Minimally cultured tumor-infiltrating lymphocytes display optimal characteristics for adoptive cell therapy.

Adoptive cell therapy (ACT) with tumor-reactive lymphocytes in patients with refractory melanoma can result in tumor regression and prolonged survival. Generating tumor-reactive lymphocyte cultures is technically difficult and resource intensive; these limitations have restricted the widespread application of ACT. Tumor-infiltrating lymphocytes (TIL) from melanoma contain tumor antigen-reactive cells. The "standard" method for producing TIL cultures for clinical administration requires extended in vitro expansion in interleukin-2, then identification of tumor-reactive cells by immunologic assays. We show here that limitations in reagents and methods during screening underrepresent the actual reactivity of TIL cultures. Furthermore, the extended culture times necessitated by the screening assays resulted in telomere shortening and reduced expression of CD27 and CD28 in the TIL cultures, properties that our prior studies showed are correlated with in vivo persistence and clinical response. We have thus developed an alternative "young" TIL method that demonstrated superior in vitro attributes compared with standard TIL. This approach uses the entire resected tumor to rapidly expand TIL for administration without in vitro testing for tumor recognition. Our observations suggest that younger TIL can have an undetermined but high level of antigen reactivity, and other advantageous attributes such as long telomeres and high levels of CD27 and CD28. We suggest that minimally cultured, unselected lymphocytes represent an alternative strategy for generating TIL cultures suitable for use in ACT that, if effective in vivo, may facilitate the widespread application of this approach to a broader population of patients with melanoma.

Enterocolitis in patients with cancer after antibody blockade of cytotoxic T-lymphocyte-associated antigen 4.

PURPOSE: Cytotoxic T-lymphocyte-associated antigen 4 (CTLA4) is an inhibitory receptor on T cells. Knocking out CTLA4 in mice causes lethal lymphoproliferation, and polymorphisms in human CTLA4 are associated with autoimmune disease. Trials of the anti-CTLA4 antibody ipilimumab (MDX-010) have resulted in durable cancer regression and immune-mediated toxicities. A report on the diagnosis, pathology, treatment, clinical outcome, and significance of the immune-mediated enterocolitis seen with ipilimumab is presented. PATIENTS AND METHODS: We treated 198 patients with metastatic melanoma (MM) or renal cell carcinoma (RCC) with ipilimumab. RESULTS: The overall objective tumor response rate was 14%. We observed several immune mediated toxicities including dermatitis, enterocolitis, hypophysitis, uveitis, hepatitis, and nephritis. Enterocolitis, defined by grade 3/4 clinical presentation and/or biopsy documentation, was the most common major toxicity (21% of patients). It presented with diarrhea, and biopsies showed both neutrophilic and lymphocytic inflammation. Most patients who developed enterocolitis responded to high-dose systemic corticosteroids. There was no evidence that steroid administration affected tumor responses. Five patients developed perforation or required colectomy. Four other patients with steroid-refractory enterocolitis appeared to respond promptly to tumor necrosis factor alpha blockade with infliximab. Objective tumor response rates in patients with enterocolitis were 36% for MM and 35% for RCC, compared with 11% and 2% in patients without enterocolitis, respectively (P = .0065 for MM and P = .0016 for RCC). CONCLUSION: CTLA4 seems to be a significant component of tolerance to tumor and in protection against immune mediated enterocolitis and these phenomena are significantly associated in cancer patients.

Nonalcoholic Fatty gallbladder disease: the influence of diet in lean and obese mice.

