Elena Rangelova

Expansion of Tumor-reactive T Cells From Patients With Pancreatic Cancer.

Generation of T lymphocytes with reactivity against cancer is a prerequisite for effective adoptive cellular therapies. We established a protocol for tumor-infiltrating lymphocytes (TILs) from patients with pancreatic ductal adenocarcinoma. Tumor samples from 17 pancreatic cancer specimens were cultured with cytokines (IL-2, IL-15, and IL-21) to expand TILs. After 10 days of culture, TILs were stimulated with an anti-CD3 antibody (OKT3) and irradiated allogeneic peripheral blood mononuclear cells. Reactivity of TILs against tumor-associated antigens (mesothelin, survivin, or NY-ESO-1) was detected by intracellular cytokine production by flow cytometry. Cytotoxicity was measured using a Chromium 51 release assay, and reactivity of TILs against autologous tumor cells was detected by INF-&ggr; production (ELISA). TIL composition was tested by CD45RA, CCR7, 4-1BB, LAG-3, PD-1, TIM3, and CTLA-4 marker analysis. TCR V&bgr; was determined by flow cytometry and TCR clonality was gauged measuring the CDR3 region length by PCR analysis and subsequent sequencing. We could reliably obtain TILs from 17/17 patients with a majority of CD8+ T cells. CD3+CD8+, CD3+CD4+, and CD3+CD4−CD8− [double-negative (DN) T cells] resided predominantly in central (CD45RA−CCR7+) and effector (CD45RA−CCR7−) memory subsets. CD8+ TILs tested uniformly positive for LAG-3 (about 100%), whereas CD4+ TILs showed only up to 12% LAG-3+ staining and PD-1 showed a broad expression pattern in TILs from different patients. TILs from individual patients recognized strongly (up to 11.9% and 8.2% in CD8+) NY-ESO-1, determined by ICS, or mesothelin, determined respectively by TNF-&agr; and IFN-&ggr; production. Twelve of 17 of CD8+ TILs showed preferential expansion of certain TCR V&bgr; families (eg, 99.2% V&bgr;13.2 in CD8+ TILs, 77% in the V&bgr;1, 65.9% in the V&bgr;22, and 63.3% in the V&bgr;14 family). TCR CDR3 analysis exhibited monoclonal or oligoclonal TCRs, some of them (eg, CD8+ V&bgr;13.2) reacting strongly against autologous tumor defined by INF-&ggr; production or by cytotoxicity. We have optimized methods for generating pancreatic cancer–specific TILs that can be used for adoptive cellular therapy of patients with pancreatic cancer.

T-Cell Therapy: Options for Infectious Diseases

The emergence of drug-resistant tuberculosis is challenging tuberculosis control worldwide. In the absence of an effective vaccine to prevent primary infection with Mycobacterium tuberculosis and tuberculosis disease, host-directed therapies may offer therapeutic options, particularly for patients with multidrug-resistant and extensively drug-resistant tuberculosis where prognosis is often limited. CD8+ and CD4+ T cells mediate antigen-specific adaptive cellular immune responses. Their use in precision immunotherapy in clinical conditions, especially in treating cancer as well as for prevention of life-threatening viral infections in allogeneic transplant recipients, demonstrated safety and clinical efficacy. We review key achievements in T-cell therapy, including the use of recombinant immune recognition molecules (eg, T-cell receptors and CD19 chimeric antigen receptors), and discuss its potential in the clinical management of patients with drug-resistant and refractory tuberculosis failing conventional therapy.

