Decreased monocyte antibody-dependent cell-mediated toxicity in stage I–II malignant melanoma

Summary Lymphocyte and monocyte antibody-dependent cell-mediated cytotoxicity (ADCC) against human red blood cells was examined in 28 stage-I-II malignant melanoma patients. Eighteen were studied at various time intervals after receiving SC Corynebacterium parvum (C. parvum); 10 were untreated. Fifteen normal age-matched controls were also studied. Monocyte ADCC was significantly decreased in untreated patients compared with controls (P<0.005) and was significantly increased above controls and untreated patients in individuals treated with C. parvum (P<0.008). No significant differences in lymphocyte ADCC were seen. Optimal enhancement of monocyte ADCC by C. parvum occurred from 2 weeks to 1 month after treatment. Significant decreases in ADCC to baseline levels occurred in patients studied from 3 to 6 months beyond treatment.     

Controlled trial of active immunotherapy in management of stage IIB malignant melanoma.

The prognosis for patients who undergo surgery for stage IIB malignant melanoma is poor. Animal studies have suggested that BCG and tumour cell vaccines given together may provide effective immunotherapy. To assess the effectiveness of this treatment 15 patients with stage IIB malignant melanoma who had their tumour excised were studied. Seven were treated conservatively, and eight were vaccinated with BCG and autologous irradiated cells. Three vaccinated patients suffered widespread recurrence within three months. All four vaccinated patients who suffered a recurrence within the first year died, while none of the three controls with recurrent disease died. In view of this alarming trend the trial was stopped after a year. BCG and the tumour cells may have enhanced the tumour growth, although there was no apparent reason for this. The results of uncontrolled or unrandomised trials that have suggested that this treatment is beneficial should be treated with scepticism.     

Smooth Muscle Antibody in Malignant Disease

Smooth muscle antibody at titres of 1/10 or more was found in 54 (67·5%) out of 80 patients with malignant disease and in 9 (20%) out of 46 controls. The incidence of S.M. antibody ranged from 18/30 (60%) in malignant melanoma to 7/8 (83%) in carcinoma of the ovary. The presence of this antibody is possibly related to changes in the malignant cell membrane.A new antibody directed at an antigen presumed to be located in bile canaliculi among other sites is described.     

Postoperative Depression of Tumour-directed Cell-mediated Immunity in Patients with Malignant Disease

Leucocytes from 46 melanoma patients, 45 breast carcinoma patients, and 95 control donors were tested by the leucocyte migration test against the supernatants of homogenates of malignant melanomas, breast carcinomas, simple breast tumours, and breasts showing simple cystic disease. By comparison with controls inhibition of migration occurred significantly more frequently when tumour patients'' leucocytes were exposed to extracts of histogenetically similar tumours.Cell-mediated immunity to tumour-associated antigens was measured in 12 patients with breast carcinoma and 12 with malignant melanoma immediately before surgical operation and in the postoperative period. All patients tested before operation showed significant inhibition of migration on contact with extracts of histogenetically similar tumours. Postoperatively the degree of leucocyte migration inhibition was reduced in all patients with melanoma and breast carcinoma. Significant inhibition of leucocyte migration returned in most patients 6-22 days after operation.     

Affinity Proteomics Exploration of Melanoma Identifies Proteins in Serum with Associations to T-Stage and Recurrence

BACKGROUND: Blood-based proteomic profiling may aid and expand our understanding of diseases and their different phenotypes. The aim of the presented study was to profile serum samples from patients with malignant melanoma using affinity proteomic assays to describe proteins in the blood stream that are associated to stage or recurrence of melanoma. MATERIAL AND METHODS: Multiplexed protein analysis was conducted using antibody suspension bead arrays. A total of 232 antibodies against 132 proteins were selected from (i) a screening with 4595 antibodies and 32 serum samples from melanoma patients and controls, (ii) antibodies used for immunohistochemistry, (iii) protein targets previously related with melanoma. The analysis was performed with 149 serum samples from patients with malignant melanoma. Antibody selectivity was then assessed by Western blot, immunocapture mass spectrometry, and epitope mapping. Lastly, indicative antibodies were applied for IHC analysis of melanoma tissues. RESULTS: Serum levels of regucalcin (RGN) and syntaxin 7 (STX7) were found to be lower in patients with both recurring tumors and a high Breslow's thickness (T-stage 3/4) compared to low thickness (T-stage 1/2) without disease recurrence. Serum levels of methylenetetrahydrofolate dehydrogenase 1-like (MTHFD1L) were instead elevated in sera of T3/4 patients with recurrence. The analysis of tissue sections with S100A6 and MTHFD1L showed positive staining in a majority of patients with melanoma, and S100A6 was significantly associated to T-stage. CONCLUSIONS: Our findings provide a starting point to further study RGN, STX7, MTHFD1L and S100A6 in serum to elucidate their involvement in melanoma progression and to assess a possible contribution to support clinical indications.     

