Human epigenetic and transcriptional T cell differentiation atlas for identifying functional T cell-specific enhancers.

The clinical benefit of T cell immunotherapies remains limited by incomplete understanding of T cell differentiation and dysfunction. We generated an epigenetic and transcriptional atlas of T cell differentiation from healthy humans that included exhausted CD8 T cells and applied this resource in three ways. First, we identified modules of gene expression and chromatin accessibility, revealing molecular coordination of differentiation after activation and between central memory and effector memory. Second, we applied this healthy molecular framework to three settings-a neoadjuvant anti-PD1 melanoma trial, a basal cell carcinoma scATAC-seq dataset, and autoimmune disease-associated SNPs-yielding insights into disease-specific biology. Third, we predicted genome-wide cis-regulatory elements and validated this approach for key effector genes using CRISPR interference, providing functional annotation and demonstrating the ability to identify targets for non-coding cellular engineering. These studies define epigenetic and transcriptional regulation of human T cells and illustrate the utility of interrogating disease in the context of a healthy T cell atlas.

Partner-developed electronic health record tools to facilitate social risk-informed care planning.

OBJECTIVE: Increased social risk data collection in health care settings presents new opportunities to apply this information to improve patient outcomes. Clinical decision support (CDS) tools can support these applications. We conducted a participatory engagement process to develop electronic health record (EHR)-based CDS tools to facilitate social risk-informed care plan adjustments in community health centers (CHCs). MATERIALS AND METHODS: We identified potential care plan adaptations through systematic reviews of hypertension and diabetes clinical guidelines. The results were used to inform an engagement process in which CHC staff and patients provided feedback on potential adjustments identified in the guideline reviews and on tool form and functions that could help CHC teams implement these suggested adjustments for patients with social risks. RESULTS: Partners universally prioritized tools for social risk screening and documentation. Additional high-priority content included adjusting medication costs and changing follow-up plans based on reported social risks. Most content recommendations reflected partners' interests in encouraging provider-patient dialogue about care plan adaptations specific to patients' social needs. Partners recommended CDS tool functions such as alerts and shortcuts to facilitate and efficiently document social risk-informed care plan adjustments. DISCUSSION AND CONCLUSION: CDS tools were designed to support CHC providers and staff to more consistently tailor care based on information about patients' social context and thereby enhance patients' ability to adhere to care plans. While such adjustments occur on an ad hoc basis in many care settings, these are among the first tools designed both to systematize and document these activities.

Diverse cutaneous side effects associated with BRAF inhibitor therapy: a clinicopathologic study.

BACKGROUND: Vemurafenib, a novel selective small molecule inhibitor of BRAF, has recently been shown to be effective in the treatment of melanomas harboring the BRAF V600E mutation. Similar to the broad-spectrum RAF inhibitor sorafenib, vemurafenib induces development of squamous cell carcinomas and keratoacanthomas as a side effect of therapy. OBJECTIVE: We sought to detail additional cutaneous adverse effects of vemurafenib and a similar BRAF inhibitor, dabrafenib. METHODS: We evaluated the clinical and histologic feature of skin side effects developing on vemurafenib or dabrafenib therapy in 14 patients. RESULTS: Eight patients developed one or more squamous cell carcinomas, and 11 patients formed benign verrucous keratoses. Eight patients developed single lesions and/or widespread eruptions with histopathologic findings of acantholytic dyskeratosis, consistent with warty dyskeratomas and Darier- or Grover-like rashes, respectively. One patient developed palmoplantar hyperkeratosis, and darkening of existing nevi and new nevi within 2 months of starting vemurafenib. Side effects presented as early as 1 week after beginning therapy, with a mean time of onset of 12.6 weeks in our cohort. LIMITATIONS: This study was limited by the small number of cases, all from a single institution. CONCLUSION: Selective BRAF inhibitor therapy is associated with the development of malignant and benign growths, including keratoacanthoma-like squamous cell carcinomas, warty dyskeratomas, and verrucous keratoses, along with widespread eruptions with histologic features of acantholytic dyskeratosis. Given the potential for malignant lesions to develop on treatment, awareness of potential adverse effects of these agents is necessary, and a low threshold for biopsy of new growths is recommended.

BAMM (BRAF Autophagy and MEK Inhibition in Melanoma): A Phase I/II Trial of Dabrafenib, Trametinib, and Hydroxychloroquine in Advanced BRAFV600-mutant Melanoma.

