Neoadjuvant-Adjuvant or Adjuvant-Only Pembrolizumab in Advanced Melanoma.

BACKGROUND: Whether pembrolizumab given both before surgery (neoadjuvant therapy) and after surgery (adjuvant therapy), as compared with pembrolizumab given as adjuvant therapy alone, would increase event-free survival among patients with resectable stage III or IV melanoma is unknown. METHODS: In a phase 2 trial, we randomly assigned patients with clinically detectable, measurable stage IIIB to IVC melanoma that was amenable to surgical resection to three doses of neoadjuvant pembrolizumab, surgery, and 15 doses of adjuvant pembrolizumab (neoadjuvant-adjuvant group) or to surgery followed by pembrolizumab (200 mg intravenously every 3 weeks for a total of 18 doses) for approximately 1 year or until disease recurred or unacceptable toxic effects developed (adjuvant-only group). The primary end point was event-free survival in the intention-to-treat population. Events were defined as disease progression or toxic effects that precluded surgery; the inability to resect all gross disease; disease progression, surgical complications, or toxic effects of treatment that precluded the initiation of adjuvant therapy within 84 days after surgery; recurrence of melanoma after surgery; or death from any cause. Safety was also evaluated. RESULTS: At a median follow-up of 14.7 months, the neoadjuvant-adjuvant group (154 patients) had significantly longer event-free survival than the adjuvant-only group (159 patients) (P = 0.004 by the log-rank test). In a landmark analysis, event-free survival at 2 years was 72% (95% confidence interval [CI], 64 to 80) in the neoadjuvant-adjuvant group and 49% (95% CI, 41 to 59) in the adjuvant-only group. The percentage of patients with treatment-related adverse events of grades 3 or higher during therapy was 12% in the neoadjuvant-adjuvant group and 14% in the adjuvant-only group. CONCLUSIONS: Among patients with resectable stage III or IV melanoma, event-free survival was significantly longer among those who received pembrolizumab both before and after surgery than among those who received adjuvant pembrolizumab alone. No new toxic effects were identified. (Funded by the National Cancer Institute and Merck Sharp and Dohme; S1801 ClinicalTrials.gov number, NCT03698019.).

Routine Computer Tomography Imaging for the Detection of Recurrences in High-Risk Melanoma Patients.

BACKGROUND: The use of routine CT imaging for surveillance in asymptomatic patients with cutaneous melanoma is controversial. We report our experience using a surveillance strategy that included CT imaging for a cohort of patients with high-risk melanoma. METHODS: A total of 466 patients with high-risk cutaneous melanoma enrolled in adjuvant immunotherapy trials were followed for tumor progression by physical examination, labs, and CT imaging as defined by protocol. Evaluations were obtained at least every 6 months for year 1, every 6 months for year 2, and then annually for the remainder of the 5-year study. Time to tumor progression, sites of recurrence, and the method of relapse detection were identified. RESULTS: The patient cohort consisted of 115 stage II patients, 328 stage III patients, and 23 patients with resected stage IV melanoma. The medium time to progression for the 225 patients who developed tumor progression was 7 months. Tumor progression was detected by patients, physician examination or routine labs, or by CT imaging alone in 27, 14, and 59% of cases respectively. Melanoma recurrences were noted to be locoregional in 36% of cases and systemic in 64% of cases. Thirty percent of patients with locoregional relapse and 75% of patients with systemic relapse were detected solely by CT imaging. CONCLUSIONS: CT imaging alone detected the majority of sites of disease progression in our patients with high-risk cutaneous melanoma. This disease was not heralded by symptoms, physical examination, or blood work. Although the benefit of the early detection of advanced melanoma is unknown, this experience is relevant because of the rapid development and availability of potentially curative immunotherapies.

Modified breast cancer model for preclinical immunotherapy studies.