Direct cytotoxicity test applied to patients in stage I and II of malignant skin melanoma

Summary Using the direct cytotoxicity test described by Takasugi and Klein (1970), 59 patients suffering from localized malignant skin melanoma (stages I and II) were investigated for cell-mediated immunity. On an average, peripheral lymphocytes obtained preoperatively from 52 patients showed a higher cytotoxicity against one of two established melanoma cell lines (RPMI 7931) as compared to lymphocytes from control persons. No significant differences were found between lymphocytotoxicity of the patients and the controls against the control cell lines.In a prospective study, 17 patients were investigated. The results obtained so far suggest a correlation between a positive reaction postoperatively and recurrence of melanoma.It is clear that the control group exerted a certain degree of nonspecific cytotoxicity depending on the target cell used (established cell lines versus short-term cultures) and the lymphocyte/target cell ratio. Furthermore, there was a day-to-day variation in the nonspecific cytotoxicity exerted by lymphocytes derived from the same control person.It is concluded that considerable refinements have to be made before the microcytotoxicity assay becomes of clinical use in the evaluation of the postoperative status and course in melanoma patients.With the technical assistance of Marianne Barfod and Vibeke AhrenkielSponsored by the Danish Cancer Society.     

Recurrent malignant melanoma: effect of adjuvant immunotherapy on survival.

Twenty-nine patients referred consecutively to a cancer clinic because of recurrent metastatic malignant melanoma were given 5 mg of Connaught Laboratories bacillus Calmette-Guérin (BCG) by multiple cutaneous puncture at weekly and later at monthly intervals. Eight were also treated with autologous tumour vaccine and three with intralesional BCG. This group was compared with a retrospective control group of 54 patients treated with surgery and radiotherapy alone after recurrence. Prognostic features such as site of primary and of first metastasis, disease-free interval, age and sex were similar in the two groups. However, the median survival from the time of first recurrence was 12 months in the control group but 21 months in the BCG-treated group. The major improvement was in patients with disease limited to the regional lymph nodes: the median survival was 16 months in the control group but over 32 months in the BCG-treated group. Autologous tumour vaccine appeared to have no effect on survival. Serial testing of immunocompetence did not offer any prognostic advantage, although the results of some tests correleated well with extent of disease.     

Hazard-Rate Analysis and Patterns of Recurrence in Early Stage Melanoma: Moving towards a Rationally Designed Surveillance Strategy

Background

While curable at early stages, few treatment options exist for advanced melanoma. Currently, no consensus exists regarding the optimal surveillance strategy for patients after resection. The objectives of this study were to identify patterns of metastatic recurrence, to determine the influence of metastatic site on survival, and to identify high-risk periods for recurrence.

Methods

A retrospective review of the Duke Melanoma Database from 1970 to 2004 was conducted that focused on patients who were initially diagnosed without metastatic disease. The time to first recurrence was computed from the date of diagnosis, and the associated hazard function was examined to determine the peak risk period of recurrence. Metastatic sites were coded by the American Joint Committee on Cancer (AJCC) system including local skin, distant skin and nodes (M1a), lung (M1b), and other distant (M1c).

Results

Of 11,615 patients initially diagnosed without metastatic disease, 4616 (40%) had at least one recurrence. Overall the risk of initial recurrence peaked at 12 months. The risk of initial recurrence at the local skin, distant skin, and nodes peaked at 8 months, and the risk at lung and other distant sites peaked at 24 months. Patients with a cutaneous or nodal recurrence had improved survival compared to other recurrence types.

Conclusions

The risk of developing recurrent melanoma peaked at one year, and the site of first recurrence had a significant impact on survival. Defining the timing and expected patterns of recurrence will be important in creating an optimized surveillance strategy for this patient population.     

