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Melanocyte differentiation antigens, such as the brown locus protein gp75, are potential biological targets for immunotherapy. We investigated whether expression of the murine gp75 cDNA mediated by an adenovirus (Ad) vector could induce melanoma rejection using this model self antigen that usually induces tolerance, and whether Ad vector-directed production of interleukin-2 (IL2) might augment this response. To evaluate this approach, Ad vectors were constructed containing the murine gp75 cDNA (Ad.gp75) and the human IL2 cDNA (Ad.IL2). Efficacy was evaluated in C57Bl/6 mice challenged i.v. with 105 B16 cells, using the number of lung metastases as the efficacy parameter. Naive control mice developed 175 ± 12 metastases by day 14. Controls receiving intranasal Ad.IL2 1 day after B16 cell injection, intraperitoneal (i.p.) mitomycin-C-treated B16 cells ± i.p. Ad.IL2 before B16 cell challenge and Ad.βgal-treated mice had similar numbers of meta- stases as controls (P > 0.1). In marked contrast, pre-immunization with intradermal Ad.gp75 provided dramatic reduction in the number of lung metastases (52 ± 7, 29% of control). Addition of regional (intranasal delivery to the lung) Ad.IL2 to intradermal Ad.gp75 preimmunization 1 day following tumor challenge provided further protection (18 ± 6, 10% of control). Depletion of CD4+ and CD8+ T-cell subsets effectively blocked the protective effect seen following immunization. Adoptive transfer of macrophage-depleted splenocytes from Ad.gp75-immunized mice similarly afforded significant protection against B16 tumor cell challenge. Further, serum obtained 21 days following Ad.gp75 immunization showed no detectable anti-gp75 antibody by immunoprecipitation. These results suggest that immunization with Ad.gp75 induces cellular immune responses that are capable of rejecting B16 melanoma in a host that is usually tolerant to gp75 antigen. [ABSTRACT FROM AUTHOR]

Background: Outcome of pseudomyxoma peritonei (PMP) after cytoreductive surgery (CRS) and hypertermic intraperitoneal chemotherapy (HIPEC) is heterogeneous even after adjusting for clinico-pathological prognostic variables. The identification of additional prognostic or even predictive biomarkers is an unmet clinical need. Patients and methods: Forty patients with mucinous appendiceal tumors and PMP were clinically eligible and had evaluable tumor samples obtained after CRS and HIPEC. We carried out next-generations sequencing (NGS) of 50 gene's hotspot regions contained in the Hotspot Cancer Panel v2 using the Ion Torrent Personal Genome Machine platform (Life Technologies). Results: KRAS and GNAS mutations were found in 72% and 52%, and their allelic frequency was below 10% in 55% and 43% of samples, respectively. KRAS and GNAS mutations were associated with worse progression-free survival (PFS) at univariate analysis (P = 0.006 and 0.011, respectively). At multivariate analysis, only KRAS mutations were independently associated with PFS (P = 0.012); GNAS mutations were not--being significantly associated with other poor prognostic features such as incomplete cytoreduction or KRAS mutations. Validation of results was carried out in an independent bi-institutional cohort of 25 patients and the prognostic effect of KRAS mutations was again confirmed in the multivariate model (P = 0.029). NGS approach allowed the discovery of other potentially druggable mutations such as those in PI3K, AKT, LKB1, FGFR3 and PDGFRA. Conclusions: Given the homogeneity of this series and the sensitivity of NGS in this low-cellularity tumor, we demonstrated for the first time a poor prognostic role of KRAS mutations. [ABSTRACT FROM AUTHOR]