BACKGROUND: Interest in immunotherapy for breast cancer is rapidly emerging, and applicable animal models that mimic human cancer are urgently needed for preclinical studies. This study aimed to improve a technique for orthotopic inoculation of syngeneic breast cancer cells to be used as a preclinical animal model for immunotherapy. MATERIALS AND METHODS: We used our previously reported murine model of orthotopic cancer cell inoculation under direct vision and compared the efficiency of tumorigenesis with tumor cells suspended in either phosphate-buffered saline or Matrigel containing varying numbers of cells. As a model for immune rejection, murine BALB/c-derived 4T1-luc2 breast cancer cells were inoculated orthotopically into both BALB/c and C57BL/6 mice. RESULTS: Matrigel-suspended cells formed larger tumors with higher efficiency than phosphate-buffered saline-suspended cells. The maximum volume of Matrigel that could be inoculated without spillage was 20 µL and 30 µL in the #2 and #4 mammary fat pads, respectively. Tumor take rates increased as the injected cell number increased. In this immune rejection model, there were no significant differences in tumor weight between the strains up to day 7, after which tumor weight decreased in C57BL/6 mice. Bioluminescence in C57BL/6 mice was also significantly less than that in BALB/c mice and increased up to day 7, then swiftly decreased thereafter. CONCLUSIONS: This improved technique of innoculating murine breast cancer cells using bioluminescence technology may be useful in evaluating the efficacy of tumor regression mediated by immune responses, as shown by an allogeneic response in C57BL/6 mice.

B-cell depletion and remissions of malignancy along with cytokine-associated toxicity in a clinical trial of anti-CD19 chimeric-antigen-receptor-transduced T cells.

We conducted a clinical trial to assess adoptive transfer of T cells genetically modified to express an anti-CD19 chimeric Ag receptor (CAR). Our clinical protocol consisted of chemotherapy followed by an infusion of anti-CD19-CAR-transduced T cells and a course of IL-2. Six of the 8 patients treated on our protocol obtained remissions of their advanced, progressive B-cell malignancies. Four of the 8 patients treated on the protocol had long-term depletion of normal polyclonal CD19(+) B-lineage cells. Cells containing the anti-CD19 CAR gene were detected in the blood of all patients. Four of the 8 treated patients had prominent elevations in serum levels of the inflammatory cytokines IFNγ and TNF. The severity of acute toxicities experienced by the patients correlated with serum IFNγ and TNF levels. The infused anti-CD19-CAR-transduced T cells were a possible source of these inflammatory cytokines because we demonstrated peripheral blood T cells that produced TNF and IFNγ ex vivo in a CD19-specific manner after anti-CD19-CAR-transduced T-cell infusions. Anti-CD19-CAR-transduced T cells have great promise to improve the treatment of B-cell malignancies because of a potent ability to eradicate CD19(+) cells in vivo; however, reversible cytokine-associated toxicities occurred after CAR-transduced T-cell infusions.

Adoptive Cell Therapy for Solid Tumors: Current Status in Melanoma and Next-Generation Therapies.

Lifileucel or TIL has recently been FDA approved for metastatic melanoma patients as first cell therapy for a solid tumor. We discuss roll-out of TIL as new SOC and other upcoming new cell therapies.

Complete genome sequence of a tenth human polyomavirus.

Nine polyomavirus (PyV) species are known to productively infect humans. The circular DNA genomes of PyVs are readily detectable using rolling circle amplification (RCA). RCA-based analysis of condyloma specimens from a patient with warts, hypogammaglobulinemia, infections, and myelokathexis (WHIM) syndrome demonstrated the presence of a tenth apparently human-tropic polyomavirus species, which we name HPyV10.

A novel nomogram for peritoneal mesothelioma predicts survival.