Inflammasome polymorphisms confer susceptibility to sporadic malignant melanoma

Genetic variants of NLRP3 and NLRP1 are known to modulate levels of pro-inflammatory cytokine interleukin (IL)-1β. The purpose of this study was to investigate the association of NLRP3/NLRP1 polymorphisms with susceptibility and clinical features of malignant melanoma in a Swedish case-control study. Common variants in NLRP3/NLRP1 were investigated in sporadic malignant melanoma patients and healthy controls followed by analysis using logistic regression. NLRP3 variant (rs35829419) was significantly more common in male patients than in controls (OR, 2.22; CI, 1.27-3.86). Upon stratification, significant association with nodular melanoma was observed (OR, 2.89; CI, 1.33-6.30), which intensified in male patients (OR 4.03, CI 1.40-11.59). The NLRP1 variant (rs12150220) was significantly more common in fair-skinned female patients (OR, 1.85; CI, 1.04-3.33) and showed strong associations with nodular melanoma (OR, 6.03; CI, 1.33-25). Our data suggest that NLRP3/NLRP1 polymorphisms are associated with melanoma susceptibility; these findings warrant validation in other independent populations.     

Identifying people at high risk of cutaneous malignant melanoma: results from a case-control study in Western Australia

To assess whether screening people at high risk of malignant melanoma would be effective in reducing the mortality from the disease data from 400 case-control pairs in a study of cutaneous malignant melanoma conducted in Western Australia during 1980-1 were used to predict the risk of melanoma in the remaining 111 pairs. All variables previously shown to be associated with a decrease or increase in the incidence of melanoma were considered for inclusion in a single conditional logistic regression model of the incidence of melanoma in the randomly chosen subset of 400 case-control pairs. Five of these variables—number of raised naevi on the arms, arrival in Australia before 10 years of age, history of non-melanocytic skin cancer, time spent outdoors in summer from the age of 10 to 24, and family history of melanoma—provided good discrimination between patients and controls in this sample and the 111 other case-control pairs. Among the 222 subjects in these other case-control pairs a group defined as being at high risk of melanoma by a risk score derived from these five variables contained 60 (54%) of the patients with melanoma but only 18 (16%) of the controls.These data suggest that in Western Australia more than half of all new patients with melanoma arise in an identifiable subpopulation constituting less than one fifth of the whole population. Identifying this subpopulation and screening it regularly for cutaneous malignant melanoma could be cost effective in reducing mortality from this disease.     

Characterization of the CD4+ and CD8+ tumor infiltrating lymphocytes propagated with bispecific monoclonal antibodies

To study the CD4+ and CD8+ tumor infiltrating lymphocytes (TIL) in the antitumor response, we propagated these subsets directly from tumor tissues with anti-CD3:anti-CD8 (CD3,8) and anti-CD3:anti-CD4 (CD3,4) bispecific mAb (BSMAB). CD3,8 BSMAB cause selective cytolysis of CD8+ lymphocytes by bridging the CD8 molecules of target lymphocytes to the CD3 molecular complex of cytolytic T lymphocytes with concurrent activation and proliferation of residual CD3+CD4+ T lymphocytes. Similarly, CD3,4 BSMAB cause selective lysis of CD4+ lymphocytes whereas concurrently activating the residual CD3+CD8+ T cells. Small tumor fragments from four malignant melanoma and three renal cell carcinoma patients were cultured in medium containing CD3,8 + IL-2, CD3,4 + IL-2, or IL-2 alone. CD3,8 led to selective propagation of the CD4+ TIL whereas CD3,4 led to selective propagation of the CD8+ TIL from each of the tumors. The phenotypes of the TIL subset cultures were generally stable when assayed over a 1 to 3 months period and after further expansion with anti-CD3 mAb or lectins. Specific 51Cr release of labeled target cells that were bridged to the CD3 molecular complexes of TIL suggested that both CD4+ and CD8+ TIL cultures have the capacity of mediating cytolysis via their Ti/CD3 TCR complexes. In addition, both CD4+ and CD8+ TIL cultures from most patients caused substantial (greater than 20%) lysis of the NK-sensitive K562 cell line. The majority of CD4+ but not CD8+ TIL cultures also produced substantial lysis of the NK-resistant Daudi cell line. Lysis of the autologous tumor by the TIL subsets was assessed in two patients with malignant melanoma. The CD8+ TIL from one tumor demonstrated cytotoxic activity against the autologous tumor but negligible lysis of allogeneic melanoma targets. In conclusion, immunocompetent CD4+ and CD8+ TIL subsets can be isolated and expanded directly from small tumor fragments of malignant melanoma and renal cell carcinoma using BSMAB. The resultant TIL subsets can be further expanded for detailed studies or for adoptive immunotherapy.     