Background: Post-operative incisional hernia (IH) is a common complication following abdominal surgery. Data regarding IH after major pancreatic surgery are limited. We aim to evaluate the long-term risk of IH following major pancreatic resection., Methods: A dual-approach study: a large multi-institutional research network (RN) was investigated for IH incidence and risk factors in propensity-score matched survivors after pancreaticoduodenectomy (PD) and distal pancreatectomy (DP), was complemented by a patient-reported questionnaire., Results: RN analysis identified 22,113 patients that underwent pancreatic surgery. 11.0% of PD patients and 8.6% of DP patients developed IH (P < 0.0001). IH rates were higher with open surgery compared with minimally invasive approaches in PD (OR = 1.56, P = 0.03) and DP (OR = 1.94, P = 0.003). BMI>35 was found to correlate with increased IH rates for PD and DP (OR = 1.87, and OR = 1.86, respectively, P < 0.0001 each), as did postoperative intraabdominal infections (P < 0.0001). Patient-based survey of 104 patients, revealed that 16 patients (15%) reported post-operative IH during the follow-up period. BMI≥30, SSI and intra-abdominal abscesses were associated with increased IH risk (P < 0.05)., Conclusion: Improved survival after pancreatic resection has led to an increased prevalence of long-term surgical sequela. In this study, we demonstrate significant rates of IH among long-term survivors and assess potential risk factors., (Copyright © 2024 International Hepato-Pancreato-Biliary Association Inc. Published by Elsevier Ltd. All rights reserved.)

Background : Pancreatic ductal adenocarcinoma (PDAC) is the third leading cause of cancer-related deaths in the United States. Previous studies have indicated that microsatellite instability and deficient mismatch repair (MMR) may be associated with improved survival in patients with pancreatic cancer. Here, we aim to investigate the impact of deficient MMR (dMMR) status on oncologic outcomes in patients after resection of PDAC and periampullary adenocarcinoma. Methods : This is a single-institution, retrospective study based on a prospectively maintained database. Pancreatic ductal adenocarcinoma (N = 342) and periampullary adenocarcinoma patients (N = 76) who underwent pancreatic resection surgery between 2016 and 2021 were included. Immunohistochemistry staining results of MMR proteins and next-generation sequencing data were recorded. Cancer-type dependent Cox regression analyses were performed to assess overall and disease-free survival, which was complemented with a 1:2 propensity-score matching for each of the cancer types in order to compare oncologic outcomes. Results : A total of 418 pancreatic cancer patients were included in the analysis. Fifteen patients (3.5%) were diagnosed as dMMR (PDAC N = 7 and periampullary adenocarcinoma N = 8). Cox regression modeling of dMMR status interaction with TNM staging and cancer type revealed that dMMR status strongly improves overall survival ( p < 0.05). After propensity-score matching, Cox regression identified dMMR status as a significant marker of improved overall survival (HR = 0.27, 95%CI 0.09-0.88, p = 0.029). Conclusions : Overall, our findings suggest that dMMR status is associated with markedly improved survival outcomes in patients after resection of pancreatic and periampullary cancer. Future large-scale studies are needed to further validate this finding.

Background: Delayed gastric emptying (DGE) is a common complication after pancreaticoduodenectomy. There remains an active debate over the effect of gastrointestinal (GI) reconstruction techniques, such as antecolic (AC) or transmesocolic (TMC) reconstruction, on DGE rates. This study compared the rates of DGE between AC reconstruction and TMC reconstruction after pylorus-preserving pancreaticoduodenectomy (PPPD) and classic pancreaticoduodenectomy (PD)., Methods: This was a retrospective analysis of a prospectively maintained pancreatic surgery database in a single, high-volume center. Demographic, perioperative, and surgical outcome data were recorded from patients who underwent a PD or PPPD between 2013 and 2021. DGE grades were classified using the International Study Group of Pancreatic Surgeons (ISGPS) criteria. Postoperatively, all patients were managed using an accelerated Whipple recovery protocol., Results: A total of 824 patients were assessed, with 303 patients undergoing AC reconstruction and 521 patients undergoing TMC reconstruction. The risk of DGE was significantly greater in patients who received an AC reconstruction than in patients who received a TMC reconstruction (odds ratio [OR], 1.51; 95% CI, 1.07-2.15; P < .05). In addition, AC reconstruction was shown to have a greater incidence of severe DGE (ISGPS grades B or C) than TMC reconstruction, with approximately a 2-fold increase in severe DGE (OR, 1.94; 95% CI, 1.10-3.45; P < .05). Logistic regression and propensity score matching have found increased DGE incidence with AC reconstruction (OR: 1.69 and 1.73, respectively; P < .05)., Conclusions: Although the correlation between GI reconstruction methods and DGE remains a subject of ongoing debate, our study indicated that TMC reconstruction may be superior to AC reconstruction in minimizing the development and severity of DGE for patients after PD., Competing Interests: Declaration of Competing Interest The authors declare no competing interests., (Copyright © 2024 Society for Surgery of the Alimentary Tract. Published by Elsevier Inc. All rights reserved.)

