Increased Tumour Infiltration of CD4+ and CD8+ T-Lymphocytes in Patients with Triple Negative Breast Cancer Suggests Susceptibility to Immune Therapy

Background: Patients with triple negative breast cancer (TNBC) have limited therapeutic options, largely because the complex tumour environment is not well-characterized. These patients are potential, but largely un-fathomed, candidates for immunotherapy. It is therefore highly relevant to characterize leukocyte complexity in TNBCs. Objective: To investigate leukocyte complexity in tumour environment of patients with TNBCs. Materials and methods: A total of 104 consecutive breast cancer patients undergoing mastectomy were recruited in the study after ethical approval. Clinico-pathological parameters were recorded and H and E staining was performed to investigate tumour morphology. Receptor status was investigated using antibodies against ER, PgR and Her-2, and patients were classified as having TNBC or non-TNBC tumours (including Luminal A, Luminal B and Her2 overexpressing tumours). Immune-cell infiltration was investigated using special stains and antibodies: α-CD3 (T-lymphocytes), α-CD20 (B-lymphocytes), α-CD4 (helper T-lymphocytes) and α-CD8 (cytotoxic T-lymphocytes). Immune cell densities were quantified as cell/ mm2 using the CAP guidelines. Results: Of the 104 breast cancer patients investigated, a total of 27 (26%) had TNBC and 77(74%) non-TNBC. Patients with TNBC showed significantly increased tumour infiltration of lymphocytes (T and B-lymphocytes) compared to the patients with non-TNBC, while myelocytic infiltration was not significantly different in the two groups. Within the TNBC group, infiltration of T-lymphocytes (equal densities of CD4+ and CD8+ T-lymphocytes) was significantly higher compared to B-lymphocytes. Conclusion: Patients with TNBC show increased lymphocytic infiltration (more T-lymphocytes compared to B-lymphocytes). This suggests higher immunogenicity of TNBCs and may indicate a higher responsiveness of these cancers to immunotherapy.     

Serum hyaluronate in malignant pleural mesothelioma

The diagnostic value of hyaluronate concentration in effusions of malignant mesothelioma has been extensively reported but no information is available about serum hyaluronate in patients with this cancer. Using a new enzymoimmunologic assay based on hyaluronate-hyaluronectin interaction, serum levels of hyaluronate were measured in 16 patients with malignant pleural mesothelioma, 50 patients with other pleural effusions, and 94 healthy blood donors. The mean serum hyaluronate level in patients with mesothelioma (mean, 750 micrograms/l; range, 29 to 5833 micrograms/l) was significantly higher than in patients with other pleural effusions (mean, 56 micrograms/l; range, 4 to 137 micrograms/l) and than in blood donors (mean, 24 micrograms/l; range, 0 to 94 micrograms/l). Comparison of serum hyaluronate values observed in mesotheliomas with the clinical course of the disease suggests that serum hyaluronate might increase only at an advanced stage of the cancer. Therefore, serum hyaluronate determination has probably no clinical value for early detection of malignant mesothelioma, but might be useful to evaluate the clinical course of this malignancy.     

A reevaluation of B-lymphocyte levels in peripheral blood from cancer patients.

B-lymphocytes were quantitated in mononuclear cell suspensions derived from the peripheral blood of patients with various nonlymphoreticular cancers. The method used was anti-IgM and anti-IgD membrane immunofluorescence. The mean percentage of circulating B-lymphocytes in 78 cancer patients tested was 5.3 +/- 4.6 with a range of 0--18%. Those values were compared with a mean of 9.4 +/- 4.0 and a range of 3--20% for 46 apparently normal individuals. The difference was highly significant (P less than or equal to 0.001). The mean percentage of B-cells in cell suspensions from 43 patients that were tested prior to treatment was 5.8 +/- 4.8 with a range of 0--18%. Very low values were observed both in the presence and absence of therapy, and a correlation with stage of disease could not be established. The low values were associated with decreased T-cell numbers and significantly increased monocyte levels. The fact that those values were significantly lower than have been reported previously for cancer patients was discussed as was the identity of the cells that previously had been counted as B-lymphocytes.     

