T cells isolated from patients with checkpoint inhibitor-resistant melanoma are functional and can mediate tumor regression.

Background: Almost half of the patients with metastatic melanoma obtain only short-term or no benefit at all from checkpoint inhibitor (CPI) immunotherapy. In this study, we investigated whether the immune system of patients progressing following CPI treatment was able to generate functional tumor-specific immune responses. Materials and methods: Tumor-infiltrating lymphocytes (TILs) were isolated and expanded from metastatic melanoma lesions which progressed during or after anti-programmed cell death protein 1 (PD)-1 and anti-Cytotoxic T-Lymphocyte Antigen 4 (CTLA-4) treatment. Tumor-specific immune responses were assessed with co-culture assays of TILs and autologous tumor cells. Results: TILs from 23 metastases of individual patients could be assessed for T cells recognition of autologous tumor cells. All metastases were progressive on or following anti-PD-1 (23/23, 100%), and the majority also after anti-CTLA-4 (17/23, 74%). Functional antitumor immune responses were detected in 19/23 patients (83%). Both CD8+ (in 18/23 patients, 78%) and CD4+ (in 16/23 patients, 70%) TILs were able to recognize autologous tumors. A large fraction of CD8+ TILs (median 23%, range 1.0%-84%) recognized tumor cells. This is similar to the cohorts of unselected patient populations with metastatic melanoma presented in previous studies. The localization of intratumoral immune infiltrates was heterogeneous among samples. In a phase I/II clinical trial, TILs were administered with lymphodepleting chemotherapy, pegIFNα2b and interleukin-2 to 12 patients with CPI-resistant melanoma. Out of 12 patients who previously failed CPI therapy, treatment with TILs resulted in two partial responses, of which one is ongoing. Conclusions: Tumor-reactive T cells appear to heavily infiltrate the tumor microenvironment of patients who failed previous CPI treatment. These patients can still respond to an infusion of unselected autologous TILs. Our results warrant further testing of novel immune re-activation strategies in melanoma patients who failed multiple CPI therapy.

Mycobacterium marinum dermatitis and panniculitis with chronic pleuritis in a captive white whale (Delphinapterus leucas) with aortic rupture.

A 16-year-old female white whale, Delphinapterus leucas, died after nearly 18 months of chronic lymphopenia and pyogranulomatous dermatitis. Necropsy revealed rupture of the aorta with hemorrhage into the cranial mediastinum and between fascial planes of the ventral neck musculature. Multiple foci of ulcerative dermatitis and panniculitis were present across the thorax and abdomen and surrounded the genital folds. In addition, there was a chronic proliferative pleuritis with over 20 liters of histiocytic exudate in the thoracic cavity. Acid-fast bacteria consistent with Mycobacterium sp. were identified in sections of skin lesions and in cytospins of pleural exudate. Cultures of pleura and 1 skin lesion collected at necropsy yielded sparse growth of an acid-fast bacillus with colony characteristics and morphology consistent with Mycobacterium marinum. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis confirmed the presence of M. marinum DNA in samples of skin. This is the first documented occurrence of mycobacteriosis in a white whale and is a unique presentation of mycobacterial dermatitis and panniculitis with chronic pleuritis in a cetacean. The improved PCR-RFLP protocol utilized in this case unifies techniques from several protocols to differentiate between species of Nocardia and rapidly growing mycobacteria clinically relevant to aquatic animals.

Identification of herpes simplex virus genital infection: comparison of a multiplex PCR assay and traditional viral isolation techniques.

