Pancreatoduodenectomy with or without prophylactic falciform ligament wrap around the hepatic artery for prevention of postpancreatectomy haemorrhage: randomized clinical trial (PANDA trial).

BACKGROUND: Postpancreatectomy haemorrhage (PPH) is a rare but potentially fatal complication after pancreatoduodenectomy. Preventive strategies are lacking with scarce data for support. The aim of this study was to investigate whether a prophylactic falciform ligament wrap around the hepatic and gastroduodenal artery can prevent PPH from these vessels. METHODS: In a randomized, controlled, multicentre trial, patients who were scheduled for elective open partial pancreatoduodenectomy with pancreatojejunostomy between 5 November 2015 and 2 April 2020 were randomly allocated in a 1 : 1 ratio to undergo pancreatoduodenectomy with (intervention) or without (control) a falciform ligament wrap around the hepatic artery. The primary endpoint was the rate of clinically relevant PPH from the hepatic artery or gastroduodenal artery stump within 3 months after pancreatoduodenectomy. Secondary endpoints were the rates of associated postoperative complications, for example postoperative pancreatic fistula (POPF) and PPH. RESULTS: Altogether, 445 patients were randomized with 222 and 223 in each group. Among the patients included in modified intention-to-treat analysis (207 in the intervention group and 210 in the control group), the primary endpoint was observed in six of 207 in the intervention group compared with 15 of 210 in the control group (2.9 versus 7.1 per cent respectively; odds ratio 0.39 (95 per cent c.i. 0.15 to 1.02); P = 0.071). Per protocol analysis showed a significant reduction in the intervention group (odds ratio 0.26 (95 per cent c.i. 0.09 to 0.80); P = 0.017). A soft pancreas texture (43 per cent) and the rate of a clinically relevant POPF were evenly (20 per cent) distributed between the groups. The rate of any clinically relevant PPH including the primary endpoint and other bleeding sites was not significantly different between intervention and control groups (9.7 versus 14.8 per cent respectively). CONCLUSION: A falciform ligament wrap may reduce PPH from the hepatic artery or gastroduodenal artery stump and should be considered during pancreatoduodenectomy. REGISTRATION NUMBER: NCT02588066 (http://www.clinicaltrials.gov).

Minichromosome Maintenance Expression Defines Slow-Growing Gastroenteropancreatic Neuroendocrine Neoplasms.

BACKGROUND: Small intestinal neuroendocrine neoplasm (SI-NEN) proliferation is quantified by Ki67 measurements which capture G1-G2M phases of the cell cycle. G0 and early G1 phases, typical of slow-growing cells, can be detected by minichromosome maintenance protein (MCM) expression. We hypothesized that these replication licensing markers may provide clinically relevant information to augment Ki67 in low-grade neuroendocrine neoplasia. METHODS: Immunohistochemical staining (IHC), Western blot analysis, quantitative polymerase chain reaction, and copy number variations of MCM2, MCM3, and Ki67 were undertaken in SI-NENs (n = 22). MCM and Ki67 expression was compared by Kaplan-Meier survival analysis (tissue microarray, independent set [n = 55]). Forty-three pancreatic NENs and 14 normal tissues were included as controls. RESULTS: In SI-NENs, MCM2 (mean: 21.2%: range: 16%-25%) and MCM3 (28.7%: 22%-34%) were detected in significantly more cells than Ki67 (2.3%: 0%-7%, P < .01). MCM2 mRNA correlated with Ki67 IHC (P < .05). MCM3 protein expression was higher in metastases (38-fold) than in normal small intestine (P = .06) and was largely absent in normal neuroendocrine cells. There was considerable variation at the MCM copy number level (0-4 copies). MCM3 expression in proliferating cells significantly predicted overall survival (P < .002). Combinations of Ki67 and MCM2/3 in algorithms differentiated low and higher proliferative lesions (overall survival: 12 vs 6.1 years, P = .06). MCM expression was not informative in pancreatic NENs. CONCLUSION: MCMs are expressed in a higher proportion of NEN cells than Ki67 in slow-growing small intestinal lesions and correlate with survival. Assessment can be used to augment Ki67 to improve prognostic classification in these low-grade tumors.