Mechanism for the recruitment of macrophages to cancer site. In vivo concentration gradient of monocyte chemotactic activity.

BACKGROUND. Tumor stroma is characterized by the development of new blood vessels, an inflammatory cell infiltration, and a fibrotic reaction. The inflammatory component of tumor stroma plays an important role in the modulation of tumor expansion. In this respect, macrophages constitute a major part of the inflammatory cell infiltration and can exert cytotoxic activity against tumor cells. The accumulation of macrophages in the vicinity of the tumor suggests their recruitment from circulating blood monocytes through the local release of chemotactic factors for monocytes. METHODS. To detect the existence of a concentration gradient of monocyte chemotactic activity (MCA) between the tumor vicinity and blood vessels, malignant pleural effusions defined by the local presence of cancer cells were evaluated for quantification of MCA. RESULTS. Unlike nonmalignant pleural effusions, malignant pleural effusions were characterized by the presence of increased levels of MCA, and in lung adenocarcinoma (a cancer with high inflammatory cell infiltration), pleural levels of MCA were significantly greater than in small cell lung carcinoma (a cancer with low inflammatory cell reaction). An MCA concentration gradient between pleural fluid and plasma was present in malignant effusions because pleural MCA levels in all cancer types were significantly greater than MCA levels in the plasma of the same patients. CONCLUSIONS. Thus, an increased local level of MCA is a feature of cancers with high inflammatory cell infiltration, and the presence of an in vivo concentration gradient of MCA suggests the direct role of this biologic activity in recruiting blood monocytes to the cancer site.     

Altered CD94/NKG2A and perforin expression reduce the cytotoxic activity in malignant pleural effusions

CD94/NKG2A is an inhibitory receptor expressed by NK cells and cytotoxic lymphocytes and, upon activation by HLA-E, downregulates the cytolytic activities of these cells thus representing a tumour immune escape mechanism.This study was aimed at assessing whether cytotoxic lymphocytes (CD8+) and NK cells from malignant pleural effusions have a deregulated expression of CD94/NKG2A.The expression of membrane CD94/NKG2A and perforin was evaluated by flow-cytometry in CD8+ and NK cells from pleural effusions and autologous peripheral blood of cancer (n = 19) and congestive heart failure (CHF) (n = 11) patients. Intracellular CD94/NKG2A expression was evaluated by flow-cytometry in pleural effusion CD8+ and NK cells from cancer patients (n = 10). Cytotoxic activity against cancer cells exerted by pleural and autologous peripheral blood T lymphocytes from cancer patients was assessed by flow-cytometry assay.Pleural CD8+ from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood and CHF pleural effusions. Reduced numbers of NK cells were present in pleural effusions from both cancer and CHF patients. Pleural NK from cancer patients showed a reduced expression of membrane CD94/NKG2A and perforin when compared to autologous peripheral blood. Pleural T lymphocytes from cancer patients exhibited a reduced cytotoxic activity against cancer cells when compared to autologous peripheral blood T lymphocytes. The intracellular expression of CD94/NKG2A in CD8+ and NK cells from cancer patients was higher than membrane expression.In conclusion, this study provides compelling evidences of new mechanisms underlying the reduced host defence against cancer within the pleural space.     