Objective: To assess whether long-term survivors of pancreatic surgery show increased risk to develop impaired bone mineral density, osteoporosis, and vitamin D deficiency., Background: Pancreatic resection poses a risk for malabsorption of fat-soluble vitamins and other micronutrients essential for bone mineralization. Here, we evaluated the long-term effects of pancreatic resection on bone mineral density (BMD) and its clinical sequelae., Methods: This was a two-pronged analysis of post-pancreatectomy patients with a follow-up period greater than 3 years comprising (1) a large, propensity score-matched, cohort study based on a multinational federated research network (FRN) and (2) a retrospective single institution review of clinical and radiographic patient data. In the FRN analysis, an initial cohort of 8,423 post-pancreatectomy patients were identified and propensity score-matched with normal controls. The primary endpoint was the 10-year risk of developing osteoporotic pathological fractures and secondary endpoints included diagnosis of osteoporosis, vitamin-D deficiency, and related therapies. The single institution retrospective analysis identified 224 patients who underwent pancreatic resection between 2005 and 2019. BMD was quantified in CT images acquired before and after surgery. BMD trends and related factors were assessed in a time-series mixed effect linear regression model., Results: A total of 8,080 propensity score-matched pairs were included in the FRN analysis. The analysis revealed a 2.4-fold increase in pathological fractures (P<0.0001) and 1.4-1.5 fold increase in osteoporosis/osteomalacia (P<0.0001) and vitamin-D deficiency (P<0.0001) in post-pancreatectomy patients. Vitamin-D supplements were more common in the pancreatectomy group (OR 1.4, 95% CI 1.28-1.53, P<0.0001), as were specific osteoporosis/osteomalacia treatments such as calcitonin, denosumab, romosozumab, abaloparatide, and teriparatide (OR 2.24, 95%CI 1.69-2.95, P<0.0001). Retrospective analysis of CT imaging revealed that BMD declined more rapidly following pancreatic resection compared to normal historical controls (P=0.015). Older age, pancreatic cancer, and pancreaticoduodenectomy were associated with increased rates of BMD loss (P<0.05, each)., Conclusions: After pancreatic resection, patients are at higher risk for BMD loss and subsequent fractures. As the cohort of pancreatic resection survivorship grows, attention will need to be paid to focused prevention efforts to reduce BMD loss, osteoporosis, and fractures in these vulnerable patients, with specific attention to the pancreatic cancer population., Competing Interests: Conflicts of interest: The authors report no conflicts of interest., (Copyright © 2024 Wolters Kluwer Health, Inc. All rights reserved.)

The KRAS proto-oncogene is a major driver of pancreatic tumorigenesis and is nearly ubiquitously mutated in pancreatic ductal adenocarcinoma (PDAC). KRAS point mutations are detected in over 90% of PDAC cases, and these mutations have been shown to be associated with worse therapy response and overall survival. Pathogenic KRAS mutations are mostly limited to codons 12, 13 and 61, with G12D, G12V, G12R, Q61H, and G13D accounting for approximately 95% of the mutant cases. Emerging data have shown the importance of specific mutant subtypes, as well as KRAS variant allele frequency on clinical prognosis. Furthermore, novel technologies and therapies are being developed to target specific mutant subtypes, with encouraging early results. In this paper, we aim to review the recent studies regarding the relative impact of specific mutant KRAS subtypes on oncologic outcomes, the application of variant allele frequency in next generation sequencing analyses, and the ongoing research into therapies targeting specific mutant KRAS subtypes.