Impact of body mass index for patients undergoing pancreaticoduodenectomy

AIM To evaluate the impact of body mass index (BMI) on short and long term results after pancreaticoduodenectomies (PD). METHODS A consecutive series of PDs performed at the Karolinska University Hospital from 2004 till 2010 were retrieved from our prospective database. The patients were divided by BMI into overweight/obese (O; BMI ≥ 25 kg/m(2)) and controls (C; BMI < 25 kg/m(2)). Demographics, peri-operative data, morbidity, mortality, pancreatic fistula (PF) rate, length of stay (LOS), hospital costs, histology, and survival were analyzed. An additional sub analysis of survival was performed in patients with a diagnosis of pancreatic ductal adenocarcinoma (PDAC) and divided in underweight, normal-weight, overweight and obese. RESULTS A total of 367 PDs were included (O = 141/C = 226). No differences were found between O and C regarding demographics, peri-operative data, costs, morbidity or mortality. O was associated with higher intra-operative blood loss (1392 ± 115 mL vs 1121 ± 83 mL; P = 0.01), rate of PF (20% vs 9.5%; P = 0.006) and marginally longer LOS (18 ± 0.9 d vs 15 ± 1.1 d; P = 0.05). An increasing risk for PF was observed with increasing BMI. The 1, 3 and 5 years survival rate was similar in O and C in PDAC (68.7%, 26.4% and 8.8% vs 66.1%, 30.9% and 17.9% respectively; P = 0.9). When the survival was analyzed using 4 different categories of BMI (underweight, normal, overweight and obese), a trend was seen toward a difference in survival, with a worse prognosis for the underweight and obese patients compared to normal weight and overweight patients. CONCLUSION Overweight increases the risk for intra-operative bleeding and PF, but do not otherwise alter short or long term outcome after PD for pancreatic cancer.

Tumor-infiltrating lymphocytes (TILs) from patients with glioma

ABSTRACT Tumor-infiltrating lymphocytes (TILs) may represent a viable source of T cells for the biological treatment of patients with gliomas. Glioma tissue was obtained from 16 patients, tumor cell lines were established, and TILs were expanded in 16/16 cases using a combination of IL-2/IL-15/IL-21. Intracellular cytokine staining (ICS, IL-2, IL-17, TNFα and IFNγ production) as well as a cytotoxicity assay was used to detect TIL reactivity against autologous tumor cells or shared tumor-associated antigens (TAAs; i.e., NY-ESO-1, Survivin or EGFRvIII). TILs were analyzed by flow cytometry, including T-cell receptor (TCR) Vβ family composition, exhaustion/activation and T-cell differentiation markers (CD45RA/CCR7). IL-2/IL-15/IL-21 expanded TILs exhibited a mixture of CD4+, CD8+, as well as CD3+ CD4−CD8− T cells with a predominant central memory CD45RA−CCR7+ phenotype. TIL showed low frequencies of T cells testing positive for PD-1, TIM-3 and CTLA-4. LAG3 tested positive in up to 30% of CD8+ TIL, with low (1.25%) frequencies in CD4+ T cells. TIL cultures exhibited preferential usage of Vβ families and recognition of autologous tumor cells defined by cytokine production and cytotoxicity. IL-2/IL-15/IL-21 expanded TILs represent a viable source for the cellular therapy of patients with gliomas.

Prediction of improved survival in patients with pancreatic cancer via IL-21 enhanced detection of mesothelin epitope-reactive T-cell responses

Most patients with pancreatic cancer present with extensive metastasis at diagnosis, with a 5-year survival rate of approximately 5%, despite chemotherapy and surgery. New treatment modalities are needed to improve survival. Mesothelin is a tumor-associated antigen (TAA) in patients with pancreatic cancer that could be used to gauge cellular immune responses directed against transformed cells since up to 100 percent of pancreatic ductal adenocarcinoma cells have been shown to strongly express mesothelin. A prospective, observational study was carried out in twenty-six, chemotherapy-naïve patients with resectable pancreatic ductal adenocarcinoma. Participants were between 48 and 81 years (median age: 64.5 years), 15 males and 11 females. All participants were clinically followed-up between 439 and 853 days post-surgery (n=14) or until death (n=12). Peripheral blood drawn on the day of surgery was stimulated with a mesothelin peptide pool (42 peptides, non-overlapping), individual mesothelin peptides, positive (anti-CD3 antibody, OKT3) and negative controls (medium) with or without adding IL-21. Kaplan-Meier estimators were used to gauge patients’ survival pattern in relation to mesothelin-specific IFN-γ responses. A survival benefit was linked with IFN-γ responses to peptides corresponding to mature mesothelin (p=0.018) and targeted recognition of the mesothelin601-615 epitope (MQEALSGTPCLLGPG) (p=0.006) in the presence of IL-21. Conversely, production of high levels of IFN-γ to OKT3 stimulation with IL-21 conditioning was associated with reduced survival of patients (p=0.016). Gauging anti-Mesothelin- directed immune responses will aid to identify patients i) in need of a more intensive clinical follow-up and ii) who may benefit from immunotherapeutic approaches targeting mesothelin.