Genital herpes simplex virus (HSV) is of major public health importance, as indicated by the marked increase in the prevalence of genital herpes over the past two decades. Viral culture has traditionally been regarded as the gold standard for diagnosis. In this study, we compared viral culture and the amplification of HSV DNA by the polymerase chain reaction (PCR) with respect to sensitivity, cost, clinical utility, and turnaround time. Patient sample swabs from 100 individuals were inoculated onto MRC-5 cells for isolation. Positive results were confirmed via a direct fluorescent antibody technique, and serotyping, when requested, was performed using HSV-1 and -2-type-specific sera. PCR techniques employed an extraction step of the same initial swab specimen, followed by PCR amplification, using a multiplex assay for HSV-1, 2 DNA. HSV-positive results were found in 32/100 samples via culture and in 36/100 samples via PCR. PCR-positive results yielded 16 (44%) patients infected with HSV-1 and 20 (56%) patients infected with HSV-2. Turnaround time for viral culture averaged 108 hours for positive results and 154 hours for negative results; PCR turnaround time averaged 24--48 hours. Laboratory cost using viral culture was $3.22 for a negative result and $6.49 for a positive result (including direct fluorescent antibody). Serotyping added $10.88 to each culture-positive test. Although laboratory costs for PCR were higher at $8.20/sample, reimbursement levels were also higher. We propose a multiplex PCR assay for diagnosis of HSV-1 and HSV-2 from patient swabs for use in a routine clinical laboratory setting. This assay offers increased sensitivity, typing, and improved turnaround time when compared with traditional viral culture techniques. Although it appears that PCR testing in a routine clinical laboratory setting is cost prohibitive compared with the case of nonserotyped viral culture, it may be very useful when clinical utility warrants distinguishing between HSV 1 and 2 and may be cost effective when reimbursement issues are examined.

Polymorphisms in the genes for coagulation factors II, V, and VII in patients with ischemic heart disease.

BACKGROUND: Cardiovascular disease remains the leading cause of mortality in the United States, accounting for approximately 33% of all deaths in this country. Of these deaths, most are due to acute myocardial infarctions (AMIs), which are associated with thrombotic coronary artery obstruction and/or occlusion. These events could potentially be due to alterations in genes coding for coagulation factors. Several polymorphisms have been described in the factor II, V, and VII genes, which may predispose one to increased risk for ischemic heart disease (IHD). OBJECTIVE: To determine if mutations in 3 coagulation factor genes could predispose an individual to increased risk for arterial thrombosis as a mechanism for developing unstable angina (UA) or AMI. METHODS: We examined 125 hospitalized patients (mean age, 53 +/- 6 years, 79 men and 46 women), including 32 with AMI, 68 with UA, and 25 noncardiac controls, for a genetic predisposition for increased risk of IHD. EDTA-anticoagulated whole blood was collected at the time of hospital admission. DNA was extracted, and the polymorphisms were detected by polymerase chain reaction amplification of these genes with subsequent restriction enzyme digestion and gel electrophoresis. RESULTS: Our results showed that 3 (9.4%), 3 (4.4%), and 1 (4%) individuals were heterozygous for prothrombin G20210A and 3 (9.4%), 5 (7.4%), and 1 (4%) individuals were heterozygous for factor V Leiden in the AMI, UA, and control groups, respectively. The following genotype frequencies for the factor VII R353Q polymorphism were identified: 25 (78.1%), 56 (82.4%), and 18 (72%) with RR and 7 (21.9%), 12 (17. 6%), and 7 (28%) with RQ in the AMI, UA, and control groups, respectively. No QQ homozygotes were identified. For the HVR4 size polymorphism, the following genotypes were identified: 3 (9.4%), 4 (5.9%), and 5 (20%) individuals with H7H7; 11 (34.4%), 33 (48.5%), and 12 (48%) with H6H7; and 18 (56.2%), 31 (45.6%), and 8 (32%) with H6H6 genotypes in the AMI, UA, and control groups, respectively. There were no H7H5 and H6H5 genotypes found in this study. CONCLUSIONS: Although the frequency differences of these polymorphisms in patients with AMI and UA were not statistically significant from those in controls, several trends are consistent with what has been reported in the literature. Although any of these or other undefined genetic abnormalities may result in IHD, it is possible that phenotypic predisposition to IHD initially presents as UA. A larger population study addressing the significance of these polymorphisms in the sequence of events that lead to IHD, including cases of UA, is warranted.