Mutations Associated with Acquired Resistance to PD-1 Blockade in Melanoma

BACKGROUND: Approximately 75% of objective responses to anti–programmed death 1 (PD-1) therapy in patients with melanoma are durable, lasting for years, but delayed relapses have been noted long after initial objective tumor regression despite continuous therapy. Mechanisms of immune escape in this context are unknown. METHODS: We analyzed biopsy samples from paired baseline and relapsing lesions in four patients with metastatic melanoma who had had an initial objective tumor regression in response to anti–PD-1 therapy (pembrolizumab) followed by disease progression months to years later. RESULTS: Whole-exome sequencing detected clonal selection and outgrowth of the acquired resistant tumors and, in two of the four patients, revealed resistance-associated loss-of-function mutations in the genes encoding interferon-receptor–associated Janus kinase 1 (JAK1) or Janus kinase 2 (JAK2), concurrent with deletion of the wild-type allele. A truncating mutation in the gene encoding the antigen-presenting protein beta-2-microglobulin (B2M) was identified in a third patient. JAK1 and JAK2 truncating mutations resulted in a lack of response to interferon gamma, including insensitivity to its antiproliferative effects on cancer cells. The B2M truncating mutation led to loss of surface expression of major histocompatibility complex class I. CONCLUSIONS: In this study, acquired resistance to PD-1 blockade immunotherapy in patients with melanoma was associated with defects in the pathways involved in interferon-receptor signaling and in antigen presentation. (Funded by the National Institutes of Health and others.

Practical guidelines for rigor and reproducibility in preclinical and clinical studies on cardioprotection

The potential for ischemic preconditioning to reduce infarct size was first recognized more than 30 years ago. Despite extension of the concept to ischemic postconditioning and remote ischemic conditioning and literally thousands of experimental studies in various species and models which identified a multitude of signaling steps, so far there is only a single and very recent study, which has unequivocally translated cardioprotection to improved clinical outcome as the primary endpoint in patients. Many potential reasons for this disappointing lack of clinical translation of cardioprotection have been proposed, including lack of rigor and reproducibility in preclinical studies, and poor design and conduct of clinical trials. There is, however, universal agreement that robust preclinical data are a mandatory prerequisite to initiate a meaningful clinical trial. In this context, it is disconcerting that the CAESAR consortium (Consortium for preclinicAl assESsment of cARdioprotective therapies) in a highly standardized multi-center approach of preclinical studies identified only ischemic preconditioning, but not nitrite or sildenafil, when given as adjunct to reperfusion, to reduce infarct size. However, ischemic preconditioning—due to its very nature—can only be used in elective interventions, and not in acute myocardial infarction. Therefore, better strategies to identify robust and reproducible strategies of cardioprotection, which can subsequently be tested in clinical trials must be developed. We refer to the recent guidelines for experimental models of myocardial ischemia and infarction, and aim to provide now practical guidelines to ensure rigor and reproducibility in preclinical and clinical studies on cardioprotection. In line with the above guideline, we define rigor as standardized state-of-the-art design, conduct and reporting of a study, which is then a prerequisite for reproducibility, i.e. replication of results by another laboratory when performing exactly the same experiment

Programmed death-ligand 1 (PD-L1) is expressed in a significant number of the uterine cervical carcinomas.

BackgroundThe programmed death-1/programmed death-ligand-1 (PD-1/PD-L1) immune regulatory axis has emerged as a promising new target for cancer therapeutics, with lasting responses seen in the treatment of metastatic renal and lung carcinomas, as well as melanomas. As tumor surface expression of PD-L1 has been found to correlate with objective responses to anti-PD-L1 immunotherapies, we investigated the expression of PD-L1 in human cervical tumors and provide an adopted scoring system for the systematic evaluation of PD-L1 staining.MethodsImmunohistochemical staining for PD-L1 expression was performed on a tissue microarray of 101 normal and neoplastic cervical tissues. Neoplastic cores were divided into three groups: squamous cell carcinoma, adenosquamous carcinoma, and endocervical adenocarcinoma. PD-L1 expression was scored based on an adopted scoring system accounting to percentage and intensity of positivity, and results provided alongside available clinical and demographic data.ResultsOverall, PD-L1 was positive in 32 of 93 (34.4%) cervical carcinomas. Subcategorically, PD-L1 was positive in 28 of 74 (37.8%) squamous cell carcinomas, two of seven (28.6%) adenosquamous carcinomas, and two of 12 (16.7%) endocervical adenocarcinomas. It was negative in six benign cervical tissues.ConclusionsThis study shows a significant expression of PD-L1 in 34.4% of cervical carcinomas and no expression of PD-L1 in benign cervical tissues. These findings suggest a role for further investigation of anti-PD-L1/PD-1 immunotherapies in the treatment of PD-L1-positive cervical tumors. In addition, our adopted scoring system will facilitate more systematic correlations between tumor reactivity and response to treatment