BACKGROUND: Malignant peritoneal mesothelioma (MPM) is a rare disease treated with cytoreductive surgery (CRS) and hyperthermic intraperitoneal chemotherapy (HIPEC). Estimation of personalized survival times can potentially guide treatment and surveillance. METHODS: We analyzed 104 patients who underwent CRS and cisplatin-based HIPEC for MPM. By means of 25 demographic, laboratory, operative, and histopathological variables, we developed a novel nomogram using machine-learned Bayesian belief networks with stepwise training, testing, and cross-validation. RESULTS: The mean peritoneal carcinomatosis index (PCI) was 15, and 66 % of patients had a completeness of cytoreduction (CC) score of 0 or 1. Eighty-seven percent of patients had epithelioid histology. The median follow-up time was 49 (1-195) months. The 3- and 5-year overall survivals (OS) were 58 and 46 %, respectively. The histological subtype, pre-CRS PCI, and preoperative serum CA-125 had the greatest impact on OS and were included in the nomogram. The mean areas under the receiver operating characteristic curve for the 10-fold cross-validation of the 3- and 5-year models were 0.77 and 0.74, respectively. The graphical calculator or nomogram uses color coding to assist the clinician in quickly estimating individualized patient-specific survival before surgery. CONCLUSIONS: Machine-learned Bayesian belief network analysis generated a novel nomogram predicting 3- and 5-year OS in patients treated with CRS and HIPEC for MPM. Pre-CRS estimation of survival times may potentially individualize patient care by influencing the use of systemic therapy and frequency of diagnostic imaging, and might prevent CRS in patients unlikely to achieve favorable outcomes despite surgical intervention.

Adoptive cell transfer for patients with metastatic melanoma: the potential and promise of cancer immunotherapy.

BACKGROUND: Current FDA-approved therapeutic options for patients with metastatic melanoma include dacarbazine, interleukin 2, ipilimumab, vemurafenib, dabrafenib, and trametinib, but long-term tumor regression using available agents remains out of reach for most patients. Adoptive cell transfer (ACT) with autologous tumor-infiltrating lymphocytes (TILs) has shown encouraging results in clinical trials, with evidence of durable ongoing complete responses in patients with advanced melanoma. Emerging techniques to engineer T-cell receptors (TCRs) or chimeric antigen receptors (CARs) using lymphocytes from peripheral blood may offer new tactics in ACT. METHODS: We reviewed the literature to provide a synopsis on the development and clinical trial results of ACT, as well as the future outlook for using ACT in patients with metastatic melanoma. RESULTS: ACT with TILs as part of a lymphodepleting regimen has been shown in clinical trials to cause objective clinical responses in approximately 40% to 72% of patients with metastatic melanoma, with up to 40% of those patients experiencing complete responses lasting up to 7 years ongoing. Pilot trials using TCR-engineered cells against melanoma-associated antigens MART-1 and gp100 and the cancer-testis antigen NY-ESO-1 have shown clinical responses in patients with melanoma. CAR cells directed against melanoma have been tested only in preclinical models; however, CAR cells targeting other histologies such as lymphoma have elicited antitumor responses in patients. CONCLUSIONS: An example of state-of-the-art personalized medicine, ACT is a potentially curative therapy for patients with metastatic melanoma. Ongoing trials aiming to simplify the regimens may allow a broader range of patients to be treated and enable ACT to be offered by academic cancer centers.

Utility of intraoperative parathyroid hormone monitoring in patients with multiple endocrine neoplasia type 1-associated primary hyperparathyroidism undergoing initial parathyroidectomy.