The obesity epidemic has contributed to an increased prevalence of gallstones and a higher percentage of chronic acalculous cholecystitis. Obesity is associated with Type II diabetes and hyperlipidemia in murine models. In addition, we have previously demonstrated that serum glucose, insulin, cholesterol, and triglycerides correlated with gallbladder contractility in murine models. However, the relative role of insulin resistance and gallbladder fat infiltration in this phenomenon remain unclear. Therefore, we tested the hypothesis that gallbladder wall lipids are related to obesity and diet and are inversely correlated with gallbladder contractility. One hundred lean control (C7BL/6J) and 36 obese leptin-deficient (Lep(ob)) 8-week-old female mice were fed either a chow diet or a 1.0% cholesterol, 15% butterfat (high-lipid) diet for four weeks. Pooled gallbladders were then analyzed for free fatty acids (FFA), phospholipids (PL), total cholesterol (TC), and triglycerides (TG). Cholesterol/phospholipid ratios were then calculated. The Lep(ob) mice fed a chow diet had significantly higher (P < 0.01) gallbladder lipids than the three other groups. The lean mice that were fed a high-lipid diet had increased (P < 0.05) gallbladder TC compared to the lean mice on a chow diet. In addition, the cholesterol/phospholipid ratio was significantly increased (P < 0.01) in the lean mice fed a high-lipid diet compared to the other three groups. Finally, the high-lipid diet decreased gallbladder FFA (P < 0.01), PL (P = 0.08), and TC (P < 0.05) in Lep(ob) mice. These data suggest that (1) obese mice have increased gallbladder lipids; (2) a high-cholesterol, high-fat diet increases gallbladder lipids and the cholesterol/phospholipid ratio in lean mice; but (3) decreases gallbladder fatty acids, phospholipids, and cholesterol in obese mice. Prior studies have documented similarly decreased gallbladder response to neurotransmitters in obese mice on a chow diet, as well as lean and obese mice on a high-lipid diet. Therefore, we conclude that leptin-deficient obesity and/or a high-fat diet causes nonalcoholic fatty gallbladder disease, which is manifested by diminished gallbladder contractility.

Diminished gallbladder motility in Rotund leptin-resistant obese mice.

BACKGROUND: Obesity is a risk factor for cholesterol gallstone formation, but the pathogenesis of this phenomenon remains unclear. Most human obesity is associated with diabetes and leptin-resistance. Previous studies from this laboratory have demonstrated that diabetic leptin-resistant (Lep(db)) obese mice have low biliary cholesterol saturation indices, enlarged gallbladders and diminished gallbladder response to neurotransmitters. Recently, a novel leptin-resistant mouse strain Lepr(db-rtnd) (Rotund) has been discovered. Rotund mice are also obese, diabetic, and have an abnormal leptin receptor. Therefore, we tested the hypothesis that leptin-resistant obese Rotund mice would have large gallbladders and reduced biliary motility. METHODS: Eight-week-old control (C57BL/6J, N=12) and Rotund leptin-resistant (Lepr(db-rnd), N=9) mice were fed a non- lithogenic diet for four weeks. Animals were fasted and underwent cholecystectomy. Gallbladder volumes were recorded, and contractile responses (N/cm(2)) to acetylcholine (10(-5) M), Neuropeptide Y (10(-8,-7,-6) M), and cholecystokinin (10(-10,-9,-8,-7) M) were measured. Results were analyzed using the Mann-Whitney Rank Sum Test. RESULTS: Compared to control mice, Rotund mice had larger body weights, higher serum glucose levels, and greater gallbladder volumes (p<0.05). Rotund gallbladders had less contractility (p<0.05)) to acetylcholine and cholecystokinin than control mice. Responses to Neuropeptide Y were also less, but not statistically significant, in the Rotund mice. CONCLUSIONS: These data suggest that leptin-resistant Rotund mice have (1) enlarged gallbladders with (2) diminished contractility compared to lean control mice. Therefore, this study confirms that leptin-resistance is associated with abnormal biliary motility and may lead to gallstone formation in leptin-resistant obesity.

Leptin-resistant obese mice do not form biliary crystals on a high cholesterol diet.

