Prognostic value of serial determinations of circulating immune complex levels in malignant melanoma

Summary A total of 50 melanoma patients free of distant metastatic disease and 54 healthy controls were analyzed for circulating immune complexes (cIC) and complement split product (C3d), using solid-phase C1q-anti-IgG radio-immunoassay (RIA), C1q-protein A RIA, and anti-C3d anti-IgG RIA for cIC detection. No significant differences in cIC and C3d levels could be demonstrated between the controls and the 31 patients with primary malignant melanoma analyzed before surgery. To evaluate the prognostic value of serial measurements, samples from the 50 patients were taken at regular intervals for 4 to 27 months (median, 20 months). Surgery was the only treatment given. Significant changes in the cIC and C3d levels were defined by reference to the changes that occured in 23 of the 54 healthy controls observed for a period of 6 to 55 months (median, 23 months). During the period of serial sampling, recurrent disease developed in 8 of the patients. In only 3 of these 8 patients (versus 10 of 42 patients without recurrence) did significant changes occur, and the changes occurred either at the same time or after the clinical diagnosis of recurrence. During the entire clinical observation period of 6 years, a total of 11 patients developed recurrences. Significant changes were only observed in 4 of these 11 patients versus 8 of 37 patients without recurrence. In conclusion, changes in cIC and/or C3d levels were not found to be indicative of early or long-term recurrence of malignant melanoma.     

On down-regulation of the immune response to metastatic malignant melanoma

Treatment of metastatic malignant melanoma with interferon alpha (IFNalpha) results in objective remission in approximately 15% of patients. In a previous investigation, we found that about 50% of the patients achieved at least minor or short-lived remissions. In some tumours extensive areas of regressive tumour change occurred. However, even in these areas remnants of tumour cells were generally found. The short duration of the immune response in some patients and the incomplete eradication of the tumour can be due either to selection of non-immunogenic tumour cells or to down-regulation of the immune reactivity to the tumour. In the present paper, the expression of the zeta chain of the T cell receptor in CD3+ lymphocytes and the expression of CD28 in CD3+, CD4+ and CD8+ lymphocytes was studied in resectable melanoma metastases from 20 treated (IFNalpha or IFNalpha in combination with cisplatinum and dacarbazine) and 16 untreated patients. A double-staining technique was used, and the occurrence and distribution of lymphocytes showing down-regulation of the zeta chain or CD28 were separately registered in different areas of the metastases: close to the tumour cells in areas of unaffected tumour growth, in areas with regressive tumour changes, in areas with marked fibrosis and in stromal areas with densely packed lymphocytes. CD3+ zeta lymphocytes were found in all metastases, but their number and distribution varied considerably. Down-regulation of the zeta chain was most often found in areas of regressive changes. In contrast, T lymphocytes infiltrating close to the tumour cells had a stronger expression of the zeta chain (P = 0.016). Down-regulation was also found in stromal areas of densely packed lymphocytes and in areas of fibrosis. The pattern of down-regulation of CD28 in various subsets of lymphocytes was similar to that of zeta chain. The same pattern of down-regulation of CD28 and the zeta chain was found in both untreated and treated patients, indicating that the down-regulation is not due to treatment but to the release of immunosuppressor factors from areas with high tumour cell density or extensive destruction of tumour cells. These results concur well with the view that IFNalpha treatment can result in immune-mediated tumour cell destruction early in the treatment period and that this immune response to the tumour can be followed by immunosuppression within a few weeks.     

Serial immune testing in surgically resected lung cancer patients

Summary Immunoregulation of phytohemagglutinin (PHA) responsiveness by glass-adherent cells and prostaglandin-synthesizing cells was serially monitored in the peripheral blood mononuclear cells (PBMC) of surgically resected stage I and stage II lung cancer patients entered on a trial of adjuvant immunotherapy. The relationship between immunoregulatory cell function, immunocompetence, and disease relapse was determined. Immunoregulatory activity was measured in PHA-stimulated cultures in the presence and absence of 2 g/ml indomethacin and in the presence and absence of glass-adherent cells. In each instance of disease relapse seen, an increase in immunoregulatory cell function to a level significantly different from normal was observed 3 months prior to or coincident with clinical confirmation of disease recurrence. This was usually associated with a decline in PHA responsiveness. In the patients who did not relapse, the levels of PHA responsiveness and immunoregulatory function persisted within normal limits throughout the course of study. Percentages and numbers of leukocytes and leukocyte subsets and delayed cutaneous hypersensitivity were also monitored in this study, but could not be consistently correlated with early changes in clinical disease status. These data suggest that the development of indomethacin-sensitive and glass-adherent suppressor cells may precede and predict for tumor recurrence in surgically resected lung cancer patients.     