BACKGROUND: Intraoperative parathyroid hormone monitoring (IOPTH) is a widely used adjunct for primary hyperparathyroidism (pHPT). However, the benefit of IOPTH in familial pHPT, such as in multiple endocrine neoplasia type I (MEN1), remains unclear. METHODS: We performed a retrospective analysis of 52 patients with MEN1-associated pHPT undergoing initial parathyroidectomy with IOPTH monitoring at our institution. Parathyroid hormone (PTH) levels were measured before skin incision and 10 min after resection of the last parathyroid gland. Variables analyzed included percent drop of PTH from baseline and the final PTH level compared to the normal reference range (RR). RESULTS: A total of 52 patients underwent initial subtotal parathyroidectomy with IOPTH. An IOPTH decrease cutoff of ≥75 % from baseline had the highest biochemical cure rate (87 %). In the remaining 13 % who met this cutoff, all had persistent pHPT, with ≥90 % drop of PTH from baseline. The remaining patients, who did not meet the ≥75 % cutoff, were cured. Follow-up was available for three of four patients with final IOPTH levels above the RR: one had persistent pHPT, two had hypoparathyroidism (50 %). When a postresection PTH level was within the RR, 88 % of patients were cured. While considered cured from pHPT, 7 % of patients in this group developed permanent hypoparathyroidism. When the final PTH level dropped below the RR, 28 % developed permanent hypoparathyroidism. CONCLUSIONS: A cutoff in IOPTH decrease of ≥75 % from baseline has the highest biochemically cure rate in patients with pHPT associated with MEN1. However, a 75 % cutoff in IOPTH decrease does not exclude persistent pHPT. The absolute IOPTH value does not accurately predict postoperative hypoparathyroidism.

Preoperative localizing studies for initial parathyroidectomy in MEN1 syndrome: is there any benefit?

BACKGROUND: The objective of the present study was to evaluate the utility of preoperative localizing studies in patients with MEN1 undergoing initial bilateral neck exploration (BNE) and parathyroidectomy for pHPT. METHODS: We performed a retrospective analysis of patients diagnosed with MEN1 who underwent initial parathyroidectomy between December 1993 and December 2010. Results of preoperative localizing studies were compared with intraoperative findings and outcome. RESULTS: Sixty patients with MEN1 (32 females and 28 males) underwent initial subtotal parathyroidectomy. The median age at the time of surgery was 33 years (range: 13-78 years). Fifty-three patients had one or more positive localizing study results. Neck ultrasonography, sestamibi scan, parathyroid protocol computed tomography scan, and neck and mediastinum magnetic resonance imaging were performed in 93, 91, 32, and 19% of patients, respectively. Fifty-three patients (88%) had cervical thymectomy. Twenty patients had 24 ectopic parathyroid glands; 18 glands were in the thymus (75%). Preoperative localizing studies identified 9 of 24 ectopic parathyroid glands (38%), including 4 ectopic glands outside the thymus in 4 patients (7%); 3 were detected by ultrasonography. There were no supernumerary glands identified on preoperative localizing studies. CONCLUSIONS: In patients with MEN1, preoperative localizing studies identified a subset of ectopic glands (38%). Preoperative localizing studies may alter the operative approach in 7% of patients. Ultrasonography can detect most ectopic parathyroid glands outside thymus. This suggests that routine preoperative localizing studies to identify ectopic and supernumerary enlarged parathyroid glands is not useful in the majority of patients with MEN1 undergoing bilateral neck exploration and subtotal parathyroidectomy with cervical thymectomy.

Rate of clinically significant postoperative pancreatic fistula in pancreatic neuroendocrine tumors.

BACKGROUND: In 2005, the International Study Group of Pancreatic Fistula (ISGPF) developed a definition and grading system for postoperative pancreatic fistula (POPF). The authors sought to determine the rate of POPF after enucleation and/or resection of pancreatic neuroendocrine tumors (PNET) and to identify clinical, surgical, or pathologic factors associated with POPF. METHODS: A retrospective analysis of pancreatic enucleations and resections performed from March 1998 to April 2010. We defined a clinically significant POPF as a grade B that required nonoperative intervention and grade C. RESULTS: One hundred twenty-two patients were identified; 62 patients had enucleations and 60 patients had resections of PNET. The rate of clinically significant POPF was 23.7 % (29/122). For pancreatic enucleation, the POPF rate was 27.4 % (17/62, 14 grade B, 3 grade C). The pancreatic resection group had a POPF rate of 20 % (12/60, 10 grade B, 2 grade C). This difference was not significant (p = 0.4). In univariate analyses, patients in the enucleation group with hereditary syndromes (p = 0.02) and non-insulinoma tumors (p = 0.02) had a higher POPF rate. Patients in the resection group with body mass index (BMI) > 25 (p < 0.01), multiple endocrine neoplasia type 1 (MEN-1; p < 0.01) and those who underwent simultaneous multiple procedures (p = 0.02) had a higher POPF rate. Multivariate analyses revealed that hereditary syndromes were able to predict POPF in the enucleation group, while having BMI > 25 and increasing lesion size were also associated with POPF in the group undergoing resection. CONCLUSIONS: We found a clinically significant POPF rate after surgery in PNET to be 23.7 % with no difference by the type of operation. Our POPF rate is comparable to that reported in the literature for pancreatic resection for other types of tumors. Certain inherited genetic diseases-von Hippel-Lindau disease (VHL) and MEN-1-were associated with higher POPF rates.