INTRODUCTION: Human obesity is associated with leptin resistance and cholesterol gallstone formation. Previously, we demonstrated that leptin-resistant (Lep(db)) obese mice fed a low cholesterol diet have enlarged gallbladders, but a decreased cholesterol saturation index, despite elevated serum cholesterol. Obese humans, however, consume a high cholesterol diet. Therefore, we hypothesized that on a high cholesterol diet, leptin-resistant mice would have cholesterol saturated bile and would form biliary crystals. METHODS: Eight-week old female lean control (n = 70) and leptin-resistant (n = 72) mice were fed a 1% cholesterol diet for 4 weeks. All animals then had cholecystectomies. Bile was collected, grouped into pools to determine cholesterol saturation index (CSI), and examined for cholesterol crystals. Serum cholesterol and leptin were also measured. RESULTS: Gallbladder volumes for Lep(db) mice were enlarged compared with the lean mice (35.8 microl versus 19.1 microl, P < 0.001), but the CSI for the Lep(db) mice was lower than for the lean animals (0.91 versus 1.15, P < 0.03). The obese animals did not form cholesterol crystals, whereas the lean animals averaged 2.2 crystals per high-powered field (hpf) (P < 0.001). Serum cholesterol and leptin were also elevated (P < 0.001) in the obese animals. CONCLUSIONS: These data suggest that Lep(db) obese mice fed a high cholesterol diet have increased gallbladder volume and decreased biliary cholesterol saturation and crystal formation despite elevated serum cholesterol compared with lean control mice. We conclude that the link among obesity, diet, and gallstone formation may not require hypersecretion of biliary cholesterol and may be related to the effects of diabetes, hyperlipidemia, or both on gallbladder motility.

Diabetes and hyperlipidemia correlate with gallbladder contractility in leptin-related murine obesity.

Obesity is associated with many comorbid conditions including diabetes, hyperlipidemia, and gallstones. However, the interaction among these modalities remains unclear. We recently demonstrated that both leptin-deficient and leptin-resistant obese mice have impaired biliary motility. These obese mice also are diabetic and hyperlipidemic. Therefore, we tested the hypothesis that serum glucose, insulin, cholesterol, and triglyceride levels would correlate with gallbladder contractility. Thirty-four lean control, 10 lean heterozygous leptin-deficient, 18 obese homozygous leptin-deficient, and 12 obese homozygous leptin-resistant mice were fed a nonlithogenic chow diet while nine lean control and nine obese homozygous leptin-deficient mice were fed a high-cholesterol diet for 4 weeks. In vitro gallbladder responses to cholecystokinin (CCK; 10(-8) mol/L), acetylcholine (ACh; 10(-5) mol/L), and neuropeptide Y (NPY; 10(-6) mol/L) were measured. Serum glucose, insulin, cholesterol, and triglyceride levels were measured from pooled serum from an additional 704 animals. Gallbladder responses were greatest for CCK, intermediate for ACh, and least for NPY. Serum glucose, insulin, cholesterol, and triglyceride levels and body weight all correlated similarly, negatively, and significantly (P<0.001) with gallbladder contractility. Hyperglycemia, insulin-resistance, hyperlipidemia, and body weight in obese mice with leptin dysfunction are associated with poor gallbladder contractility, which in turn may contribute to the association between obesity and gallstone formation.

Gallbladder motility in agouti-yellow and leptin-resistant obese mice.

BACKGROUND: Obesity is a polygenic disorder that is associated with gallstone disease. We have previously shown that leptin deficiency in obese mice correlates with decreased gallbladder motility, suggesting that leptin plays a role in the link between gallstone disease and obesity. However, most obese humans are leptin-resistant, and relatively few are leptin-deficient. To confirm that leptin dysfunction is responsible for impaired gallbladder motility in obese mice, we hypothesized that leptin-resistant obese mice (Lep(db)) would have abnormal gallbladder motility while obese mice with intact leptin function (Agouti Yellow, A(y)) would have normal gallbladder motility. MATERIALS AND METHODS: Eighteen lean control (C57BL/6J), 10 A(y) and 12 Lep(db) female mice were fasted overnight, weighed, and livers and gallbladders were harvested. Liver weights and gallbladder volumes were measured. Gallbladder contractile responses (N/cm(2)) to acetylcholine (10(-5)M), neuropeptide Y (10(-8,-7,-6) M) and cholecystokinin (10(-10,-9,-8,-7)M) were determined in muscle bath chambers. Results were analyzed by analysis of various (ANOVA) and with the Mann-Whitney Rank Sum Test. RESULTS: Both Agouti yellow (A(y)) and leptin-resistant (Lep(db)) obese mice had body weights, liver weights and gallbladder volumes that were significantly greater (P < 0.01) than lean control mice. Leptin-resistant obese mice had gallbladder responses to acetylcholine, neuropeptide Y and cholecystokinin that were significantly less (P < 0.01) than both lean control and Agouti yellow obese mice. CONCLUSIONS: These data suggest that (1). leptin-resistant obese mice (Lep(db)) have abnormal gallbladder motility and (2). obese mice with normal leptin metabolism (A(y)) have normal gallbladder response to neurotransmitters. We conclude that leptin represents a link between obesity, gallbladder motility and gallstone formation.