High levels of IgA-containing circulating immune complex and secretory IgA in Kawasaki disease

Sera of patients with Kawasaki disease were studied for the levels of IgA-containing (C3-fixing) circulating immune complexes (IgA-CIC), IgG-containing (IgG-)CIC, total IgA, secretory IgA, and complement component (C3) by means of enzyme-linked immunosorbent assays or single radial immunodiffusion methods. There was significantly high level of IgA-CIC, but not IgG-CIC. The levels of total IgA, secretory IgA, and C3 were significantly elevated. Significantly high levels of secretory IgA were found in 22 (51%) of 43 patients. The proportion of secretory IgA to total IgA also increased. These abnormalities in the IgA system may play a role in Kawasaki disease.     

Molecular markers in circulating tumour cells from metastatic colorectal cancer patients

The prognosis of metastatic cancer patients is still largely affected by treatment failure, mainly due to drug resistance. The hypothesis that chemotherapy might miss circulating tumour cells (CTCs) and particularly a subpopulation of more aggressive, stem‐like CTCs, characterized by multidrug resistance, has been recently raised. We investigated the prognostic value of drug resistance and stemness markers in CTCs from metastatic colorectal cancer patients treated with oxaliplatin (L‐OHP) and 5‐fluoruracil (5‐FU) based regimens. Forty patients with metastatic colorectal cancer were enrolled. CTCs were isolated from peripheral blood and analysed for the expression of aldheyde dehydrogenase 1 (ALDH1), CD44, CD133 (used as markers of stemness), multidrug resistance related protein 5 (MRP5 used as marker of resistance to 5‐FU and L‐OHP) and survivin (used as a marker of apoptosis resistance). CTCs were found in 27/40 (67%) patients. No correlation was found between the expression of either CD44 and CD133 in CTCs and the outcome of patients, while a statistically significant shorter progression‐free survival was found in patients with CTCs positive for the expression of ALDH1, survivin and MRP5. These results support the idea that isolating survivin and MRP5⁺ CTCs may help in the selection of metastatic colorectal cancer patients resistant to standard 5‐FU and L‐OHP based chemotherapy, for which alternative regimens may be appropriate.     

Serial tests of the change in concentration of circulating immune complexes in healthy subjects and in tumor patients

Authors performed by using various tests serial examinations of the circulating immune complex (IC) concentration in healthy persons, untreated rats, as well as in patients with multiple myeloma or leukemia, and in immunocytoma bearing rats. A characteristic fluctuation of the IC values has been observed in the untreated rats and the healthy persons. The discrepant IC values obtained in the identical sample with the different tests suggest the heterogeneity of IC components. As compared to healthy individuals a significantly higher fluctuation of IC values has been found characteristic in immunocytoma. Authors analyzed the composition of IC and discussed the possible importance of the tumor specific antigens and the natural antibodies reacting with them.     

Recovery of a cell surface fetal antigen from circulating immune complexes of melanoma patients

Summary A well-characterized 69.5×10³ dalton glycoprotein fetal antigen (FA), isolated from the spent culture medium of a melanoma cell line, UCLA-SO-14 (M14), was utilized to characterize the antigen component of circulating immune complexes (CIC) from melanoma patients. Ten serum samples from five patients with stage II melanoma at 1 and 4 months prior to the clinical detection of recurrent disease were selected for study. The CIC were dissociated with low pH and ultrafiltered through a 100¢10³ dalton exclusion limit membrane. The low pH treatment resulted in an increase in antibody titer in eight of ten serum samples. The antibody activity in membrane immunofluorescence was quantitatively inhibited by the filtered antigen fraction and purified FA, suggesting the presence of anti-FA antibodies in the treated serum, which possibly were complexed with FA in the untreated sample. As determined by competitive inhibition in an enzyme-linked immunosorbent assay, the filtrate (antigen fraction) contained an antigen that was immunologically similar to FA. These results clearly demonstrate that FA, expressed on the cell surface of melanoma cells, is present in CIC of selected melanoma patients.Supported in part by NIH grants CA 30019, CA 12582, CA 09010, CA 29605 awarded by DHSS     

Highly sensitive proximity mediated immunoassay reveals HER2 status conversion in the circulating tumor cells of metastatic breast cancer patients

Background

Strawberries (Fragaria ananassa) reproduce asexually through stolons, which have strong tendencies to form adventitious roots at their second node. Understanding how the development of the proximal (I-1) and distal (I-2) internodes of stolons differ should facilitate nursery cultivation of strawberries.

Results

Herein, we compared the proteomic profiles of the strawberry stolon I-1 and I-2 internodes. Proteins extracted from the internodes were separated by two-dimensional gel electrophoresis, and 164 I-1 protein spots and 200 I-2 protein spots were examined further. Using mass spectrometry and database searches, 38 I-1 and 52 I-2 proteins were identified and categorized (8 and 10 groups, respectively) according to their cellular compartmentalization and functionality. Many of the identified proteins are enzymes necessary for carbohydrate metabolism and photosynthesis. Furthermore, identification of proteins that interact revealed that many of the I-2 proteins form a dynamic network during development. Finally, given our results, we present a mechanistic scheme for adventitious root formation of new clonal plants at the second node.

