Gregory A. Daniels

Full-Length mRNA-Seq from single cell levels of RNA and individual circulating tumor cells

Genome-wide transcriptome analyses are routinely used to monitor tissue-, disease- and cell type–specific gene expression, but it has been technically challenging to generate expression profiles from single cells. Here we describe a robust mRNA-Seq protocol (Smart-Seq) that is applicable down to single cell levels. Compared with existing methods, Smart-Seq has improved read coverage across transcripts, which enhances detailed analyses of alternative transcript isoforms and identification of single-nucleotide polymorphisms. We determined the sensitivity and quantitative accuracy of Smart-Seq for single-cell transcriptomics by evaluating it on total RNA dilution series. We found that although gene expression estimates from single cells have increased noise, hundreds of differentially expressed genes could be identified using few cells per cell type. Applying Smart-Seq to circulating tumor cells from melanomas, we identified distinct gene expression patterns, including candidate biomarkers for melanoma circulating tumor cells. Our protocol will be useful for addressing fundamental biological problems requiring genome-wide transcriptome profiling in rare cells.

Talimogene Laherparepvec Improves Durable Response Rate in Patients With Advanced Melanoma

PURPOSE Talimogene laherparepvec (T-VEC) is a herpes simplex virus type 1-derived oncolytic immunotherapy designed to selectively replicate within tumors and produce granulocyte macrophage colony-stimulating factor (GM-CSF) to enhance systemic antitumor immune responses. T-VEC was compared with GM-CSF in patients with unresected stage IIIB to IV melanoma in a randomized open-label phase III trial. PATIENTS AND METHODS Patients with injectable melanoma that was not surgically resectable were randomly assigned at a two-to-one ratio to intralesional T-VEC or subcutaneous GM-CSF. The primary end point was durable response rate (DRR; objective response lasting continuously ≥ 6 months) per independent assessment. Key secondary end points included overall survival (OS) and overall response rate. RESULTS Among 436 patients randomly assigned, DRR was significantly higher with T-VEC (16.3%; 95% CI, 12.1% to 20.5%) than GM-CSF (2.1%; 95% CI, 0% to 4.5%]; odds ratio, 8.9; P < .001). Overall response rate was also higher in the T-VEC arm (26.4%; 95% CI, 21.4% to 31.5% v 5.7%; 95% CI, 1.9% to 9.5%). Median OS was 23.3 months (95% CI, 19.5 to 29.6 months) with T-VEC and 18.9 months (95% CI, 16.0 to 23.7 months) with GM-CSF (hazard ratio, 0.79; 95% CI, 0.62 to 1.00; P = .051). T-VEC efficacy was most pronounced in patients with stage IIIB, IIIC, or IVM1a disease and in patients with treatment-naive disease. The most common adverse events (AEs) with T-VEC were fatigue, chills, and pyrexia. The only grade 3 or 4 AE occurring in ≥ 2% of T-VEC-treated patients was cellulitis (2.1%). No fatal treatment-related AEs occurred. CONCLUSION T-VEC is the first oncolytic immunotherapy to demonstrate therapeutic benefit against melanoma in a phase III clinical trial. T-VEC was well tolerated and resulted in a higher DRR (P < .001) and longer median OS (P = .051), particularly in untreated patients or those with stage IIIB, IIIC, or IVM1a disease. T-VEC represents a novel potential therapy for patients with metastatic melanoma.

Phase II Clinical Trial of a Granulocyte-Macrophage Colony-Stimulating Factor–Encoding, Second-Generation Oncolytic Herpesvirus in Patients With Unresectable Metastatic Melanoma

PURPOSE Treatment options for metastatic melanoma are limited. We conducted this phase II trial to assess the efficacy of JS1/34.5-/47-/granulocyte-macrophage colony-stimulating factor (GM-CSF) in stages IIIc and IV disease. PATIENTS AND METHODS Treatment involved intratumoral injection of up to 4 mL of 10(6) pfu/mL of JS1/34.5-/47-/GM-CSF followed 3 weeks later by up to 4 mL of 10(8) pfu/mL every 2 weeks for up to 24 treatments. Clinical activity (by RECIST [Response Evaluation Criteria in Solid Tumors]), survival, and safety parameters were monitored. RESULTS Fifty patients (stages IIIc, n = 10; IVM1a, n = 16; IVM1b, n = 4; IVM1c, n = 20) received a median of six injection sets; 74% of patients had received one or more nonsurgical prior therapies for active disease, including dacarbazine/temozolomide or interleukin-2 (IL-2). Adverse effects were limited primarily to transient flu-like symptoms. The overall response rate by RECIST was 26% (complete response [CR], n = 8; partial response [PR], n = 5), and regression of both injected and distant (including visceral) lesions occurred. Ninety-two percent of the responses had been maintained for 7 to 31 months. Ten additional patients had stable disease (SD) for greater than 3 months, and two additional patients had surgical CR. On an extension protocol, two patients subsequently achieved CR by 24 months (one previously PR, one previously SD), and one achieved surgical CR (previously PR). Overall survival was 58% at 1 year and 52% at 24 months. CONCLUSION The 26% response rate, with durability in both injected and uninjected lesions including visceral sites, together with the survival rates, are evidence of systemic effectiveness. This effectiveness, combined with a limited toxicity profile, warrants additional evaluation of JS1/34.5-/47-/GM-CSF in metastatic melanoma. A US Food and Drug Administration-approved phase III investigation is underway.