Leptin-resistant obese mice have paradoxically low biliary cholesterol saturation.

BACKGROUND: Human obesity is associated with leptin resistance, elevated serum glucose and lipids, hepatic steatosis, and cholesterol gallstone formation. These gallstones are thought to result from hypersecretion of biliary cholesterol as well as biliary stasis. Leptin-resistant Lep(db) obese mice, which are known to have elevated serum leptin, glucose, and lipids, as well as hepatic steatosis, should be an appropriate model for human gallstone formation. Therefore, we tested the hypothesis that leptin-resistant mice would have increased gallbladder volume, biliary cholesterol saturation, and cholesterol crystal formation. METHODS: Sixty lean control mice and 60 Lep(db) obese mice on a low cholesterol chow diet were studied. Gallbladder volumes were measured and bile was pooled to calculate cholesterol saturation index. Serum cholesterol, glucose, and leptin levels were determined from pooled serum. Hepatic fat vacuoles were counted. Bile from a second group of 90 lean control and 59 obese mice was observed microscopically for cholesterol crystal formation. RESULTS: Leptin-resistant obese mice have significantly higher serum cholesterol, glucose, and leptin levels, hepatic fat vacuoles, and gallbladder volume than lean control mice. However, biliary cholesterol saturation index and cholesterol crystal formation were significantly diminished in the obese mice. CONCLUSIONS: These data suggest that leptin-resistant Lep(db) obese mice have (1) increased gallbladder volume, (2) decreased biliary cholesterol saturation despite elevated serum cholesterol and hepatic steatosis, and (3) decreased in vitro cholesterol crystal formation. We conclude that the link between obesity and gallstone formation does not require hypersecretion of biliary cholesterol.

Improved survival in resected biliary malignancies.

BACKGROUND: For many years the prognosis for patients with biliary malignancies has been poor. However, recent advances in radiology and laparoscopy have improved staging, and active biliary stent management may improve outcome in these patients. In the past the goal with surgery was to excise all gross tumor. Now, the surgical goal is to achieve negative microscopic margins even if a major hepatic resection is required. Similarly, chemotherapy or radiation was frequently given in isolation, but chemoradiation has become the standard. Therefore, the aim of this analysis was to determine whether survival has improved with better staging, active stent management, more aggressive surgery, and chemoradiation. METHODS: From 1990 through 2001, 140 patients with biliary malignancies were treated at the Medical College of Wisconsin. One hundred eleven malignancies were cholangiocarcinomas (intrahepatic, 22%; perihilar, 65%; and distal, 13%), and 29 were gallbladder (GB) cancers. Eighty-six of the 140 patients (61%) underwent exploration (intrahepatic, 58%; perihilar, 57%; distal, 67%, and GB, 72%). Forty-four of these 86 patients (51%) underwent resection (intrahepatic, 64%; perihilar, 41%; distal, 70%; and GB, 52%). Chemoradiation with confocal radiation, 5-fluorouracil, and gemcitabine was used more frequently in the patients resected since 1998. RESULTS: Thirty-day operative mortality was 4%. In the resected patients (n = 44) the 5-year actuarial survival was 31% and the median survival was 27.8 months. Patients resected between 1998 and 2001 (n = 25) had a median survival longer than 44 months with a 3-year actuarial survival of 70% as compared to patients resected between 1990 and 1997 (n = 19), who had a median survival of 13 months and a 3-year actuarial survival of 21% (P <.01). CONCLUSIONS: These data suggest that (1) approximately one third of patients with biliary malignancies have resectable disease and (2) surgery in carefully selected patients with adjuvant chemoradiation has improved survival in resected patients. We suspect that a combination of improved staging, active biliary stenting, safe but extensive surgery to obtain negative margins, and newer techniques for chemoradiation have resulted in improved outcomes for patients with biliary malignancies.