PURPOSE: Autophagy is a resistance mechanism to BRAF/MEK inhibition in BRAFV600-mutant melanoma. Here we used hydroxychloroquine (HCQ) to inhibit autophagy in combination with dabrafenib 150 mg twice daily and trametinib 2 mg every day (D+T). PATIENTS AND METHODS: We conducted a phase I/II clinical trial in four centers of HCQ + D+T in patients with advanced BRAFV600-mutant melanoma. The primary objectives were the recommended phase II dose (RP2D) and the one-year progression-free survival (PFS) rate of >53%. RESULTS: Thirty-four patients were evaluable for one-year PFS rate. Patient demographics were as follows: elevated lactate dehydrogenase: 47%; stage IV M1c/M1d: 52%; prior immunotherapy: 50%. In phase I, there was no dose-limiting toxicity. HCQ 600 mg orally twice daily with D+T was the RP2D. The one-year PFS rate was 48.2% [95% confidence interval (CI), 31.0%-65.5%], median PFS was 11.2 months (95% CI, 5.4-16.9 months), and response rate (RR) was 85% (95% CI, 64%-95%). The complete RR was 41% and median overall survival (OS) was 26.5 months. In a patient with elevated LDH (n = 16), the RR was 88% and median PFS and OS were 7.3 and 22 months, respectively. CONCLUSIONS: HCQ + D+T was well tolerated and produced a high RR but did not meet criteria for success for the one-year PFS rate. There was a high proportion of patients with pretreated and elevated LDH, an increasingly common demographic in patients receiving targeted therapy. In this difficult-to-treat population, the RR and PFS were encouraging. A randomized trial of D+T + HCQ or placebo in patients with BRAFV600-mutant melanoma with elevated LDH and previous immunotherapy is being conducted.

A single dose of neoadjuvant PD-1 blockade predicts clinical outcomes in resectable melanoma.

Immunologic responses to anti-PD-1 therapy in melanoma patients occur rapidly with pharmacodynamic T cell responses detectable in blood by 3 weeks. It is unclear, however, whether these early blood-based observations translate to the tumor microenvironment. We conducted a study of neoadjuvant/adjuvant anti-PD-1 therapy in stage III/IV melanoma. We hypothesized that immune reinvigoration in the tumor would be detectable at 3 weeks and that this response would correlate with disease-free survival. We identified a rapid and potent anti-tumor response, with 8 of 27 patients experiencing a complete or major pathological response after a single dose of anti-PD-1, all of whom remain disease free. These rapid pathologic and clinical responses were associated with accumulation of exhausted CD8 T cells in the tumor at 3 weeks, with reinvigoration in the blood observed as early as 1 week. Transcriptional analysis demonstrated a pretreatment immune signature (neoadjuvant response signature) that was associated with clinical benefit. In contrast, patients with disease recurrence displayed mechanisms of resistance including immune suppression, mutational escape, and/or tumor evolution. Neoadjuvant anti-PD-1 treatment is effective in high-risk resectable stage III/IV melanoma. Pathological response and immunological analyses after a single neoadjuvant dose can be used to predict clinical outcome and to dissect underlying mechanisms in checkpoint blockade.

Radiofrequency ablation of primary lung cancer: results from an ablate and resect pilot study.

STUDY OBJECTIVES: The role of radiofrequency ablation (RFA) for primary lung cancer remains poorly defined. The purpose of this "ablate and resect" pilot study was to evaluate the safety of performing RFA in patients with primary non-small cell lung cancer (NSCLC) and to characterize the histologic changes in tumor tissue following such ablation. DESIGN: This prospective study was undertaken at a single institution, and 10 patients were accrued from June 2002 to June 2003. Eligible patients included those with clinical stage I or II disease. RFA of the tumor was performed through a standard thoracotomy followed by conventional lobectomy and lymph node dissection. Extent of cell death was determined histologically. MEASUREMENTS AND RESULTS: Following the exclusion of two patients, the treated portions of eight tumors were examined for tumor cell viability. Gross inspection and routine histologic staining could not reliably identify the "immediately ablated" tissue. However, using a supravital staining technique, the treated areas from seven of the eight tumors (87.5%) demonstrated > 80% nonviability (100% nonviability was noted in the treated areas from three of the eight tumors). No bleeding or thermal complications were noted at the time of RFA, and none of the patients had skin burns at the electrode dispersive pad sites. CONCLUSIONS: RFA of primary NSCLC is feasible and can be performed safely in the setting of an open thoracotomy. Complete tumor cell necrosis, as determined by supravital staining, was noted in the treated areas from three of eight tumors (37.5%). Such complete ablation was observed in the treated areas from smaller tumors (< 2 cm), whereas the treated areas from larger tumors demonstrated incomplete ablation. Additional investigation with histopathologic correlation is needed to fully assess the long-term efficacy of RFA for NSCLC.

The association of cytokeratin-only-positive sentinel lymph nodes and subsequent metastases in breast cancer.