CD14/TLR4 priming potentially recalibrates and exerts anti-tumor efficacy in tumor associated macrophages in a mouse model of pancreatic carcinoma.

Pancreatic cancer is the fourth major cause of cancer related deaths in the world and 5 year survival is below 5%. Among various tumor directed therapies, stimulation of Toll-like receptors (TLR) has shown promising effects in various tumor models. However, pancreatic cancer cells frequently express these receptors themselves and their stimulation (TLR 2 and/or 4 particularly) within tumor microenvironment is known to potentially enhance tumor cell proliferation and cancer progression. Consistent stimulation of tumor associated macrophages (TAMs), in particular with tumor derived TLR ligand within the tumor microenvironment promotes cancer related inflammation, which is sterile, non-immunogenic and carcinogenic in nature. In view of this, recalibrating of TAM has the potential to induce immunogenic inflammation. Consistent with this, we provide experimental evidence for the first time in this study that priming of TAMs with TLR4 ligend (LPS) alone or in combination with IFN-γ not only recalibrates pancreatic tumor cells induced M2 polarization, but also confers anti-tumor potential in TAMs. Most interestingly, reduced tumor growth in macrophage depleted animals suggests that macrophage directed approaches are important for the management of pancreatic tumors.

Low doses of gamma irradiation potentially modifies immunosuppressive tumor microenvironment by retuning tumor-associated macrophages: lesson from insulinoma.

Tumor infiltrating iNOS+ macrophages under the influence of immunosuppressive tumor microenvironment gets polarized to tumor-promoting and immunosuppressive macrophages, known as tumor-associated macrophages (TAM). Their recruitment and increased density in the plethora of tumors has been associated with poor prognosis in cancer patients. Therefore, retuning of TAM to M1 phenotype would be a key for effective immunotherapy. Radiotherapy has been a potential non-invasive strategy to improve cancer immunotherapy and tumor immune rejection. Irradiation of late-stage tumor-bearing Rip1-Tag5 mice twice with 2 Gy dose resulted in profound changes in the inflammatory tumor micromilieu, characterized by induction of M1-associated effecter cytokines as well as reduction in protumorigenic and M2-associated effecter cytokines. Similarly, in vitro irradiation of macrophages with 2 Gy dose-induced expression of iNOS, NO, NFκBpp65, pSTAT3 and proinflammatory cytokines secretion while downregulating p38MAPK which are involved in iNOS translation and acquisition of an M1-like phenotype. Enhancement of various M2 effecter cytokines and angiogenic reprogramming in iNOs+ macrophage depleted tumors and their subsequent reduction by 2 Gy dose in Rip1-Tag5 transgenic mice furthermore demonstrated a critical role of peritumoral macrophages in the course of gamma irradiation mediated M1 retuning of insulinoma.

Autotransplantation of parathyroid grafts into the tibialis anterior muscle after parathyroidectomy: a novel autotransplantation site.

BACKGROUND: Surgical management of renal secondary hyperparathyroidism (sHPT) is varying. Total parathyroidectomy with heterotopic autotransplantation (TPTX + AT) is one of the standard surgical procedures in sHPT, but there is no consensus about the optimal site for graft insertion. At the surgical department of the University Hospital of Heidelberg we prefer the autotransplantation into the tibialis anterior muscle. The aim of this study was to assess the long-term function of the auto-transplanted parathyroid tissue in this type of surgical procedure. METHODS: The function of the autograft of 42 patients was assessed 8.2 ± 2.5 years after surgery, using a modified Casanova-test of the leg bearing the parathyroid tissue. Ischemic blockage was induced by tourniquet and the levels of parathyroid hormone (PTH) were assessed during the test. RESULTS: At the point of assessment, the ischemic blockage led to a significant reduction in the concentration of PTH (≥50% of the baseline value) in 19 patients (45%) indicating well-functioning autografts. In 11 patients (26%), ischemic blockage did not cause any change in the concentration of PTH (≤20% of the baseline value), indicating functioning residual parathyroid tissue from another site. The source of PTH production was classified as unidentifiable in five patients (12%). Two patients had developed graft-dependent recurrent HPT (5%) without therapeutic consequences and three patients suffered from persistent symptomatic hypoparathyroidism (7%). CONCLUSIONS: These results indicate that TPTX + AT into the tibialis anterior muscle is a successful surgical treatment for renal HPT and that the modified Casanova-test is a suitable diagnostic tool for autografts function.