Evaluation of nodal patterns for melanoma of the ear

Treatment of malignant melanoma of the external ear presents unique challenges. Because of the significant debate regarding the efficacy and validity of using sentinel lymph node mapping for the treatment of ear melanomas, data for a population of patients with melanomas of the ear who underwent surgical excision and reconstruction were reviewed to determine the efficacy of sentinel node mapping. A retrospective chart review of cases treated by a single surgical oncologist was performed. All patients who were treated for malignant melanomas and required reconstruction of the external ear by the plastic surgical service between 1995 and 2001 were identified. Nineteen patients were selected, of whom nine underwent sentinel node mapping. The average age of the patients was 65.2 years. Evaluation of melanoma depth, medical history, surgical margins, lymph node metastasis, and recurrence was performed. Lymphoscintigraphy with technetium-99-sulfur colloid and 1% Lymphazurin (isosulfan blue; Zenith Parenterals, Rosemont, Ill.) demonstrated widely variable lymphatic drainage patterns. The lower tail of the parotid gland and the upper cervical area were the two most common locations. The average number of sentinel nodes identified and removed was 3.7. The average Breslow thickness for these patients was 2.3 mm. None of these patients demonstrated micrometastatic disease in their sentinel nodes. The most common reconstructive procedure after surgical resection was the use of rotational advancement flaps. Localization of radioactivity, as detected with external technetium-99 scanning, was the most reliable method for detection of the sentinel lymph node basins and the individual nodes. The average value for the primary injection site was 8375 counts per second, and the average value for the nodes removed was 973.5 counts per second. Of the nine patients who underwent sentinel lymph node mapping, only one, with an initial lesion depth of 5 mm, developed a local recurrence. The average follow-up period in this study was 21 months (range, 12 to 79 months). All patients in this study were evaluated at least 1 year after the initial surgical resection. Patients were monitored by the same surgical oncologist every 3 months for the first 2 years. Little can be found in the literature regarding the efficacy of sentinel node biopsies for ear melanomas. Larger studies are indicated; however, it seems that this method is practical for designing therapeutic methods for patients with melanoma of the ear.     

Adjuvant BCG immunotherapy for stage I and II malignant melanoma.

Initial adjuvant immunotherapy trials have demonstrated a greater disease-free interval in patients treated with bacille Calmette-Guérin (BCG) compared with historical controls. In this study 149 patients at high risk of recurrence after surgical treatment of local or regional malignant melanoma were given BCG for 2 years and were followed up for a median of 28 months from the start of immunotherapy. The 36 patients in the comparison group had a higher rate of recurrence than the patients treated with BCG, and the rate in the treatment group was close to that reported from a similar study at the University of California at Los Angeles. The relatively long disease-free interval for the high-risk comparison patients in this study suggests that the control groups at other centres may have included patients with unrecognized additional risk. The rates of survival in the Canadian treatment group were also comparable to those reported by other centres. However, reports of a favourable BCG-mediated pattern of recurrence could not be confirmed. Therefore, the routine use of adjuvant BCG immunotherapy is not recommended.     

T lymphocyte subsets in patients with malignant melanoma

Summary T lymphocyte subset profiles were determined by monoclonal antibodies on cryopreserved peripheral blood lymphocytes from 57 patients with malignant melanoma and 19 healthy controls. Quantitation of percentages of total T cells (OKT3.PAN), helper (OKT4.IND) or suppressor (OKT8.SUP) cells, and the ratio of helper/suppressor subsets revealed no correlation of these markers with stage of disease or clinical outcome. A sequential study of these markers on peripheral blood lymphocytes from three stage I melanoma patients with subsequent recurrent disease showed no fluctuations that could be correlated to tumor progression. This study indicates that there is no systemic imbalance in T cell subsets in malignant melanoma and that quantitation of these subsets cannot predict the clinical course of this disease.     

Immunologic function during adjuvant BCG immunotherapy for malignant melanoma: Induction of anergy

Summary Serial tests of immunological function were performed on 28 patients participating in a randomized controlled clinical trial of adjuvant Tice-stain BCG immunotherapy administered by tine technique for malignant melanoma. Cryopreserved lymphocyte samples obtained prior to study entry and at 3 and 6 months there-after were tested by mixed lymphocyte culture (MLC), cell-mediated lympholysis (CML), antibody-dependent cell-mediated cytotoxicity (K cell), and natural killing (NK cell) assays, the last two assays being performed with the Chang cell line. Delayed-type hypersensitivity (DTH) skin tests to recall antigens were performed at the same intervals.At entry to the study in vitro lymphocyte reactivity of patients was similar to that of normal controls, and most (75%) of the patients reacted to at least one recall antigen. Serial lymphocyte reactivity measured by the in vitro tests was not different in the BCG and control groups, but BCG treatment was associated with a marked, statistically significant (P<0.01) reduction in DTH skin test reactivity. BCG therapy was not shown to delay recurrence of the disease.     