Expression of immune checkpoint regulators, cytotoxic T lymphocyte antigen 4 (CTLA-4) and programmed death-ligand 1 (PD-L1), in female breast carcinomas

<div><p>Background</p><p>Immune checkpoint regulators, <i>cytotoxic T lymphocyte antigen 4</i> (CTLA-4) and the <i>programmed cell death protein-1/programmed death-ligand 1</i> (PD-1/PD-L1) have emerged as promising new targets for cancer therapeutics. While tumor expression of PD-L1 has been shown to have objective responses to anti-PD-L1 immunotherapies, the clinical implications of CTLA-4 expression in tumor cells or immune cells in the tumor microenvironment is still controversial. We investigated the expression of CTLA-4 and PD-L1 in human breast tumors and provided a scoring system for the systematic evaluation of CTLA-4 staining.</p><p>Methods</p><p>Immunohistochemical staining for PD-L1 and CTLA-4 expression was performed on a tissue microarray of 102 cores, which included normal and neoplastic breast tissues. Neoplastic cores were divided into four groups: <i>Ductal carcinoma in situ</i> (DCIS), <i>invasive ductal carcinoma</i> (IDC), <i>invasive lobular carcinoma</i> (ILC) and <i>invasive tubular carcinoma</i> (ITC). PD-L1 and CTLA-4 expressions were scored based on a system which accounted for the percentage and intensity of positivity and results provided in conjunction with available clinical and demographic data.</p><p>Results</p><p>Overall, CTLA-4 was over-expressed in 49 of 93 (52.7%) breast tumors. Subcategorically, CTLA-4 was positive in 3 of 8 (37.5%) ductal carcinoma in situ, 40 of 73 (55%) of invasive ductal carcinomas, 4 of 10 (40%) of invasive lobular carcinomas and 2 of 2 (100%) of invasive tubular carcinomas. All 6 normal breast tissues were interpreted as negative for CTLA-4 staining. Only 4.1% of the invasive ductal carcinomas were positive for PD-L1 reactivity and the remaining carcinomas stained negative.</p><p>Conclusions</p><p>This study shows a significant overexpression of CTLA-4 in >50% of breast carcinomas with no such overexpression of CTLA-4 in benign breast tissues. PDL-1 staining is seen in only a small number of invasive ductal carcinomas (4.1%). These findings suggest the need for further investigation of anti-CTLA-4 and anti-PD-L1 immunotherapies and their efficacy in the treatment of breast carcinomas with overexpression of these immune modulators. In addition, the proposed scoring system will facilitate a more systematic correlation between tumor reactivity and clinical outcome which can be applied to all intracytoplasmic tumor markers.</p></div

Summary of the <i>normal</i> breast tissue cores with the CTLA-4 reaction, score, and the interpretation.

<p>Summary of the <i>normal</i> breast tissue cores with the CTLA-4 reaction, score, and the interpretation.</p

PD-L1 in IDC (40x objective).

<p>Photomicrographs of an invasive ductal carcinoma are displayed. <b>B</b> shows PD-L1 reaction involving cell membranes of the tumor cells with large open nuclear chromatin. The reaction was observed in 20% of the tumor cells in this core. Small mononuclear cells are also present in between the tumor cells. A hematoxylin & eosin stain photomicrograph of the same tumor is shown in panel <b>A</b> (#37; <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0195958#pone.0195958.t003" target="_blank">Table 3</a>).</p