T cells targeting carcinoembryonic antigen can mediate regression of metastatic colorectal cancer but induce severe transient colitis.

Autologous T lymphocytes genetically engineered to express a murine T cell receptor (TCR) against human carcinoembryonic antigen (CEA) were administered to three patients with metastatic colorectal cancer refractory to standard treatments. All patients experienced profound decreases in serum CEA levels (74-99%), and one patient had an objective regression of cancer metastatic to the lung and liver. However, a severe transient inflammatory colitis that represented a dose limiting toxicity was induced in all three patients. This report represents the first example of objective regression of metastatic colorectal cancer mediated by adoptive T cell transfer and illustrates the successful use of a TCR, raised in human leukocyte antigen (HLA) transgenic mice, against a human tumor associated antigen. It also emphasizes the destructive power of small numbers of highly avid T cells and the limitations of using CEA as a target for cancer immunotherapy.

Efficacy and safety of lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, in patients with advanced melanoma after progression on immune checkpoint inhibitors and targeted therapies: pooled analysis of consecutive cohorts of the C-144-01 study.

BACKGROUND: Patients with advanced melanoma have limited treatment options after progression on immune checkpoint inhibitors (ICI). Lifileucel, a one-time autologous tumor-infiltrating lymphocyte (TIL) cell therapy, demonstrated an investigator-assessed objective response rate (ORR) of 36% in 66 patients who progressed after ICI and targeted therapy. Herein, we report independent review committee (IRC)-assessed outcomes of 153 patients treated with lifileucel in a large multicenter Phase 2 cell therapy trial in melanoma. METHODS: Eligible patients had advanced melanoma that progressed after ICI and targeted therapy, where appropriate. Melanoma lesions were resected (resected tumor diameter ≥1.5 cm) and shipped to a central good manufacturing practice facility for 22-day lifileucel manufacturing. Patients received a non-myeloablative lymphodepletion regimen, a single lifileucel infusion, and up to six doses of high-dose interleukin-2. The primary endpoint was IRC-assessed ORR (Response Evaluation Criteria in Solid Tumors V.1.1). RESULTS: The Full Analysis Set consisted of 153 patients treated with lifileucel, including longer-term follow-up on the 66 patients previously reported. Patients had received a median of 3.0 lines of prior therapy (81.7% received both anti-programmed cell death protein 1 and anti-cytotoxic lymphocyte-associated protein 4) and had high disease burden at baseline (median target lesion sum of diameters (SOD): 97.8 mm; lactate dehydrogenase (LDH) >upper limit of normal: 54.2%). ORR was 31.4% (95% CI: 24.1% to 39.4%), with 8 complete responses and 40 partial responses. Median duration of response was not reached at a median study follow-up of 27.6 months, with 41.7% of the responses maintained for ≥18 months. Median overall survival and progression-free survival were 13.9 and 4.1 months, respectively. Multivariable analyses adjusted for Eastern Cooperative Oncology Group performance status demonstrated that elevated LDH and target lesion SOD >median were independently correlated with ORR (p=0.008); patients with normal LDH and SOD

Sentinel lymph node biopsy for melanoma: indications and rationale.