Lymphocyte response to pokeweed mitogen in chronic lymphocytic leukemia

The response of lymphocyte subpopulations to pokeweed mitogen (PWM) was studied in normal volunteers and patients with B-cell chronic lymphocytic leukemia (CLL). Since unfractionated peripheral blood mononuclear (PBM) cells from CLL patients consist of a markedly increased proportion of B-lymphocytes and a decreased proportion of T-lymphocytes, enriched fractions of CLL B-cells and CLL T-cells were cultured in 1:1 proportions in autologous and allogeneic combinations with normal B-cell and T-cell-enriched fractions. Cultures containing normal B-cells with either autologous or allogeneic normal T-cells responded well to PWM. CLL T-cells were capable of providing a helper function for both proliferation and differentiation of normal B-cells, which was not significantly different from that provided by allogeneic normal T-cells. CLL B-lymphocytes were unresponsive to PWM when cultured in the presence of either autologous CLL T-lymphocytes or allogeneic normal T-lymphocytes. The responsiveness of CLL B-cells was not restored by the addition of normal peripheral blood monocytes to the cultures. These experiments indicate that there is an intrinsic B-cell defect which prevents CLL B-lymphocytes from responding to PWM.     

Malignant pleural effusions: meaning of pleural-fluid pH determination

In 36 patients with malignant pleural effusions, we determined the pH and the glucose concentration of the pleural fluid. Twenty-one of 36 patients (58.3%) had a low pH (less than 7.30) and 15 had a normal pH (greater than or equal to 7.30; 7.13 +/- 0.12 vs. 7.37 +/- 0.05; p less than 0.0005). The patients with low pH had significantly lower glucose concentrations than those with normal pH (2.7 +/- 1.4 vs. 6.3 +/- 2.9 mmol/l; p less than 0.0005). Twenty-one of 34 patients (61.7%) had a glucose concentration lower than a cut-off value of 4.4 mmol/l; of these, 17 (81%) had a low pH. The mean survival in the low-pH group was 4.8 +/- 4.4 months, whereas the mean survival in the normal-pH group was 5 +/- 8 months (p greater than 0.4). Twelve of 36 patients (33.3%) were treated with intrapleural Corynebacterium parvum (CBP) injections. Fourteen of 21 low-pH patients (66.6%) survived more than 2 months, and 4 of them are still alive. Six of 15 normal-pH patients (40%) survived more than 2 months, and 1 of them is still alive. Three of the 5 living patients were treated with CBP (2 in the low-pH group and 1 in the normal-pH groups). Our results confirm that pH and glucose concentrations in the pleural fluid of patients with malignant effusions are frequently low. However, the survival and the response to CBP pleurodesis in patients with low-pH malignant effusions are the same as those in patients with normal-pH malignant effusions.     

Evaluation of pleural CYFRA 21-1 and carcinoembryonic antigen in the diagnosis of malignant pleural effusions.

CYFRA 21-1 assay, measuring cytokeratin 19 fragments, was compared with carcinoembryonic antigen (CEA) assay, as an addition to cytological analysis for the diagnosis of malignant effusions. Both markers were determined with commercial enzyme immunoassays in pleural fluid from 196 patients. Cytological analysis and/or pleural biopsy confirmed the malignant origin of the effusion in 99 patients (76 carcinomas, nine pleural mesotheliomas and 14 non-epithelial malignancies). Effusions were confirmed as benign in 97 patients (33 cardiac failures, 39 infectious diseases--including 12 tuberculosis-- and 25 miscellaneous effusions). Both markers were significantly higher in malignant than in benign effusions. All the patients with non-epithelial malignancies presented CYFRA and CEA values lower than the 95% diagnostic specificity thresholds (100 and 6 ng ml(-1) respectively). The diagnostic sensitivity in the group of carcinomas and mesotheliomas was similar for CYFRA (58.8%) and CEA (64.7%). However, CEA had a significantly higher sensitivity in carcinomas (72.4% vs 55.3%), while CYFRA had a clearly higher sensitivity in mesotheliomas (89.9% vs 0%). Interestingly, 12 out of the 16 malignant effusions with a negative cytology were CEA and/or CYFRA positive. Regarding their high diagnostic sensitivity and their complementarity, CEA and CYFRA appear to be very useful for the diagnosis of malignant pleural effusions when cytology is negative.     