Conclusions

Comparative proteomic analysis of I-1 and I-2 proteins revealed that the ubiquitin-proteasome pathway and sugar-hormone pathways might be important during adventitious root formation at the second node of new clonal plants.     

Fine needle aspiration cytology of myxoid metastatic malignant melanoma

The macroscopic and microscopic findings in a fine needle aspirate of a myxoid metastatic malignant melanoma are presented. The macroscopically mucoid material and the microscopically normal appearance of the cells in the myxomatous background were pitfalls while the presence of a melanin-positive pigment was a clue to the correct diagnosis.     

Evidence for immune defects in breast and lung cancer patients

Immunosuppression is often identified in cancer patients. The aim of this study was to evaluate several immune parameters for patients with breast and lung cancer. Immunophenotyping analysis showed that the cancer patients investigated had significantly lower absolute numbers of peripheral blood lymphocytes than controls. The immunosuppression was more evident for the breast cancer subgroup. The most severe immune defect noticed was the marked impairment of IFN- secretion. A shift toward the Th2 phenotype as revealed by assessment of intracellular level of IFN- and IL-4 was also noticed. The secretion of proinflammatory cytokines IL-1 and TNF- in whole blood cultures was not impaired. Although the proportion of activated cells was slightly lower than in the control group, our results showed that both peripheral T lymphocytes and NK cells of cancer patients could be induced to express early activation marker CD69 after ex vivo mitogen stimulation. In conclusion, our study revealed several immune defects in cancer patients. This suggests that an appropriate immunotherapeutical approach might be used to restore compromised immune functions with beneficial effects on both antitumor and general immunity.     

Circulating immune complexes in patients with breast cancer.

In a retrospective study in women with breast cancer circulating immune complex levels were measured by radioimmunoprecipitation with 125I-Clq. Before operation all the patients showed plasma immune complex levels significantly higher than those in controls. Twelve months after mastectomy patients identified clinicopathologically as having a good prognosis had almost normal levels of immune complexes. By contrast, patients with detectable dissemination on diagnosis or those who died within 22 months after mastectomy had significantly raised plasma levels. The tumour-specific nature of the immune complexes detected remains to be shown and suggestions about the applicability of this test not only for prognosis but also for monitoring the course of malignant diseases need to be confirmed by further investigations.     

Prognostic value of assays for circulating immune complexes and natural cytotoxicity in malignant skin melanoma (stages I and II)

Summary The aim of the present study was to determine the possible prognostic value of immune complex determinations and estimations of natural cytotoxicity in melanoma patients. Circulating immune complexes were assayed in 46 patients suffering from malignant skin melanoma (stages I and II) by a direct complement consumption (CC) test, a polyethylene glycol (PEG) precipitation CC assay and a solid-phase Clq-protein A-binding assay. The presence of both IgG- and IgM-containing complexes in IC-positive melanoma sera was confirmed by an Ig-class-specific precipitation-immunoradiometric assay. Results based on this assay and reduction-alkylation data indicated that IgM-containing IC were responsible for a major part of the IC activity recorded in the CC-assays.Soluble immune complex activity, as measured by the direct CC test and the PEG-CC assay, correlated with relapse, whereas the expected inverse relationship between natural cytotoxicity and recurrence was not established. This finding may be explained by the apparent predominance of IgM-containing immune complexes in patients subsequently showing relapse.     

Antigens in immune complexes from patients with breast cancer

Summary Sera and effusion fluids of patients with breast cancer (BC) contain immune complexes (IC). Antigens present in these complexes were isolated as follows: a pool of effusions from patients with BC was fractionated with ammonium sulfate. The proteins precipitating at 40% saturation were further fractionated by filtration through a Sephadex G-200 column. The material recovered in the first peak (molecules larger than monomeric IgG) was brought to pH 3.0 to dissociate the IC, and the mixture was filtered through a column of Sephacryl S-300 at pH 3.0. Proteins smaller than monomeric IgG were collected, radioiodinated, and used as antigens (¹²⁵Ag) to search for corresponding antibodies in sera of patients with BC (BCS) and of healthy individuals (NHS). ¹²⁵Ag was reacted with the sera and the immune complexes obtained were precipitated with an antiserum to human Ig and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Both NHS and BCS contained antibodies against two antigens; one of these appeared as a strong band of 17KD, the other as a doublet of approximately 25KD. It is concluded that some of the proteins in the IC from patients with BC are auto-antigens. No BC-specific antigens were identified.