Background: The purpose of this study was to examine the relationship between various respiratory conditions, including hypercapnic respiratory disease, and a multitude of resected pancreatic lesions., Methods: This retrospective case-control study queried a prospectively maintained database of patients who underwent pancreaticoduodenectomy between January 2015 and October 2021. Patient data, including smoking history, medical history, and pathology reports, were recorded. Patients with no smoking history and no concomitant respiratory conditions were designated as the control group., Results: A total of 723 patients with complete clinical and pathological data were identified. Male current smokers showed increased rates of PDAC (OR 2.33, 95% CI 1.07-5.08, p = 0.039). Male patients with COPD had a markedly increased association with IPMN (OR 3.02, CI 1.08-8.41, p = 0.039), while females with obstructive sleep apnea had a four-fold increase in risk of IPMN compared to women in the control group (OR 3.89, CI 1.46-10.37, p = 0.009). Surprisingly, female patients with asthma had a decreased incidence of pancreatic and periampullary adenocarcinoma (OR 0.36, 95% CI 0.18-0.71. p < 0.01)., Conclusion: This large cohort study reveals possible links between respiratory pathologies and various pancreatic mass-forming lesions.

Background: Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and lethal cancer. Hypercapnic tumor microenvironments were previously shown to promote cancer chemoresistance. In this study, we aimed to investigate the impact of tissue hypercapnia on PDAC prognosis., Study Design: PDAC cancer-cell lines were cultured in normocapnic (5% CO 2 ) and hypercapnic conditions (10% CO 2 ). RNA was extracted, and whole-exome transcriptome was sequenced. Differentially expressed genes were identified and used to construct a "hypercapnic gene set." PDAC transcriptomic patient data from the Tumor Cancer Genome Atlas was used to calculate single-sample gene set enrichment scores based on each patient's tissue expression of the hypercapnic gene set. Tissue hypercapnic scores (HSs) in PDAC patients (TMN stages Ia-IIb) were determined and correlated with clinicopathological parameters and overall survival., Results: A cohort of 135 resected stage I-II PDAC patients were assessed in this study. The average age was 65 ± 11.0 years, and the male:female ratio was 74:61. Median overall survival was 19.5 ± 1.4 months. High HSs were associated with increased tumor stage (p < 0.05) and higher lymph-node ratio (p < 0.05). In active smokers, high HS also correlated with smoking pack-years (p < 0.05). Cox regression analysis revealed high HS to be an independent prognostic factor for overall survival (hazard ratio [HR] 2.66, p = 0.004), along with lymph-node ratio (HR 4.2, p = 0.002) and age at diagnosis (HR 2.63, p = 0.01)., Conclusions: The pancreatic tumor microenvironment plays an integral role in tumor aggressiveness, and our previous in vitro data suggest that hypercapnia promotes an aggressive, more resistant phenotype. Herein, we show that in early-stage pancreatic cancer, hypercapnic tissue signatures corresponded with a worse overall survival., (Copyright © 2023 by the American College of Surgeons. Published by Wolters Kluwer Health, Inc. All rights reserved.)

Background: Next-generation sequencing (NGS) provides information on genetic mutations and mutant allele frequency in tumor specimens. We investigated the prognostic significance of KRAS mutant allele frequency in patients with right-sided pancreatic ductal adenocarcinoma (PDAC) treated with surgical resection., Methods: A retrospective study reviewed patients who underwent surgical resection for PDAC and analyzed tumors with an in-house mutational panel. Microdissected samples were studied using an NGS-based assay to detect over 200 hotspot mutations in 42 genes (Pan42) commonly involved in PDAC., Results: A total of 144 PDAC right-sided surgical patients with a Pan42 panel were evaluated between 2015 and 2020; 121 patients (84%) harbored a KRAS mutation. Detected mutant allele frequencies were categorized as less than 20% (low mKRAS, n = 92) or greater than or equal to 20% (high mKRAS, n = 29). High mKRAS (KRAS ≥ 20%) patients were noted to have shorter disease-free survival after surgery (11.5 ± 2.1 vs. 19.5 ± 3.5 months, p = 0.03), more advanced tumor stage (p = 0.02), larger tumors (3.6 vs. 2.7 cm, p = 0.001), greater tumor cellularity (26% vs. 18%, p = 0.001), and higher rate of distant recurrence (p = 0.03) than low mKRAS patients., Conclusion: This study demonstrates the importance of KRAS mutant allele frequency on pathological characteristics and prognosis in right-sided PDAC treated with surgery., (© 2022 Wiley Periodicals LLC.)