INTRODUCTION: The purpose of this study was to better characterize the clinical significance of cytokeratin immunohistochemistry (IHC)-only-positive lymph node metastases among patients with breast cancer. METHODS: We performed a retrospective review of 334 patients who underwent sentinel lymph node (SLN) biopsy from 1 February 1997 through 31 July 2001. SLN biopsies were evaluated using standard hematoxylin and eosin (H&E) techniques. If H&E was negative, cytokeratin IHC was performed. We then evaluated the incidence of subsequent regional and distant metastatic disease. RESULTS: Cytokeratin IHC was performed on 183 sentinel node biopsies from 180 patients comprising a total of 427 sentinel lymph nodes. The procedures included lumpectomy and SLN biopsy (n = 83), mastectomy with SLN biopsy (n = 7), lumpectomy with SLN biopsy and completion axillary dissection (n = 80), and modified radical mastectomy with SLN biopsy and completion axillary dissection (n = 13). Cytokeratin IHC was negative in 175 axillary specimens and positive in 8 (4.4%) from 8 different patients. In these eight specimens, deeper sections with subsequent H&E staining additionally identified micrometastasis in four patients. Three of these 8 patients (37.5%) developed distant metastatic disease compared with 1 of the 172 patients (0.6%) with negative cytokeratin IHC (P < .001). Additionally, one of the cytokeratin-positive patients developed regional nodal metastasis compared with none of the 172 cytokeratin-negative patients. CONCLUSIONS: Cytokeratin IHC provides a clinically relevant adjunct to H&E staining for evaluating sentinel lymph nodes in breast cancer. These data suggest that patients with cytokeratin-positive sentinel nodes are at increased risk for development of regional and distant metastatic disease.

Phase II Trial of Temozolomide and Sorafenib in Advanced Melanoma Patients with or without Brain Metastases.

PURPOSE: The combination of the oral alkylating agent temozolomide and the oral multikinase inhibitor sorafenib was evaluated in advanced melanoma patients. EXPERIMENTAL DESIGN: Patients with metastatic melanoma (n = 167) were treated on four arms. All patients received sorafenib at 400 mg p.o. twice daily without interruption. Patients without brain metastases or prior temozolomide were randomized between arm A: extended dosing of temozolomide (75 mg/m(2) temozolomide daily for 6 of every 8 weeks) and arm B: standard dosing (150 mg/m(2) temozolomide daily for 5 of every 28 days). Patients previously treated with temozolomide were enrolled on arm C: extended dosing of temozolomide. Patients with brain metastases and no prior temozolomide were assigned to arm D: standard dosing. The primary end point was 6-month progression-free survival (PFS) rate. Secondary end points included response rate, toxicity rates, and the rates of BRAF or NRAS mutations. RESULTS: The 6-month PFS rate for arms A, B, C, and D were 50%, 40%, 11%, and 23%. The median PFS for patients on arm A, B, C, and D was 5.9, 4.2, 2.2, and 3.5 months, respectively. No significant differences were observed between arms A and B in 6-month PFS rate, median PFS, or response rates. Treatment was well tolerated in all arms. No significant differences in toxicity were observed between arms A and B except for more grade 3 to 4 lymphopenia in arm A. CONCLUSION: Temozolomide plus sorafenib was well tolerated and showed activity in melanoma patients without prior history of temozolomide. The activity of this combination regimen warrants further investigation. (Clin Cancer Res 2009;15(24):7711-8).

Colon carcinoma cells induce CXCL11-dependent migration of CXCR3-expressing cytotoxic T lymphocytes in organotypic culture.

Adoptive immunotherapy of cancer patients with cytolytic T lymphocytes (CTL) has been hampered by the inability of the CTL to home into tumors in vivo. Chemokines can attract T lymphocytes to the tumor site, as demonstrated in animal models, but the role of chemokines in T-lymphocyte trafficking toward human tumor cells is relatively unexplored. In the present study, the role of chemokines and their receptors in the migration of a colon carcinoma (CC) patient's CTL toward autologous tumor cells has been studied in a novel three-dimensional organotypic CC culture. CTL migration was mediated by chemokine receptor CXCR3 expressed by the CTL and CXCL11 chemokine secreted by the tumor cells. Excess CXCL11 or antibodies to CXCL11 or CXCR3 inhibited migration of CTL to tumor cells. T cell and tumor cell analyses for CXCR3 and CXCL11 expression, respectively, in ten additional CC samples, may suggest their involvement in other CC patients. Our studies, together with previous studies indicating angiostatic activity of CXCL11, suggest that CXCL11 may be useful as an immunotherapeutic agent for cancer patients when transduced into tumor cells or fused to tumor antigen-specific Ab.