Serum amyloid A in carcinoma of the lung

Serum concentrations of serum amyloid A protein (SAA), peripheral blood lymphocytes (PBL) mitogenic response to phytohemagglutinin (PHA) and Concanavalin A (Con A), numbers of circulating T- and B-lymphocytes and length of survival after diagnosis were measured in 50 patients with cancer of the lung. SAA levels were significantly elevated when compared to 50 control subjects (P less than 0.001), but did not correlate with state of tumor spread at the time of diagnosis. Mitogenic responses of PBL from the cancer patients to PHA and Con A were significantly depressed (P less than 0.001), but also did not predict state of metastatic spread. The percentage of circulating T-lymphocytes was also significantly depressed in cancer patients when compared to controls. In six patients who survived tumor-free for greater than 2.5 years, SAA serum concentrations returned to normal. Statistical analysis showed a significant correlation between SAA serum concentrations and PBL mitogenic response to Con A. In addition, both high SAA concentrations and depressed PBL responses to Con A correlated with shortened survival. Therefore, these parameters may be of value in evaluating prognosis in patients with lung cancer. In addition, serial monitoring of SAA concentrations may be of value in evaluating recurrence or cure of lung cancer.     

Tumor markers in pleural effusion diagnosis

In order to discriminate between malignant and benign effusions, the values of carcinoembryonic antigen (CEA), ferritin, beta2-microglobulin (BMG), acid-soluble glycoprotein (ASP), tissue polypeptide antigen (TPA), adenosine deaminase (ADA), and immunosuppressive acidic protein (IAP) were measured in the pleural fluid of 54 patients with lung cancer, 20 with malignancies other than lung cancer, 18 with tuberculous pleurisy, and 22 with benign diseases other than tuberculosis. CEA levels in malignant effusions were significantly higher than those in benign effusions. At a cutoff level of 5 ng/ml, 68% of the patients with lung cancer and 44% of the patients with other malignancies showed elevated pleural fluid CEA levels. In 13 lung cancer cases with negative pleural fluid cytology, nine cases had elevated pleural fluid CEA levels. The mean pleural fluid BMG level of patients with benign diseases was significantly higher than that of patients with malignant diseases, but there was a marked overlap between those with malignant and benign diseases. No significant differences were found in the pleural fluid ferritin, ASP, TPA, and IAP levels between malignant and benign conditions. ASP and IAP pleural fluid levels showed significant correlations with the pleural fluid C-reactive protein (CRP) concentrations suggesting that they also reflect inflammatory activity. The mean ADA activity in tuberculous effusion was significantly higher than that resulting from other causes of pleural effusion.     

Ratios of pleural fluid to serum immunoglobulins in malignant pleural effusions

Pleural fluid and serum protein electrophoresis and quantitative immunoglobulin measurements were carried out in patients with pleural effusions. The mean pleural fluid/serum ratios of IgA, IgG, and IgM were elevated in patients with malignant pleural effusions compared with patients with nonmalignant pleural effusions (P less than 0.04). The sensitivity of a pleural/serum IgA, IgG ratio P greater than 0.6 was 46%, 69%, respectively, and for IgM ratio greater than 0.5 was 28%. The specificity for these same ratios was 89%, 74%, and 100% respectively.     

T- and B-lymphocyte counts and their function in patients with primary liver cancer and alveococcosis

The levels of T- and B-lymphocytes were assayed and their functional activity studied in 23 patients with primary cancer and 21 cases of alveococcosis of liver. The lowered levels of T-lymphocytes in both groups and the decreased C3-fraction of B-lymphocytes in primary cancer of the liver were observed. Considerable changes in serum immunoglobulin system, i. e. an increased level of all immunoglobulins (G, A and M), are reported.     

Induction of DNA repair synthesis in human monocytes/B-lymphocytes compared with T-lymphocytes after exposure to N-acetoxy-N-acetylaminofluorene and dimethylsulfate in vitro.