Castleman disease (CD) includes a group of rare and heterogeneous disorders with characteristic lymph node histopathological abnormalities. CD can occur in a single lymph node station, which is referred to as unicentric CD (UCD). CD can also involve multicentric lymphadenopathy and inflammatory symptoms (multicentric CD [MCD]). MCD includes human herpesvirus-8 (HHV-8)-associated MCD, POEMS-associated MCD, and HHV-8-/idiopathic MCD (iMCD). The first-ever diagnostic and treatment guidelines were recently developed for iMCD by an international expert consortium convened by the Castleman Disease Collaborative Network (CDCN). The focus of this report is to establish similar guidelines for the management of UCD. To this purpose, an international working group of 42 experts from 10 countries was convened to establish consensus recommendations based on review of treatment in published cases of UCD, the CDCN ACCELERATE registry, and expert opinion. Complete surgical resection is often curative and is therefore the preferred first-line therapy, if possible. The management of unresectable UCD is more challenging. Existing evidence supports that asymptomatic unresectable UCD may be observed. The anti-interleukin-6 monoclonal antibody siltuximab should be considered for unresectable UCD patients with an inflammatory syndrome. Unresectable UCD that is symptomatic as a result of compression of vital neighboring structures may be rendered amenable to resection by medical therapy (eg, rituximab, steroids), radiotherapy, or embolization. Further research is needed in UCD patients with persisting constitutional symptoms despite complete excision and normal laboratory markers. We hope that these guidelines will improve outcomes in UCD and help treating physicians decide the best therapeutic approach for their patients., (© 2020 by The American Society of Hematology.)

We have employed computer-based molecular modeling approaches to design peptides from the ras-p21 and p53 proteins that either induce tumor cell reversion to the untransformed phenotype or induce tumor cell necrosis without affecting normal cells. For rasp21, we have computed and superimposed the average low energy structures for the wild-type protein and oncogenic forms of this protein and found that specific domains change conformation in the oncogenic proteins. We have synthesized peptides corresponding to these and found that ras peptides, 35-47 (PNC-7) and 96-110 (PNC-2), block oncogenic ras-p21-induced oocyte maturation but have no effect on insulin-induced oocyte maturation that requires activation of endogenous wild-type ras-p21. These results show signal transduction pathway differences between oncogenic and activated wild-type ras-p21. Both peptides, attached to a membrane-penetrating peptide (membrane residency peptide or MRP), either induce phenotypic reversion to the untransformed phenotype or tumor cell necrosis of several ras-transformed cell lines, but have no effect on the growth of normal cells. Using other computational methods, we have designed two peptides, PNC-27 and 28, containing HDM-2-protein-binding domain sequences from p53 linked on their C-termini to the MRP that induce pore formation in the membranes of a wide range of cancer cells but not any normal cells tested. This is due to the expression of HDM-2 in the cancer cell membrane that does not occur in normal cells. These peptides eradicate a highly malignant tumor in nude mice with no apparent side effects. Both ras and p53 peptides show promise as anti-tumor agents in humans.

In a study of interactions between the raf-MEK-MAPK (ERK) and JNK-jun pathways, we found previously that JNK can induce phosphorylation of raf but not vice versa. In this study, we investigate the nature of the JNK-induced phosphorylation of raf. In in vitro experiments in which immunobead-bound raf is phosphorylated by activated JNK, we find strong phosphorylation signals at raf-Ser259 and Ser338. The Ser259 phosphorylation is surprising since it is associated with inhibition of migration of raf to the cell membrane where it can interact with ras-p21. We also find that in oocytes induced to mature with oncogenic ras-p21, which induces high levels of phosphorylated JNK and MAPK, the same pattern of phosphorylation of raf occurs. In contrast, in oocytes induced to mature with insulin, which requires activation of wild-type ras-p21, phosphorylation of raf-Ser338 but not raf-Ser259 occurs. In oncogenic ras-transformed human pancreatic cancer MIA-PaCa-2 cells, phosphorylation of both raf serines occurs. Treatment of these cells with the ras peptide, PNC-2 attached to a penetrating sequence that blocks JNK and MAPK phosphorylation and induces tumor cell necrosis, results in a marked decrease in phosphorylation of raf-Ser259, but not that of raf-Ser338. These results suggest that oncogenic ras-p21 induces phosphorylation of both raf-Ser259 and Ser338 and that raf-Ser 259 phosphorylation may be effected by activated JNK.