Neutral amino acid transport systems in animal cells: Potential targets of oncogene action and regulators of cellular growth

Amino acid uptake by a tissue cell in a multicellular organism appears to be one of several determinants of growth rate. While a multiplicity of transport systems for amino acids exists for the uptake of these essential nutrients, only a few of these have been subject to careful and critical analysis (see section B). Of these systems, one particular system, the system A transporter, has been found to be regulated by a wide variety of external stimuli and conditions (section C), and in some cells, its activity correlates reasonably well with growth state (section D). System A is thus one of the few identified transport systems which appears to be a target of protooncogene and oncogene action and regulator of cellular growth (section E). In this review, the evidence for this postulate is presented and evaluated.

A simple method to cure established tumors by inflammatory killing of normal cells

We describe a simple technology used to cure an established metastatic disease. Intradermal injection of plasmid DNA encoding a transcriptionally targeted cytotoxic gene, along with hsp70, not only promoted tissue-specific, inflammatory killing of normal melanocytes, but also induced a CD8+ T-cell–dependent, antigen-specific response in mice that eradicated systemically established B16 tumors. This CD8+ T cell response was subsequently suppressed in vivo within a few days. The data demonstrate that deliberate destruction of normal tissue can be exploited to generate immunity against a malignant disease originating from that tissue. This approach obviates the need to identify tumor antigens and does not require complex isolation of tumor cells or their derivatives. In addition, it provides a model system for studying the mechanisms underlying the etiology and control of autoimmune diseases. Finally, despite targeting normal tissue, therapy could be separated from development of overt autoimmune symptoms, suggesting that the strategy may be valuable against tumors derived from both non-essential and essential tissue types.*Note: In the version of this article originally published online, the name of one of the authors was spelled incorrectly. Mayoi Lai should be Maoyi Lai. This mistake has been corrected in the HTML version and will appear correctly in print.

Tumor Mutational Burden as an Independent Predictor of Response to Immunotherapy in Diverse Cancers

Immunotherapy induces durable responses in a subset of patients with cancer. High tumor mutational burden (TMB) may be a response biomarker for PD-1/PD-L1 blockade in tumors such as melanoma and non–small cell lung cancer (NSCLC). Our aim was to examine the relationship between TMB and outcome in diverse cancers treated with various immunotherapies. We reviewed data on 1,638 patients who had undergone comprehensive genomic profiling and had TMB assessment. Immunotherapy-treated patients (N = 151) were analyzed for response rate (RR), progression-free survival (PFS), and overall survival (OS). Higher TMB was independently associated with better outcome parameters (multivariable analysis). The RR for patients with high (≥20 mutations/mb) versus low to intermediate TMB was 22/38 (58%) versus 23/113 (20%; P = 0.0001); median PFS, 12.8 months vs. 3.3 months (P ≤ 0.0001); median OS, not reached versus 16.3 months (P = 0.0036). Results were similar when anti-PD-1/PD-L1 monotherapy was analyzed (N = 102 patients), with a linear correlation between higher TMB and favorable outcome parameters; the median TMB for responders versus nonresponders treated with anti-PD-1/PD-L1 monotherapy was 18.0 versus 5.0 mutations/mb (P < 0.0001). Interestingly, anti-CTLA4/anti-PD-1/PD-L1 combinations versus anti-PD-1/PD-L1 monotherapy was selected as a factor independent of TMB for predicting better RR (77% vs. 21%; P = 0.004) and PFS (P = 0.024). Higher TMB predicts favorable outcome to PD-1/PD-L1 blockade across diverse tumors. Benefit from dual checkpoint blockade did not show a similarly strong dependence on TMB. Mol Cancer Ther; 16(11); 2598–608. ©2017 AACR.