We have explored the induction of DNA repair synthesis in monocyte/B- and T-lymphocyte enriched cell fractions from 12 different human mononuclear blood cell populations. Unscheduled DNA synthesis was measured in monocyte/B- and T-cells after exposure to the DNA-damaging agents dimethylsulfate (DMS) and N-acetoxy-N-acetylaminofluorene in vitro. Also, the binding of DMS to DNA was measured. An increased DNA repair synthesis was measured in monocyte/B-lymphocytes after induction of the two different types of DNA lesions, whereas no induction of unscheduled DNA synthesis was observed in T-lymphocytes. A significantly higher DMS-DNA binding was also observed in monocyte/B-lymphocytes when compared with T-lymphocytes. Specific characterization of mononuclear blood cell populations used in biomonitoring of DNA adducts and repair is recommended.     

Suppression of B-lymphocyte function by T-lymphocytes in patients with advanced lung cancer

The ability of B-lymphocytes to produce immunoglobulins in response to pokeweek mitogen stimulation was studied in 21 untreated stage III lung cancer patients by culture of their mononuclear cells in vitro. The number of immunoglobulin-producing cells was significantly lower in 20 of the 21 patients when compared to responses shown by normal control subjects. In contrast, the proliferative responses of many of the patients were within the normal range. When the T-lymphocytes of these patients were irradiated with 1,250 rad to eliminate the suppressor T-cell activity and then cultured with autologous B-cells, the number of immunoglobulin-producing cells was enhanced to the normal range in 7 of the 18 patients. These results indicate that B-cell function is impaired in most patients with advanced lung cancer. They also suggest that, in addition to suppression by radiosensitive suppressor T-cells, other mechanisms are involved in the observed B-cell functional abnormality.     

Vascular endothelial growth factor levels and induction of permeability in malignant pleural effusions.

Vascular endothelial growth factor (VEGF) is an important mediator of angiogenesis and vascular permeability. We hypothesized that malignant pleural effusions may contain high levels of VEGF protein as well as other cytokines implicated in these processes. Pleural effusions cytologically proven to be malignant were collected from 39 patients with various types of cancer, and VEGF, interleukin-8, and angiogenin levels in the effusions were determined by immunoassay. Negative controls were nonmalignant ascites and serum samples from healthy individuals. VEGF levels were significantly higher than those of control samples in pleural effusions secondary to breast, mesothelioma, and non-small cell lung cancer and when all malignant pleural effusion samples were pooled. Neither interleukin-8 nor angiogenin levels were elevated in malignant pleural effusions relative to the control samples. Vascular permeability, which was measured by using the Miles assay in nude mice, was increased proportionately with VEGF levels in the malignant pleural effusions; this increase in permeability induced by injection of recombinant VEGF or the malignant effusions was reduced by pretreating the mice with a VEGF receptor antibody.     

Lymphokine-activated killer induction and its regulation by macrophages in malignant pleural effusions

Mononuclear cells (MNC) from pleural effusions and peripheral blood of 18 patients with primary lung cancer with malignant pleural effusion were studied. Pleural and blood MNC generated lymphokine-activated killer (LAK) activity similarly when cultured for 4 days with an optimal concentration of interleukin 2 (IL-2). Highly purified lymphocytes (greater than 98%) and monocyte-macrophages (greater than 90%) were isolated by discontinuous Percoll gradient centrifugation from pleural and blood MNC. Pleural macrophages, as well as blood monocytes, showed significant augmenting effects on in vitro LAK cell induction from pleural and blood lymphocytes by IL-2. During daily intrapleural administration of IL-2, significant induction of LAK activity in vivo was observed after 3 days, but then this LAK activity in pleural MNC decreased almost to zero by day 15. Daily injections of IL-2 resulted in reduction in the up-regulation of LAK induction by pleural macrophages and also in increases in the levels of soluble IL-2 receptors in pleural effusions. These findings indicate that in vivo LAK induction of lymphocytes in malignant effusions by IL-2 may be regulated by macrophages in the effusions.     