Purpose: We investigated the effects of two peptides from the ras-p21 protein, corresponding to residues 35-47 (PNC-7) and 96-110 (PNC-2), on two ras-transformed human cancer cell lines, HT1080 fibrosarcoma and MIAPaCa-2 pancreatic cancer cell lines. In prior studies, we found that both peptides block oncogenic, but not insulin-activated wild-type, ras-p21-induced oocyte maturation. When linked to a transporter penetratin peptide, these peptides induce reversion of ras-transformed rat pancreatic cancer cells (TUC-3) to the untransformed phenotype., Methods: These peptides and a control peptide, linked to a penetratin peptide, were incubated with each cell lines. Cell counts were obtained over several weeks. The cause of cell death was determined by measuring caspase as an indicator of apoptosis and lactate dehydrogenase (LDH) as marker of necrosis. Since both peptides block the phosphorylation of jun-N-terminal kinase (JNK) in oocytes, we blotted cell lysates of the two cancer cell lines for the levels of phosphorylated JNK to determine if the peptides reduced these levels., Results: We find that both peptides, but not control peptides linked to the penetratin sequence, induce phenotypic reversion of the HT-1080 cell line but cause tumor cell necrosis of the MIA-PaCa-2 cell line. On the other hand, neither peptide has any effect on the viability of an untransformed pancreatic acinar cell line, BMRPA1. We find that, while total JNK levels remain constant during peptide treatment, phosphorylated JNK levels decrease dramatically, consistent with the mechanisms of action of these peptides., Conclusion: We conclude that these peptides block tumor but not normal cell growth likely by blocking oncogenic ras-p21-induced phosphorylation of JNK, an essential step on the oncogenic ras-p21-protein pathway. These peptides are therefore promising as possible anti-tumor agents.

We have previously found that oncogenic ras-p21 and insulin, which activates wild-type ras-21 protein, both induce Xenopus laevis oocyte maturation that is dependent on activation of raf. However, oncogenic ras-p21 utilizes raf-dependent activation of the two classic raf targets, MEK and MAP kinase (MAPK or ERK) while insulin-activated wild-type ras-p21 does not depend on activation of these two kinases. Utilizing a microarray containing the entire Xenopus genome, we discovered two dual specificity kinases, T-Cell Origin Protein Kinase (TOPK), known to bind to raf and the nuclear kinase, DYRK1A, that are expressed at much higher levels in insulin-matured oocytes. Using SiRNA's directed against expression of both of these proteins, we now show that each inhibits insulin-but not oncogenic ras-p21-induced oocyte maturation. Control siRNA's have no effect on either agent in induction of maturation. We find that each SiRNA "knocks down" expression of its target protein while not affecting expression of the other protein. These results suggest that both proteins are required for maturation induced by wild-type, but not oncogenic, ras-p21. They also suggest that oncogenic and wild-type ras-p21 utilize pathways that become divergent downstream of raf. On the basis of these findings, we propose a model for two signal transduction pathways by oncogenic and activated wild-type ras-p21 showing points of overlap and divergence.