Is robotic pancreatectomy indicated for patients with pancreatic cancer

Minimally invasive pancreatic surgery (MIPS) is becoming more popular in the recent years. Robotic pancreatic surgery represents an important step, mostly for Whipple procedures, in order to reproduce the same surgical steps performed in open surgery and to facilitate the access to MIPS even by surgeons without a large experience in laparoscopic surgery. The short-term results of pancreatic robotic surgery are comparable with the one obtained by the traditional open surgery, even if the peri-operative costs, still represent a problem. Very few data are currently available regarding the long-term oncologic outcome of robotic pancreatectomy for pancreatic cancer. For this reason, more and larger studies, with a long follow-up are necessary to define the oncologic role and safety of robotic pancreatectomies.

AB117. P092. Safety of intraoperative pancreatoscopy for the investigation of main pancreatic duct involvement and assessment of skip lesions in operated main duct (MD) involving IPMNs: a feasibility study

Background: Current management of main duct (MD)involving intraductal papillary mucinous neoplasm (IPMN) is driven by intra-operative frozen section. However, data regarding the clinical utility of this approach are discordant. In fact, frozen section of pancreatic resection margin can’t detect skip lesions within MPD and this might imply incomplete resections and short term recurrences after resection. Peroral pancreatoscopy is a promising tool to investigate the MPD but is technically highly skill demanding and therefore difficult to use on a large scale. The application of intraoperative pancreatoscopy might be able to bypass this problem but data about its safety are currently lacking. This study aims to assess the safety of intraoperative pancreatoscopy. Methods: Retrospective cohort analysis of patients undergoing surgical resection for MD-involving IPMN. All indications for surgery were decided according to the European Guidelines for the management of pancreatic cystic tumors. Data about characteristics of patients, type of surgery, mortality and length of hospital stay, overall complications and procedure related preoperative complications (pancreatitis, perforations) were recorded. Results: From 2015 to 2016 22 patients, 10 (45%) male, median age 67 (45–82 years) underwent surgical resection for MD-involving IPMN and intraoperative pancreatoscopy. Overall complications were reported in 9 (40%) of patients. 1 patient (4.5%) underwent reoperation for incisional hernia, 1 (4.5%) developed pancreatic fistula, 2 (9%) had GI bleeding requiring endoscopy. None of the patients developed procedure related morbidity and mortality. The mean length of hospital stay was 15.36 days. Conclusions: Intraoperative pancreatoscopy in the investigation of IPMN patients with dilated MPD is a feasible and safe procedure.

Enhanced tumor-infiltrating lymphocytes (eTIL) for cellular therapy of patients with pancreatic cancer or glioblastoma

Meeting abstracts The generation of T lymphocytes with specific reactivity against autologous tumor cells is a prerequisite for effective targeted cellular therapies. We established a protocol for tumor infiltrating lymphocytes (TILs) cultures from small biopsies or surgically resected material

IL-2, IL-15 and IL-21 expand T cells for targeted adoptive therapy

Meeting abstracts Expansion of antigen-specific T cells, from peripheral blood specific for tumor-associated antigens (TAAs) is a prerequisite for the advanced cellular therapy. Such antigen-specific T cells should express a Th1-functional phenotype and are able to enter tumor-tissue. We identified

The Use of LigaSure™ Does not Affect Histologic Margin Assessment in Pancreatoduodenectomy (PD) Specimens

CONTEXT LigaSure™ is considered safe in performing pancreaticoduodenectomy (PD). However, no data are available regarding the possible damage of tissues at the resection margins and the impact thereof on histologic margin assessment. OBJECTIVE This study compares the degree of histologic damage to the resection margins when using LigaSure™ (Group 1) or traditional ligature (Group 2). METHODS Both groups included 8 consecutive patients who underwent PD at Karolinska Institute in December 2013 (Group 1) or earlier (Group 2) by the same surgeon (M.D.C.). The quality of tissues at the circumferential margins was compared between both groups by scoring for three different kinds of damage: tissue fragmentation, hemorrhage, and cell damage. RESULTS The mean score for fragmentation was 1.3 (Group 1) versus 1.7 (Group 2; P=0.1). For hemorrhage the mean score was 0.8 (Group 1) versus 1.5 (Group 2; P=0.04). The mean score for cell damage was 1.4 (Group 1) compared to 1.2 (Group 2; P=0.1). CONCLUSIONS LigaSure™ does not cause tissue damage that could affect histologic margin assessment in PD specimens.