BACKGROUND: The disease status of regional lymph nodes is the most important prognostic indicator for patients with melanoma. Sentinel lymph node biopsy (SLNB) was developed as a technique to surgically assess the regional lymph nodes and spare node-negative patients unnecessary and potentially morbid complete lymphadenectomies. METHODS: We reviewed the literature on SLNB for cutaneous melanoma to provide insight into the rationale for the current widespread use of SLNB. RESULTS: Multiple studies show that the status of the SLN is an important prognostic indicator. Those with positive SLNs have significantly decreased disease-free and melanoma-specific survival compared with those who have negative SLNs. In the Multicenter Selective Lymphadenectomy Trial I (MSLT-I), in which patients with intermediate-thickness melanoma were randomized to SLNB (and immediate completion lymphadenectomy if the SLN was positive) vs observation (and a lymphadenectomy only after presenting with clinically evident recurrence), the 5-year survival rate was 72.3% for patients with positive sentinel nodes and 90.2% for those with negative sentinel nodes (P < .001). Although overall survival was not increased in patients who underwent SLNB compared with those who were randomized to observation, patients who underwent SLNB had a significantly increased 5-year disease-free survival rate compared with those who underwent observation alone (78.3% in the biopsy group and 73.1% in the observation group; P = .009). For those with nodal metastases, patients who underwent SLNB and immediate lymphadenectomy had an increased overall 5-year survival rate compared with those who had lymphadenectomy only after presenting with clinically evident disease (72.3% vs 52.4%; P = .004). Moreover, other studies show that for patients with thin melanomas or= 0.76 mm and increased mitotic rate have been shown to be associated with an increased incidence of SLN metastases. CONCLUSIONS: SLNB provides important prognostic and staging data with minimal morbidity and can be used to identify regional node-negative patients who would not benefit from a complete nodal dissection. In our opinion, SLNB should be performed on most patients (with acceptable surgical and anesthesia risk) who have melanomas with a Breslow depth >or= 0.76 mm.

CTLA-4 blockade with monoclonal antibodies in patients with metastatic cancer: surgical issues.

BACKGROUND: CTLA-4 (cytotoxic T lymphocyte-associated antigen 4) is a modulatory receptor on T cells involved in downregulating T cell activation. In animal models, CTLA-4 blockade abrogates tolerance to "self" antigens, resulting in the augmentation of antitumor immunity and induction of autoimmunity. CTLA-4 blockade by means of monoclonal antibodies (ipilimumab and tremelimumab) has been evaluated in multiple clinical trials in patients with metastatic cancer, mainly those with melanoma and renal cell cancer. METHODS: We examine available literature and ongoing clinical trials with ipilimumab and tremelimumab and review our own experience with patients treated with CTLA-4 blockade, with an emphasis on issues of direct relevance to surgical oncologists. RESULTS: CTLA-4 blockade can cause durable tumor regression in patients with metastatic melanoma and other solid tumors. Grade III/IV autoimmune toxicity has been frequently encountered in clinical trials and includes enterocolitis, dermatitis, hypophysitis, uveitis, and hepatitis. Enterocolitis is the most common immune-related adverse event and may cause severe diarrhea requiring intravenous hydration, high-dose corticosteroids, and blockade of tumor necrosis factor alpha with infliximab. Most patients respond to medical treatment, but up to 12% with grade III/IV enterocolitis develop perforation or bleeding that requires colectomy. CONCLUSIONS: As more patients are enrolled onto clinical trials involving ipilimumab and tremelimumab, an increasing number of surgeons may be involved in the care of these patients who develop treatment-related complications. In this report, we review the rationale for CTLA-4 blockade and review selected clinical studies published so far with ipilimumab and tremelimumab. We offer guidelines on the management of patients who develop enterocolitis.

Autoimmunity correlates with tumor regression in patients with metastatic melanoma treated with anti-cytotoxic T-lymphocyte antigen-4.