Objective: The current study compares outcome after resection of papillary hilar cholangiocarcinoma to that of the more common nodular-sclerosing subtype., Methods: Clinical, radiologic, histopathologic, and survival data on all patients with hilar cholangiocarcinoma were analyzed. Resected tumors were reexamined and classified as nodular-sclerosing (no component of papillary carcinoma) or papillary (any component of papillary carcinoma); for papillary tumors, the proportion of invasive carcinoma present was determined. Differences in the clinical behavior and histopathologic features of nodular-sclerosing and papillary tumors were assessed., Results: From January 1991 to November 2003, 279 patients were evaluated, 154 men (55.2%) and 125 women (44.8%), with a mean age of 65.4 +/- 0.7 years (median = 68, range 23-87 years). Of the 215 patients explored, 106 (49.5%) underwent a complete gross resection. An en bloc partial hepatectomy (n = 87) and an R0 resection (n = 82) were independent predictors of favorable outcome. Operative mortality was 7.5% but was 2.8% over the last 4 years of the study, and there were no operative deaths in the last 33 consecutive resections. Twenty-five resected tumors (23.6%) contained a papillary component: 12 were minimally or noninvasive (<10% invasive cancer) and 13 had an invasive component ranging from 10% to 95% (> or =10%). Patients with papillary and nodular-sclerosing tumors had similar demographics, operative procedures, and proportion of R0 resections. By contrast, papillary tumors were significantly larger, more often well-differentiated, and earlier stage. Disease-specific survival after resection of papillary tumors (55.7 months) was greater than after resection of nodular-sclerosing lesions (33.5 months, P = 0.013). The papillary phenotype was an independent predictor of survival, although the benefit was more pronounced for less invasive tumors., Conclusions: The presence of a component of papillary carcinoma is more common than previous reports have suggested and is an important determinant of survival after resection of hilar cholangiocarcinoma.

Posttransplantation allograft malignancy of donor origin is a rare complication after liver transplantation. In the case described, subjective fevers and nonspecific abdominal complaints nearly 6 months following cadaveric liver transplantation in a young woman prompted an evaluation which was remarkable for a large central liver mass. A poorly differentiated squamous cell carcinoma was diagnosed, but was unresectable at exploration. The tumor was confined to the liver. Histocompatibility testing using polymerase chain reaction (PCR) amplification techniques identified both donor and recipient HLA alleles. The patient was treated with chemoembolization, systemic chemotherapy and cessation of immunosuppression. Repeat biopsy 2 months later showed the tumor to be completely necrotic. With decompensated liver disease, she was relisted and retransplanted. More than 2 years later she remains disease-free with complete pathological remission. This is the only reported case of squamous cell carcinoma of donor origin arising in a transplanted liver.

Tyrosinase-related proteins-1 and -2 (gp75/TRP-1 and TRP-2) are melanosomal membrane glycoproteins recognized by antibodies and T-cells from patients with melanoma. Xenogeneic DNA immunization against gp75/TRP-1 generates antibody-dependent tumor immunity and autoimmune depigmentation. In contrast xenogeneic TRP-2 DNA immunization induces immunity mediated by CD8+ T-cells. The role of IFN-gamma in the generation of tumor immunity and autoimmune depigmentation in these two models was investigated. No tumor protection and minimal depigmentation was observed after immunization with human TRP-2 DNA in mice deficient in IFN-gamma ligand. Repletion with recombinant murine IFN-gamma restored tumor immunity. Experiments using IL4 deficient mice demonstrated that tumor immunity was unaffected but that autoimmune depigmentation was potentially accelerated, consistent with down-modulation of autoimmunity against TRP-2 by IL4. In contrast, IFN-gamma was not required for the generation of immunity to gp75/TRP-1. In fact, exogenous IFN-gamma ablated autoantibody responses against gp75/TRP-1 after xenogeneic DNA immunization, consistent with a down-regulatory effect of IFN-gamma. These results show that immunity to TRP-2 following DNA immunization uses an IFN-gamma-dependent Th1 pathway, but immunity to gp75/TRP-1 is down-regulated by IFN-gamma.

Background: Sott tissue sarcomas (STS) of the groin may present a difficult problem because or misdiagnosis as groin hernia and proximity to major neurovascular structures. We evaluated our management and survival in a large cohort of patients., Study Design: Patients treated between July 1, 1982 and July 1, 1998 with primary or recurrent STS of the groin were included. Groin sarcomas were defined as those tumors within 5 cm of the inguinal crease. Patient, tumor, clinical, and survival data were analyzed using a log rank test and Cox regression., Results: We treated and followed 88 patients with STS of the groin. The median age was 52 years (range 16 to 86 years) and 55 patients (63%) were male. Disease-specific survival was 72% at 5 years. Tumors tended to be larger than 5 cm (52%), deep (72%), and high-grade (60%). Unfavorable prognostic factors for disease-specific survival were high grade (p < 0.001), neurovascular invasion (p < 0.001), positive margin (p < 0.01), deep depth (p < 0.01), and selection for adjuvant therapy (p < 0.005). Multivariate analysis indicated age greater than 50 years (p < 0.05), high grade (p < 0.001), neurovascular invasion (p < 0.001), and positive microscopic margins (p < 0.001). Fourteen patients (16%) were diagnosed with STS at hernia operation then went on to a definitive operation with no impact on survival. Seventeen patients (19%) had involvement of a major vessel or nerve, and 5 of these ultimately required amputations, 3 for local recurrence., Conclusions: High grade, neurovascular invasion, and positive microscopic margins are associated with poor outcomes. The biology of these tumors is similar to other extremity STS, and similar principles of management apply. Even with neurovascular involvement, most patients with primary groin STS do not require amputation.

