Sézary cells. IL-2 production was lower than in normal cells. The addition of PHA or PHA plus TPA led to an increase in IL-2 production. Also IFN-gamma production was marked lower than in normal controls but increased after 7-day culture in exogenous IL-2. In conclusion in this case the Sézary cells may represent a neoplastic expansion of the CD3+, CD4+, CD5+, Leu-7+, Leu-11- subpopulation which is equivalent to the 2-4% of the Leu-7+ population in normal lymphocytes.

In Vitro and In Vivo Effect of Thymostimulin in Melanoma Patients

In addition to skin tests, numerous in vitro methods have been employed for the evaluation of cell-mediated immunity in melanoma. Mitogens such as phytohemagglutinin (PHA), PPD and concanavalin A (ConA) have proved to be of little use in most cases of melanoma.1–5 Also, lymphocyte microcytotoxicity tests, while displaying a certain difference between metastatic and non-metastatic patients, have provided inconsistent results with respect to prognosis.6–9

ALK expression favorably impacts the prognosis of NRAS‑mutated metastatic melanomas

Recent studies reported the expression of anaplastic lymphoma kinase (ALK) in malignant melanomas. The aim of this study was to investigate whether ALK expression is associated with specific clinical and molecular characteristics of melanoma metastases, and to evaluate its correlation with survival outcomes. Seventy-one patients with metastatic melanoma were investigated. Clinical features and survival outcomes were analyzed and correlated to ALK expression, as detected by immunohistochemistry and reverse transcription-quantitative polymerase chain reaction, and to the mutational status of BRAF, KRAS, NRAS, and PIK3CA. No translocations or ALK alternative isoforms were identified. ALK expression was mainly detected in NRAS mutated metastatic lesions. Interestingly, among NRAS-mutated patients, ALK positive samples displayed a significantly more favorable outcome in terms of disease specific survival, as compared to ALK negative ones. In conclusion, we suggest that ALK positive/NRAS mutated metastases represent a specific subset of metastatic melanomas, associated with a better prognosis. Validation of these observations in larger cohorts could contribute to understand the molecular events cooperating to melanoma progression, in addition to open new perspectives in the clinical and therapeutic management of this subgroup of patients.