Stathmin in pancreatic neuroendocrine neoplasms: a marker of proliferation and PI3K signaling.

Chromosome 1p35-36, which encodes tumor suppressors and mitotic checkpoint control genes, is commonly altered in human malignancies. One gene at this locus, stathmin 1 (STMN1), is involved in cell cycle progression and metastasis. We hypothesized that increased STMN1 expression may play a role in pancreatic neuroendocrine neoplasm (pNEN) malignancy. We investigated stathmin copy number variation, mRNA, and protein expression using PCR-Taqman Copy Number Assays, Q-PCR, Western blot, and immunohistochemistry. A mechanistic role for stathmin in proliferation was assessed in the BON cell line under growth-restrictive conditions and siRNA silencing. Furthermore, its role in PI3K signaling pathway activation was evaluated using pharmacological inhibitors. mRNA (p = 0.0001) and protein (p < 0.05) were overexpressed in pNENs. Expression was associated with pNEN tumor extension (p < 0.05), size (p < 0.01), and Ki67 expression (p < 0.01). Serum depletion decreased Ki67 expression (p < 0.01) as well as Ser38 phosphorylation (p < 0.05) in BON cells. STMN1 knockdown (siRNA) decreased proliferation (p < 0.05), and PI3K inhibitors directly inhibited proliferation via stathmin inactivation (dephosphorylation p < 0.01). We identified that stathmin was overexpressed and associated with pathological parameters in pancreatic NENs. We postulate that STMN1 overexpression and phosphorylation result in a loss of cell cycle mitotic checkpoint control and may render tumors amenable to PI3K inhibitory therapy.

A mechanistic role for the chromatin modulator, NAP1L1, in pancreatic neuroendocrine neoplasm proliferation and metastases.

BACKGROUND: The chromatin remodeler NAP1L1, which is upregulated in small intestinal neuroendocrine neoplasms (NENs), has been implicated in cell cycle progression. As p57(Kip2) (CDKN1C), a negative regulator of proliferation and a tumor suppressor, is controlled by members of the NAP1 family, we tested the hypothesis that NAP1L1 may have a mechanistic role in regulating pancreatic NEN proliferation through regulation of p57(Kip2). RESULTS: NAP1L1 silencing (siRNA and shRNA/lipofectamine approach) decreased proliferation through inhibition of mechanistic (mammalian) target of rapamycin pathway proteins and their phosphorylation (p < 0.05) in the pancreatic neuroendocrine neoplasm cell line BON in vitro (p < 0.0001) and resulted in significantly smaller (p < 0.05) and lighter (p < 0.05) tumors in the orthotopic pancreatic NEN mouse model. Methylation of the p57 (Kip2) promoter was decreased by NAP1L1 silencing (p < 0.05), and expression of p57(Kip2) (transcript and protein) was upregulated. For methylation of the p57 (Kip2) promoter, NAP1L1 bound directly to the promoter (-164 to +21, chromatin immunoprecipitation). In 43 pancreatic NEN samples (38 primaries and 5 metastasis), NAP1L1 was over-expressed in metastasis (p < 0.001), expression which was inversely correlated with p57(Kip2) (p < 0.01) on mRNA and protein levels. Menin was not differentially expressed. CONCLUSION: NAP1L1 is over-expressed in pancreatic neuroendocrine neoplasm metastases and epigenetically promotes cell proliferation through regulation of p57 (Kip2) promoter methylation.

Maximizing time from the constraining European Working Time Directive (EWTD): The Heidelberg New Working Time Model.