不同剂量糖皮质激素治疗老年哮喘急性发作的对比研究

目的:探讨不同剂量吸入型糖皮质激素(ICS)治疗老年哮喘急性发作的疗效及安全性分析。方法:将108例老年哮喘急性发作患者随机分为A组(34例)、B组(38例)和C组(36例),分别吸入布地奈德200μg/d、400μg/d、800μg/d;治疗3个月后再随机分为低剂量组(200μg/d,n=)和高剂量组(400μg/d)进行维持治疗,并随访观察12个月。结果:治疗3个月后,三组的临床症状评分、肺功能较治疗前均有显著改善,差异有统计学意义(P<0.05);B组、C组治疗后的临床症状评分、肺功能及症状消失时间均显著优于A组,而B组、C组之间差异无统计学意义(P>0.05);A组和B组不良反应的发生率分别为5.9%,10.5%,显著低于C组30.6%,差异有统计学意义(P<0.05);维持治疗期间,低剂量组复发率为21.2%,高剂量组为20.4%,差异无统计学意义(P>0.05)。结论:老年哮喘急性发作患者给予ICS 400μg/d治疗剂量及200μg/d维持剂量即可取得较好的治疗效果,可减少不良反应,提高患者的耐受性。     

Prognostic value of cytologic examination of peritoneal washings in patients with endometrial carcinoma

Peritoneal pelvic washings from 54 women with pathologic stage I endometrial carcinoma were evaluated in a blind retrospective fashion for the concentration of malignant cells present. None of the 42 patients with normal washings developed recurrence after a median disease-free survival of 36 months. Of the 12 patients with adenocarcinoma in the washings, 4 had high concentrations of malignant cells (greater than 1000 cells/100 ml sample), and all 4 died as a consequence of carcinoma within two years. The remaining eight patients had lower concentrations of malignant cells in the washings (less than 1000 cells/100 ml sample), and six of these patients had no evidence of disease after 37 to 64 months. Cox's nonparametric statistical model showed that increasing concentrations of adenocarcinoma cells in washings significantly shortened the time to recurrence of disease. The abundance of malignant cells has prognostic importance in identifying those patients with pathologic stage I disease who may require more aggressive therapy.     

Topical mitomycin C and radiation induce conjunctival DNA-polyploidy.

INTRODUCTION: Atypical cell changes often occur following treatment of premalignant or malignant conjunctival neoplasias with topical mitomycin C (MMC) and/or radiation. These reactive, non-neoplastic alterations of the conjunctival epithelium can be a differential diagnostic problem. Our aim was to investigate changes in the nuclear DNA-distribution of conjunctival epithelial cells after MMC- and radiation therapy by DNA-image-cytometry. METHODS: Conjunctival brush smears were obtained from 13 patients (13 eyes) with squamous cell carcinomas and six patients (6 eyes) with conjunctival malignant melanomas in situ before, during and after treatment. The patients were treated with MMC-drops (0.02% or 0.04%) alone (n=12), with radiation therapy (n=3) or both (n=4). At first, the obtained brush smears were evaluated by cytology. Secondly, after Feulgen restaining, the DNA content of reactively changed cells was determined using the AutoCyte-QUIC-DNA workstation. RESULTS: We observed euploid DNA-polyploidy and cytomorphological changes in all patients (19/19). We considered these alterations as reactive to treatment. Four patients showed their greatest DNA-stemline at 4c and 15 patients at 8c. This effect was observed during and following MMC-drops and/or radiation and remained stable in 94% of all patients after a mean follow-up of 22.5 months (SD 15.4). In five cases image cytometry additionally demonstrated DNA-stemline aneuploidy as an evidence of tumor recurrence. CONCLUSION: Measurements of DNA-content revealed euploid polyploidisation of morphological suspicious but benign squamous cells which is the biologic correlate of well known secondary morphologic changes following topical chemotherapy and/or radiation. DNA-image-cytometry is a useful tool in the differention of euploid polyploidization as a sign of reactive cell changes following treatment and tumor recurrences.     

Changes in serum immunoglobulins in subjects with acute myocardial infarct and essential hypertension

20 (12 men and 8 women) acute myocardial infarction (AMI) patients and 17 (14 men and 3 women) patients with arterial hypertension (II degrees stage according to OMS) in comparison to controls age and sex matched, were studied, serum IgA, IgG, IgM were evaluated with radial immunodiffusion and serum IgE with RIA. Ho significant changes ef immunoglobulins were observed between hypertensive patients and controls; whereas a significant increase of IgM, IgG and IgE, with out changes of IgA, were shown in AMI patients. Serum Ig and IgM were significantly augmented in AMI patients in comparison to hypertensive patients.