Effect of IL-6 elevation in malignant pleural effusion on hyperfibrinogenemia in lung cancer patients

BACKGROUND: The involvements of interleukin-6 (IL-6) and fibrinogen in cancer development were elucidated independently, irrespective of IL-6 activity to induce fibrinogen. This study was undertaken to clarify the clinicopathological association of these molecules in lung cancer patients with malignant pleurisy. METHODS: IL-6, fibrinogen and the related molecules in blood and pleural effusion of 38 patients were assayed at 3-day intervals. RESULTS: IL-6 levels were elevated in sera of 27 cases (71.1%) and in all the effusions with mean values of 20.5 and 9970.5 pg/ml, respectively. Their correlation in 22 cases who were examined on the same day was statistically strong (r = 0.902, p < 0.0001). Occasional elevations of tumor necrosis factor-alpha were independent of IL-6 elevation. Levels of plasma fibrinogen, fibrin(ogen) degradation products (FDP) and C-reactive protein (CRP) were more frequently elevated in the IL-6-elevated cases than those without IL-6 elevation. In all pleural effusions, fibrinogen levels were significantly decreased to <150 mg/dl with large elevations of FDP level. Immunocytologically, IL-6 was detected in cancer cells in 16 cases of adenocarcinoma in addition to host pleural cells, but its cellular positivity was not reflected in the IL-6 level in each pleural effusion. CONCLUSION: Compared with lung cancer patients without malignant pleurisy, IL-6, fibrinogen, FDP and CRP levels in patients with malignant pleurisy were increased more frequently in their peripheral blood. These were basically attributed to systemic leakage of IL-6 from the affected pleural cavity, in which plasma fibrinogen induced in response to serum IL-6 was exudated and degraded predominantly to FDP.     

The cellular immune status in normal subjects, patients with and without neoplasms]

The distribution of lymphatic subpopulations in the peripheral blood of patients with malignant solid tumors has been studied in two steps. Firstly, it was investigated whether there are changes in the distribution of blood lymphoid cell populations in tumor patients (n = 101) compared to normal individuals (n = 39) and whether there is a correlation between the distribution pattern and the clinical stage of the disease. There was found a significant reduction of T-lymphocytes in patients with cancer, which was marked in the advanced tumor disease (n = 34). In a further step the influence of curative tumor-resection on the lymphatic subpopulations was studied, when 26 operated tumor patients were compared with 23 operated patients without neoplasia. In the group with non-malignant diseases there could not be found a significant pre- and postoperative difference concerning the T- and B-lymphocyte counts. Tumor patients showed after resection of the tumor a significant increase of the absolute and relative number of preoperatively reduced T-lymphocytes. The pathophysiological possibilities for the phenomena of a reversible reduction of T-lymphocytes by the curative resection of the tumor are discussed.     

Modulation of lymphocyte proliferation and immunoglobulin production by transferrin-gallium

Gallium resembles iron with respect to transferrin (Tf) binding and cellular uptake via Tf receptors. We have previously shown that transferrin-gallium (Tf-Ga) complexes interfere with the cellular incorporation of iron and inhibit the proliferation of HL60 cells. Since mitogen-stimulated peripheral blood lymphocytes express Tf receptors, we examined the effect of Tf-Ga on lymphocyte proliferation and on immunoglobulin synthesis by B-lymphocytes. Tf-Ga inhibited phytohemagglutinin, pokeweed mitogen, and tetanus toxoid-stimulated lymphocyte proliferation by greater than 50%, an effect which appeared to be cytostatic rather than cytotoxic. In cocultures of T-lymphocytes or CD4+ T-lymphocytes and B-lymphocytes, Tf-Ga also inhibited pokeweed mitogen-stimulated immunoglobulin production by 84 to 100%. Tf-Ga inhibited both T-independent Epstein Barr virus-stimulated B-lymphocyte proliferation and immunoglobulin production; however, these effects appeared to be independent of each other, since immunoglobulin production was inhibited by 75% by a concentration of Tf-Ga which did not uniformly inhibit proliferation. Tf-Ga is capable of targeting Tf receptor-bearing T- and B-lymphocytes and interfering with their proliferation and function. Such effects may be of relevance to patients being treated with this metal. The potential immunosuppressive activity of gallium warrants further investigation.