BACKGROUND: The introduction of the European Working Time Directive (EWTD) has greatly reduced training hours of surgical residents, which translates into 30% less surgical and clinical experience. Such a dramatic drop in attendance has serious implications such compromised quality of medical care. As the surgical department of the University of Heidelberg, our goal was to establish a model that was compliant with the EWTD while avoiding reduction in quality of patient care and surgical training. METHODS: We first performed workload analyses and performance statistics for all working areas of our department (operation theater, emergency room, specialized consultations, surgical wards and on-call duties) using personal interviews, time cards, medical documentation software as well as data of the financial- and personnel-controlling sector of our administration. Using that information, we specifically designed an EWTD-compatible work model and implemented it. RESULTS: Surgical wards and operating rooms (ORs) were not compliant with the EWTD. Between 5 pm and 8 pm, three ORs were still operating two-thirds of the time. By creating an extended work shift (7:30 am-7:30 pm), we effectively reduced the workload to less than 49% from 4 pm and 8 am, allowing the combination of an eight-hour working day with a 16-hour on call duty; thus, maximizing surgical resident training and ensuring patient continuity of care while maintaining EDTW guidelines. CONCLUSION: A precise workload analysis is the key to success. The Heidelberg New Working Time Model provides a legal model, which, by avoiding rotating work shifts, assures quality of patient care and surgical training.

Low-dose irradiation programs macrophage differentiation to an iNOS⁺/M1 phenotype that orchestrates effective T cell immunotherapy.

Inefficient T cell migration is a major limitation of cancer immunotherapy. Targeted activation of the tumor microenvironment may overcome this barrier. We demonstrate that neoadjuvant local low-dose gamma irradiation (LDI) causes normalization of aberrant vasculature and efficient recruitment of tumor-specific T cells in human pancreatic carcinomas and T-cell-mediated tumor rejection and prolonged survival in otherwise immune refractory spontaneous and xenotransplant mouse tumor models. LDI (local or pre-adoptive-transfer) programs the differentiation of iNOS⁺ M1 macrophages that orchestrate CTL recruitment into and killing within solid tumors through iNOS by inducing endothelial activation and the expression of TH1 chemokines and by suppressing the production of angiogenic, immunosuppressive, and tumor growth factors.

ENERgized vessel sealing systems versus CONventional hemostasis techniques in thyroid surgery--the ENERCON systematic review and network meta-analysis.

PURPOSE: Energized vessel-sealing systems have been proposed to save operation time and reduce post-operative complications. The aim of the present systematic review was to compare operation time and postoperative morbidity for ultrasonic and electrothermal bipolar-activated devices with conventional hemostasis techniques and with each other in open thyroidectomy. METHODS: A systematic literature search (MEDLINE, Cochrane Library, EMBASE and ISI Web of Science) was performed to identify randomised controlled trials (RCTs) comparing conventional hemostasis techniques, ultrasonic devices (Harmonic® scalpel) and/or electrothermal bipolar-activated vessel sealing systems (Ligasure®) during open thyroidectomy. For the primary endpoint (operation time), a network meta-analysis with Bayesian random effects model was performed. Pairwise meta-analyses with random effects were calculated for primary and secondary endpoints. RESULTS: One hundred sixteen publications were evaluated for eligibility; 35 RCTs (4,061 patients) were included. There was considerable methodological and clinical heterogeneity of included trials. The Harmonic scalpel significantly reduced operation time compared with conventional techniques (22.26 min, 22.7 min in the inconsistency model). The use of Ligasure significantly reduced operation time in total thyroidectomy (13.84 min in the consistency model, 12.18 min in the inconsistency model). In direct comparison, operations with the Harmonic scalpel were faster than with Ligasure (8.42 min in the consistency model, 2.45 min in the inconsistency model). The rates of recurrent nerve palsy and postoperative hypocalcaemia did not significantly differ in the intervention groups. CONCLUSIONS: This meta-analysis shows superiority of ultrasonic devices in terms of operation time compared with conventional hemostasis techniques in thyroid surgery, with no detriment to safety outcomes.

Is there a link between the calcium sensing receptor and Hirschsprung's disease? A mutational analysis.