PURPOSE: Previously, we reported our experience treating 14 patients with metastatic melanoma using a fully human antibody to cytotoxic T-lymphocyte antigen-4 (anti-CTLA-4) in conjunction with peptide vaccination. We have now treated 56 patients to evaluate two different dose schedules of anti-CTLA-4 and to explore the relationship between autoimmunity and tumor regression. PATIENTS AND METHODS: A total of 56 patients with progressive stage IV melanoma were enrolled onto the study. All had Karnofsky performance status > or = 60% with no prior history of autoimmunity. Twenty-nine patients received 3 mg/kg anti-CTLA-4 every 3 weeks, whereas 27 received 3 mg/kg as their initial dose with subsequent doses reduced to 1 mg/kg every 3 weeks. In both cohorts patients received concomitant vaccination with two modified HLA-A*0201-restricted peptides from the gp100 melanoma-associated antigen, gp100:209-217(210M) and gp100:280-288(288V). RESULTS: Two patients achieved a complete response (ongoing at 30 and 31 months, respectively) and five patients achieved a partial response (durations of 4, 6, 25+, 26+, and 34+ months, respectively), for an overall objective response rate of 13%. Tumor regression was seen in lung, liver, brain, lymph nodes, and subcutaneous sites. Of 14 patients with grade 3/4 autoimmune toxicity, five (36%) experienced a clinical response compared with only two responses in the 42 patients (5%) with no autoimmune toxicity (P = .008). There were no significant differences in response rate or toxicity between the two dose schedules. CONCLUSION: Administration of anti-CTLA-4 monoclonal antibody plus peptide vaccination can cause durable objective responses, which correlate with the induction of autoimmunity, in patients with metastatic melanoma.

Targeting vaccinia to solid tumors with local hyperthermia.

We have previously demonstrated that mutant vaccinia viruses target tumors in vivo after systemic delivery, and they have potential as vectors for tumor-directed gene therapy. We hypothesized that hyperthermia may augment vaccinia delivery to tumors after systemic injection, as hyperthermia increases the permeability of the endothelial vasculature to nanoparticles. In our in vitro experiments, we have shown that hyperthermia does not alter tumor cells' susceptibility to the intrinsic cytopathogenicity of the vaccinia virus compared with normothermic controls. Hyperthermia also does not change the viral infectivity or the level of viral marker gene expression when compared with normothermia. In an in vitro model of endothelial cell monolayer permeability, we have demonstrated that hyperthermia increases the permeability of the monolayer to vaccinia virus and that this phenomenon is completely reversible. In vivo we have demonstrated that the tumors that were treated with systemic vaccinia under conditions of hyperthermia (41.5 degrees C for 30 min) had significantly higher levels of vaccinia marker gene activity (>100-fold) than those treated under normothermic conditions (p < 0.05) and that this effect was specific to tumor. We also demonstrated that mice with 1 cm subcutaneous tumors treated with a systemically delivered, conditionally replicating vaccinia under conditions of hyperthermia had complete tumor regression in 50% and significantly improved antitumor response, compared with normothermic viral-treated controls (mean tumor volume of 110 mm(3) vs 3169 mm(3), 13 days after treatment) and compared with hyperthermic, nonvirally treated control animals (p < 0.0001). Regional hyperthermia improves vaccinia targeting to tumors, and thereby enhances the antitumor response.

Tumor-infiltrating lymphocytes genetically engineered with an inducible gene encoding interleukin-12 for the immunotherapy of metastatic melanoma.