Potent Selection of Antigen Loss Variants of B16 Melanoma following Inflammatory Killing of Melanocytes In vivo

We have reported that i.d. injection of plasmids encoding hsp70 and a suicide gene transcriptionally targeted to melanocytes generates specific proinflammatory killing of melanocytes. The resulting CD8+ T cell response eradicates systemically established B16 tumors. Here, we studied the consequences of that CD8+ T cell response on the phenotype of preexisting tumor. In suboptimal protocols, the T cell response selected B16 variants, which grow extremely aggressively, are amelanotic and have lost expression of the tyrosinase and tyrosinase-related protein 2 (TRP-2) antigens. However, expression of other melanoma-associated antigens, such as gp100, was not affected. Antigen loss could be reversed by long-term growth in culture away from immune-selective pressures or within 96 hours by treatment with the demethylating agent 5-azacytidine (5-Aza). When transplanted back into syngeneic animals, variants were very poorly controlled by further vaccination. However, a combination of vaccination with 5-Aza to reactivate antigen expression in tumors in situ generated highly significant improvements in therapy over treatment with vaccine or 5-Aza alone. These data show that inflammatory killing of normal cells activates a potent T cell response targeted against a specific subset of self-antigens but can also lead to the immunoselection of tumor variants. Moreover, our data indicate that emergence of antigen loss variants may often be due to reversible epigenetic mechanisms within the tumor cells. Therefore, combination therapy using vaccination and systemic treatment with 5-Aza or other demethylating agents may have significant therapeutic benefits for antitumor immunotherapy.

Adoptive Transfer of MART-1 T-Cell Receptor Transgenic Lymphocytes and Dendritic Cell Vaccination in Patients with Metastatic Melanoma

Purpose: It has been demonstrated that large numbers of tumor-specific T cells for adoptive cell transfer (ACT) can be manufactured by retroviral genetic engineering of autologous peripheral blood lymphocytes and expanding them over several weeks. In mouse models, this therapy is optimized when administered with dendritic cell (DC) vaccination. We developed a short 1-week manufacture protocol to determine the feasibility, safety, and antitumor efficacy of this double cell therapy. Experimental Design: A clinical trial (NCT00910650) adoptively transferring MART-1 T-cell receptor (TCR) transgenic lymphocytes together with MART-1 peptide-pulsed DC vaccination in HLA-A2.1 patients with metastatic melanoma. Autologous TCR transgenic cells were manufactured in 6 to 7 days using retroviral vector gene transfer, and reinfused with (n = 10) or without (n = 3) prior cryopreservation. Results: A total of 14 patients with metastatic melanoma were enrolled and 9 of 13 treated patients (69%) showed evidence of tumor regression. Peripheral blood reconstitution with MART-1–specific T cells peaked within 2 weeks of ACT, indicating rapid in vivo expansion. Administration of freshly manufactured TCR transgenic T cells resulted in a higher persistence of MART-1–specific T cells in the blood as compared with cryopreserved. Evidence that DC vaccination could cause further in vivo expansion was only observed with ACT using noncryopreserved T cells. Conclusion: Double cell therapy with ACT of TCR-engineered T cells with a very short ex vivo manipulation and DC vaccines is feasible and results in antitumor activity, but improvements are needed to maintain tumor responses. Clin Cancer Res; 20(9); 2457–65. ©2014 AACR.

Melanoma, version 4.2014: Featured updates to the NCCN guidelines

The NCCN Guidelines for Melanoma provide multidisciplinary recommendations on the clinical management of patients with melanoma. This NCCN Guidelines Insights report highlights notable recent updates. Foremost of these is the exciting addition of the novel agents ipilimumab and vemurafenib for treatment of advanced melanoma. The NCCN panel also included imatinib as a treatment for KIT-mutated tumors and pegylated interferon alfa-2b as an option for adjuvant therapy. Also important are revisions to the initial stratification of early-stage lesions based on the risk of sentinel lymph node metastases, and revised recommendations on the use of sentinel lymph node biopsy for low-risk groups. Finally, the NCCN panel reached clinical consensus on clarifying the role of imaging in the workup of patients with melanoma.

Killing of Normal Melanocytes, Combined with Heat Shock Protein 70 and CD40L Expression, Cures Large Established Melanomas

Previously, we showed that nine intradermal injections of a plasmid in which the HSVtk suicide gene is expressed from a melanocyte-specific promoter (Tyr-HSVtk), combined with a plasmid expressing heat shock protein 70 (CMV-hsp70), along with systemic ganciclovir, kills normal melanocytes and raises a CD8+ T cell response that is potent enough to eradicate small, 3-day established B16 tumors. We show in this study that, in that regimen, hsp70 acts as a potent immune adjuvant through TLR-4 signaling and local induction of TNF-α. hsp70 is required for migration of APC resident in the skin to the draining lymph nodes to present Ags, derived from the killing of normal melanocytes, to naive T cells. The addition of a plasmid expressing CD40L increased therapeutic efficacy, such that only six plasmid injections were now required to cure large, 9-day established tumors. Generation of potent immunological memory against rechallenge in cured mice accompanied these therapeutic gains, as did induction of aggressive autoimmune symptoms. Expression of CD40L, along with hsp70, increased both the frequency and activity of T cells activated against melanocyte-derived Ags. In this way, addition of CD40L to the hsp70-induced inflammatory killing of melanocytes can be used to cure large established tumors and to confer immunological memory against tumor cells, although a concomitant increase in autoimmune sequelae also is produced.