INTRODUCTION: Hirschsprung's disease (HD) is a congenital malformation of the hindgut characterized by the absence of enteric ganglion cells in the submucosal and myenteric plexuses. Hirschsprung's disease is routinely treated by resection of the aganglionic bowel and pull-through procedure of the proximal ganglionated bowel to the anorectal region. Occasionally, after resection of the aganglionic bowel, HD patients still experience persistent disturbances in gut motility. The etiology of HD as well as the underlying pathomechanism for postoperative disturbances in gut motility is unclear. Molecular analysis of putative candidate genes may help to clarify the pathophysiology of these conditions. In the present study, we investigated the correlation between mutations and polymorphisms in the calcium sensing receptor (CaSR) and HD patients. METHODS: Mutational screening of the CaSR coding sequence was performed via polymerase chain reaction and direct sequencing of the amplified samples from 63 HD patients and 100 controls. RESULTS: We identified 3 common polymorphisms (p.A986S, p.G990R, and p.Q1011E) residing in exon 7 and 1 polymorphism in intron 5 (IVS 5-88 T>C) of the CaSR gene. Overall, 16 patients (25%) and 25 controls (25%) were carriers of the p.A986S polymorphism (P = 1). The incidence of p.R990G polymorphism of patients was twice as high as in the control group (P = .17). Furthermore, we verified a 4 times higher incidence of p.Q1011E polymorphism carriers in patients compared to the control group (P = .1). CONCLUSION: We found a higher incidence of R990G and Q1011E polymorphisms in HD patients compared to controls. However, there was no statistically significant association between HD and the 3 polymorphic variants in the intracellular signaling region of CaSR. It will be important to further investigate genetic variations in CaSR in more patient cohorts to better characterize the function of this gene and to establish a correlation between CaSR variants and HD.

The clinical implications and biologic relevance of neurofilament expression in gastroenteropancreatic neuroendocrine neoplasms.

BACKGROUND: Although gastroenteropancreatic neuroendocrine neoplasms (GEP-NENs) exhibit widely divergent behavior, limited biologic information (apart from Ki-67) is available to characterize malignancy. Therefore, the identification of alternative biomarkers is a key unmet need. Given the role of internexin alpha (INA) in neuronal development, the authors assessed its function in neuroendocrine cell systems and the clinical implications of its expression as a GEP-NEN biomarker. METHODS: Functional assays were undertaken to investigate the mechanistic role of INA in the pancreatic BON cell line. Expression levels of INA were investigated in 50 pancreatic NENs (43 primaries, 7 metastases), 43 small intestinal NENs (25 primaries, 18 metastases), normal pancreas (n = 10), small intestinal mucosa (n = 16), normal enterochromaffin (EC) cells (n = 9), mouse xenografts (n = 4) and NEN cell lines (n = 6) using quantitative polymerase chain reaction, Western blot, and immunostaining analyses. RESULTS: In BON cells, decreased levels of INA messenger RNA and protein were associated with the inhibition of both proliferation and mitogen-activated protein kinase (MAPK) signaling. INA was not expressed in normal neuroendocrine cells but was overexpressed (from 2-fold to 42-fold) in NEN cell lines and murine xenografts. In pancreatic NENs, INA was overexpressed compared with pancreatic adenocarcinomas and normal pancreas (27-fold [P = .0001], and 9-fold [P = .02], respectively). INA transcripts were correlated positively with Ki-67 (correlation coefficient [r] = 0.5; P < .0001) and chromogranin A (r = 0.59; P < .0001). INA distinguished between primary tumors and metastases (P = .02), and its expression was correlated with tumor size, infiltration, and grade (P < .05). CONCLUSIONS: INA is a novel NEN biomarker, and its expression was associated with MAPK signaling and proliferation. In clinical samples, elevated INA was correlated with Ki-67 and identified malignancy. INA may provide additional biologic information relevant to delineation of both pancreatic NEN tumor phenotypes and clinical behavior.

A randomized controlled trial to investigate the influence of low dose radiotherapy on immune stimulatory effects in liver metastases of colorectal cancer.