PURPOSE: Infusion of interleukin-12 (IL12) can mediate antitumor immunity in animal models, yet its systemic administration to patients with cancer results in minimal efficacy and severe toxicity. Here, we evaluated the antitumor activity of adoptively transferred human tumor-infiltrating lymphocytes (TILs) genetically engineered to secrete single-chain IL12 selectively at the tumor site. EXPERIMENTAL DESIGN: Thirty-three patients with metastatic melanoma were treated in a cell dose-escalation trial of autologous TILs transduced with a gene encoding a single-chain IL12 driven by a nuclear factor of the activated T cells promoter (NFAT.IL12). No IL2 was administered. RESULTS: The administration of 0.001 to 0.1 × 10(9) NFAT.IL12-transduced TILs to 17 patients resulted in a single, objective response (5.9%). However, at doses between 0.3 and 3 × 10(9) cells, 10 of 16 patients (63%) exhibited objective clinical responses. The responses tended to be short, and the administered IL12-producing cells rarely persisted at 1 month. Increasing cell doses were associated with high serum levels of IL12 and IFNγ as well as clinical toxicities, including liver dysfunction, high fevers, and sporadic life-threatening hemodynamic instability. CONCLUSIONS: In this first-in-man trial, administration of TILs transduced with an inducible IL12 gene mediated tumor responses in the absence of IL2 administration using cell doses 10- to 100-fold lower than conventional TILs. However, due to toxicities, likely attributable to the secreted IL12, further refinement will be necessary before this approach can be safely used in the treatment of cancer patients.

Chemotherapy-refractory diffuse large B-cell lymphoma and indolent B-cell malignancies can be effectively treated with autologous T cells expressing an anti-CD19 chimeric antigen receptor.

PURPOSE: T cells can be genetically modified to express an anti-CD19 chimeric antigen receptor (CAR). We assessed the safety and efficacy of administering autologous anti-CD19 CAR T cells to patients with advanced CD19(+) B-cell malignancies. PATIENTS AND METHODS: We treated 15 patients with advanced B-cell malignancies. Nine patients had diffuse large B-cell lymphoma (DLBCL), two had indolent lymphomas, and four had chronic lymphocytic leukemia. Patients received a conditioning chemotherapy regimen of cyclophosphamide and fludarabine followed by a single infusion of anti-CD19 CAR T cells. RESULTS: Of 15 patients, eight achieved complete remissions (CRs), four achieved partial remissions, one had stable lymphoma, and two were not evaluable for response. CRs were obtained by four of seven evaluable patients with chemotherapy-refractory DLBCL; three of these four CRs are ongoing, with durations ranging from 9 to 22 months. Acute toxicities including fever, hypotension, delirium, and other neurologic toxicities occurred in some patients after infusion of anti-CD19 CAR T cells; these toxicities resolved within 3 weeks after cell infusion. One patient died suddenly as a result of an unknown cause 16 days after cell infusion. CAR T cells were detected in the blood of patients at peak levels, ranging from nine to 777 CAR-positive T cells/µL. CONCLUSION: This is the first report to our knowledge of successful treatment of DLBCL with anti-CD19 CAR T cells. These results demonstrate the feasibility and effectiveness of treating chemotherapy-refractory B-cell malignancies with anti-CD19 CAR T cells. The numerous remissions obtained provide strong support for further development of this approach.

Parathyroid hormone-related protein and hypercalcemia in patients with metastatic melanoma: case report and review.

Hypercalcemia associated with malignancy has been attributed to osteolytic processes secondary to bony metastases and to humoral factors causing increased bone resorption and decreased renal excretion of calcium. Parathyroid hormone-related protein (PTH-rP) is a humoral factor that has been associated with hypercalcemia in renal cell carcinoma, squamous cell carcinoma, and bladder carcinoma. Hypercalcemia does occur in patients with melanoma; however, few studies have reported on hypercalcemia in these patients, and even fewer have described a direct connection to PTH-rP. We here report a patient with stage IV malignant melanoma presenting with severe hypercalcemia associated with elevated PTH-rP levels. Immunohistochemistry showed strong expression of PTH-rP in biopsy of the patient's subcutaneous masses. In addition, we found a 4.9% incidence of hypercalcemia in 1,146 consecutive patients treated for metastatic melanoma at the Surgery Branch of the National Cancer Institute between January 1, 1988 and March 31, 2000. Thus, PTH-rP may play a significant role in severe hypercalcemia in patients with metastatic melanoma. The discovery of PTH-rP and relevant literature will also be reviewed.