BACKGROUND: Insufficient migration and activation of tumor specific effector T cells in the tumor is one of the main reasons for inadequate host anti-tumor immune response. External radiation seems to induce inflammation and activate the immune response. This phase I/II clinical trial aims to evaluate whether low dose single fraction radiotherapy can improve T cell associated antitumor immune response in patients with colorectal liver metastases. METHODS/DESIGN: This is an investigator-initiated, prospective randomised, 4-armed, controlled Phase I/II trial. Patients undergoing elective hepatic resection due to colorectal cancer liver metastasis will be enrolled in the study. Patients will receive 0 Gy, 0.5 Gy, 2 Gy or 5 Gy radiation targeted to their liver metastasis. Radiation will be applied by external beam radiotherapy using a 6 MV linear accelerator (Linac) with intensity modulated radiotherapy (IMRT) technique two days prior to surgical resection. All patients admitted to the Department of General-, Visceral-, and Transplantion Surgery, University of Heidelberg for elective hepatic resection are consecutively screened for eligibility into this trial, and written informed consent is obtained before inclusion. The primary objective is to assess the effect of active local external beam radiation dose on, tumor infiltrating T cells as a surrogate parameter for antitumor activity. Secondary objectives include radiogenic treatment toxicity, postoperative morbidity and mortality, local tumor control and recurrence patterns, survival and quality of life. Furthermore, frequencies of systemic tumor reactive T cells in blood and bone marrow will be correlated with clinical outcome. DISCUSSION: This is a randomized controlled patient blinded trial to assess the safety and efficiency of low dose radiotherapy on metastasis infiltrating T cells and thus potentially enhance the antitumor immune response. TRIAL REGISTRATION: ClinicalTrials.gov: NCT01191632.

Inactivating calcium-sensing receptor mutations in patients with primary hyperparathyroidism.

OBJECTIVE: Primary hyperparathyroidism (HPT) is characterised by autonomous secretion of PTH from enlarged parathyroid glands leading, in most patients, to asymptomatic hypercalcaemia. Familial hypocalciuric hypercalcaemia (FHH) is an autosomal dominant disorder caused by inactivating mutations in the calcium-sensing receptor (CaSR) gene; it is characterised by lifelong and usually asymptomatic hypercalcaemia. Establishing the correct diagnosis is important because surgery can be curative in HPT, but ineffective in FHH. There is overlap in the diagnostic criteria for the two disorders and some patients carrying inactivating mutations in the CaSR gene, which is suggestive of FHH, also have HPT with hyperplastic parathyroid glands or adenomas. DESIGN AND PATIENTS CaSR gene mutations were analysed and clinical and biochemical parameters evaluated in 139 consecutive outpatients presenting with hypercalcaemia and suspected of having HPT. RESULTS: Six different mutations of the CaSR gene were found in eight patients. In four patients, classical FHH was suspected based on clinical and biochemical results and was confirmed by the CaSR mutations. In the other four patients, HPT was diagnosed based on the biochemical profile or symptoms; in these four patients, the parathyroids were operated on and single adenomas were histologically confirmed. In all four patients, serum calcium decreased postoperatively; and in three patients, serum calcium normalised postoperatively. The CaSR mutations in these patients were R25X, E250K and Q926R. CONCLUSION: The coexistence of HPT and FHH in four of 139 patients suggests a pathogenetic role of CaSR mutations in HPT. Despite also having a CaSR mutation, these patients benefited from parathyroid surgery.

Concomitant tumor and autoantigen vaccination supports renal cell carcinoma rejection.

Efficient tumor vaccination frequently requires adjuvant. Concomitant induction of an autoimmune response is discussed as a means to strengthen a weak tumor Ag-specific response. We asked whether the efficacy of dendritic cell (DC) vaccination with the renal cell carcinoma Ags MAGE-A9 (MAGE9) and G250 could be strengthened by covaccination with the renal cell carcinoma autoantigen GOLGA4. BALB/c mice were vaccinated with DC loaded with MHC class I-binding peptides of MAGE9 or G250 or tumor lysate, which sufficed for rejection of low-dose RENCA-MAGE9 and RENCA-G250 tumor grafts, but only retarded tumor growth at 200 times the tumor dose at which 100% of animals will develop a tumor. Instead, 75-100% of mice prevaccinated concomitantly with Salmonella typhimurium transformed with GOLGA4 cDNA in a eukaryotic expression vector rejected 200 times the tumor dose at which 100% of animals will develop tumor. In a therapeutic setting, the survival rate increased from 20-40% by covaccination with S. typhimurium-GOLGA4. Autoantigen covaccination significantly strengthened tumor Ag-specific CD4(+) and CD8(+) T cell expansion, particularly in peptide-loaded DC-vaccinated mice. Covaccination was accompanied by an increase in inflammatory cytokines, boosted IL-12 and IFN-gamma expression, and promoted a high tumor Ag-specific CTL response. Concomitant autoantigen vaccination also supported CCR6, CXCR3, and CXCR4 upregulation and T cell recruitment into the tumor. It did not affect regulatory T cells, but slightly increased myeloid-derived suppressor cells. Thus, tumor cell eradication was efficiently strengthened by concomitant induction of an immune response against a tumor Ag and an autoantigen expressed by the tumor cell. Activation of autoantigen-specific Th cells strongly supports tumor-specific Th cells and thereby CTL activation.

Rapid T cell-based identification of human tumor tissue antigens by automated two-dimensional protein fractionation.

Identifying the antigens that have the potential to trigger endogenous antitumor responses in an individual cancer patient is likely to enhance the efficacy of cancer immunotherapy, but current methodologies do not efficiently identify such antigens. This study describes what we believe to be a new method of comprehensively identifying candidate tissue antigens that spontaneously cause T cell responses in disease situations. We used the newly developed automated, two-dimensional chromatography system PF2D to fractionate the proteome of human tumor tissues and tested protein fractions for recognition by preexisting tumor-specific CD4+ Th cells and CTLs. Applying this method using mice transgenic for a TCR that recognizes an OVA peptide presented by MHC class I, we demonstrated efficient separation, processing, and cross-presentation to CD8+ T cells by DCs of OVA expressed by the OVA-transfected mouse lymphoma RMA-OVA. Applying this method to human tumor tissues, we identified MUC1 and EGFR as tumor-associated antigens selectively recognized by T cells in patients with head and neck cancer. Finally, in an exemplary patient with a malignant brain tumor, we detected CD4+ and CD8+ T cell responses against two novel antigens, transthyretin and calgranulin B/S100A9, which were expressed in tumor and endothelial cells. The immunogenicity of these antigens was confirmed in 4 of 10 other brain tumor patients. This fast and inexpensive method therefore appears suitable for identifying candidate T cell antigens in various disease situations, such as autoimmune and malignant diseases, without being restricted to expression by a certain cell type or HLA allele.

Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

BACKGROUND & AIMS: Chronic pancreatitis is characterized by alternating phases of acute inflammation and quiescent disease. Involvement of T-cell responses has been suggested, but pancreatitis-specific T cells have not been described. METHODS: We characterized T-cell responses against pancreatitis, pancreatic carcinoma-associated antigens, and tetanus toxoid in the bone marrow, blood, and/or pancreatitis lesions of patients with pancreatitis, pancreatic cancer, and healthy individuals. T cells were functionally characterized by antigen-dependent secretion of interferon (IFN)-gamma, interleukin (Il)-4, and IL-10, which indicate type 1, type 2, or regulatory T-cell responses, respectively. Regulatory T cells were characterized by multicolor flow cytometry. Isolated regulatory T cells were tested for their capacity to recognize pancreatitis-associated antigens and to suppress conventional T cells in an antigen-dependent manner. T cell-derived cytokines in tissue lesions were quantified by enzyme-linked immunosorbent assay. RESULTS: Chronic pancreatitis patients showed similar to pancreatic cancer patients and healthy individuals type 1 T-cell responses against tetanus toxoid; however, they exhibited strong IL-10-based T-cell responses against pancreatitis-associated but not pancreatic carcinoma-associated antigens. T cells from pancreatic cancer patients responded to pancreatic cancer-associated but not pancreatitis-associated antigens with IFN-gamma secretion. Pancreatitis-specific IL-10 responses were mediated by IL-10(+)IFN-gamma(-)FoxP3(+) regulatory T cells, which were expanded in the blood, bone marrow, and pancreatitis lesions and possessed the potential to suppress the proliferation of autologous conventional T cells in an antigen-specific manner. Pancreatitis lesions, in comparison with pancreatic carcinomas, contained increased concentrations of IL-10 and reduced levels of IFN-gamma, suggesting pancreatitis-specific activity of regulatory T cells in situ. CONCLUSIONS: Chronic pancreatitis is associated with disease-specific regulatory T-cell responses.

Role of tumor endothelium in CD4+ CD25+ regulatory T cell infiltration of human pancreatic carcinoma.

BACKGROUND: Regulatory T (Treg) cells have been detected in human carcinomas and may play a role in preventing the rejection of malignant cells. METHODS: We quantified Treg cells and the expression of the addressins and the respective ligands that attract them in blood and in human pancreatic tumors and adjacent nonmalignant tissues from 47 patients. The capacity of Treg cells to adhere to and transmigrate through autologous endothelial cells was tested in vitro using spheroid adhesion assays and in vivo using a xenotransplant NOD/SCID model and in the presence and absence of antibodies to addressins. All statistical tests were two-sided. RESULTS: More Treg cells infiltrated pancreatic carcinomas than adjacent nonmalignant pancreatic tissues (120 cells per mm2 versus 80 cells per mm2, difference = 40 cells per mm2, 95% confidence interval [CI] = 21.2 cells per mm2 to 52.1 cells per mm2; P<.001). In contrast to conventional CD4+ T cells, more blood-derived Treg cells adhered to (1.0% versus 5.2%, difference = 4.2%, 95% CI = 2.7% to 5.6%; P<.001) and transmigrated through (3332 cells versus 4976 cells, difference = 1644 cells, 95% CI = 708 cells to 2580 cells; P = .008) autologous tumor-derived endothelial cells in vitro and in vivo (458 cells versus 605 cells, difference = 147 cells, 95% CI = 50.8 to 237.2 cells; P = .04). Tumor-derived endothelial cells expressed higher levels of addressins--including mucosal adressin cell adhesion molecule-1 (MAdCAM-1), vascular cell adhesion molecule-1 (VCAM-1), CD62-E, and CD166--than endothelial cells from normal tissue. Experiments using antibodies to addressins showed that transmigration was mediated by interactions of addressins, including MAdCAM-1, VCAM-1, CD62-E, and CD166 with their respective ligands, beta7 integrin, CD62L, and CD166, which were expressed specifically on Treg cells. CONCLUSIONS: Tumor-induced expression of addressins on the surface of endothelial cells allows a selective transmigration of Treg cells from peripheral blood to tumor tissues.

Wnt-signaling and apoptosis after neoadjuvant short-term radiotherapy for rectal cancer.

Recent surgical concepts for primary rectal cancer include the combination of surgery and short-term neoadjuvant radiotherapy (STNR). This is usually given in a dose of 25 Gy over five days in order to reduce local recurrence rates. Clinical studies have shown that local recurrence is found in some patients despite STNR. We identified molecular patterns of the Wnt- and apoptosis pathways as well as expression of junction-associated molecules in rectal cancer specimens of patients who received STNR and in those who did not. Expression patterns were examined by immunohistochemistry and molecular techniques such as LightCycler RT-PCR and Western blot analysis in 25 sporadic rectal adenocarcinoma specimens derived from STNR-patients or non-pretreated donors, respectively. The molecular pattern in response to STNR was heterogeneous and was reflected by responders who show activation of apoptosis and cellular remodeling, whereas the group of non-responders from STNR did not show such reaction and was very similar to untreated controls. Enhanced expression of beta-catenin was generally mediated by STNR, but exclusively in the responder group impaired expression of c-Myc and junction-associated molecules as well as cleavage of poly-ADP-ribose polymerase and of the caspase substrate cytokeratin 19 were found. The molecular profile suggests that STNR interferes with Wnt-signaling and c-Myc expression. STNR in its present form is not suitable to fully complete the sequence of apoptosis in all rectal adenocarcinomas.