Innovations for the development of cancer vaccines are emerging from advances in molecular immunology and cancer biology (1). Of these, DNA-based vaccination has become a powerful and potentially versatile method for eliciting an immune response against cancer. One method for DNA immunization involves the delivery of plasmid DNA by particle bombardment. Originally developed for plant hybridization, this approach has proven to be readily transferable to mammalian applications (2-3). Otherwise known as the gene gun, this method allows for the introduction of exogenous "naked" DNA into skin (4) (Fig. 1). Fig. 1. Photograph of the helium-driven gene gun. This is a hand-held device attached to a high pressure helium line and electrical source which operates the trigger. As shown, plastic bullets containing gold particles coated with plasmid DNA are measured and cut to fit within a cartridge. The cartridge, filled with 12 bullets, is easily placed within the barrel of the gun.

Background: Xenogeneic DNA immunization can exploit small differences in expressed protein sequence resulting in immune recognition of self-molecules. We hypothesized that immunizing mice with xenogeneic DNA coding for the human melanosomal membrane glycoprotein gp100 would overcome immune ignorance or tolerance and result in tumor immunity. We also investigated the immunologic mechanisms of the antitumor immunity., Methods: C57BL/6 mice were immunized with DNA coding for human gp100, mouse gp100, or control vector by gene gun. After immunization, mice were challenged with a syngeneic melanoma expressing gp100, and tumor growth was analyzed. Mice deficient in major histocompatibility complex class I or class II molecules were similarly studied to assess the immunologic mechanism of the tumor protection., Results: There was significant tumor protection after vaccination with xenogeneic human gp100 DNA. Class I, but not class II, major histocompatibility complex molecules were required for tumor immunity. In addition, mice immunized with human gp100 demonstrated autoimmunity manifested as coat color depigmentation., Conclusions: Immunization with xenogeneic DNA coding for the melanosomal glycoprotein gp100 results in tumor protection and autoimmune depigmentation. These results show that xenogeneic DNA vaccines can lead to cancer immunity without CD4(+) T-cell help with potential implications for rational vaccine design.

Background: Despite optimal surgical therapy for patients with dermatofibrosarcoma protuberans (DFSP), some patients still continue to develop local recurrence. The authors' objective was to identify and analyze clinicopathologic factors for disease free survival in a large group of patients who were followed prospectively at a single institution., Methods: Prospectively collected data and pathology slides were available for review from 159 patients with primary or recurrent DFSP who underwent treatment between July 1950 and July 1998. The study group was comprised of patients with either the "classic" form of DFSP or the fibrosarcomatous "high grade" variant of DFSP (FS-DFSP). Patient, tumor, pathologic, and treatment factors were analyzed using the log rank test for univariate influence and Cox regression analysis for multivariate influence. Local recurrence free survival was determined by the Kaplan-Meier actuarial method., Results: Of the 159 patients who comprised the current study group, 134 (84%) had the classic form of DFSP. The FS-DFSP variant was found in the remaining 25 patients (16%). The overall 5-year local recurrence free survival rate was 75%, with a median follow-up of 4. 75 years. The 5-year recurrence free survival rate for each group was 81% and 28%, respectively. On univariate analysis, age > 50 years, very close (< 1 mm) to positive microscopic margins, FS-DFSP variant, high mitotic rate, and increased cellularity were unfavorable prognostic factors. Multivariate analysis determined very close (< 1 mm) to positive microscopic margins and FS-DFSP variant to be independent adverse prognostic factors. For the 34 patients who developed a recurrence after surgical resection (21%), the median time to local recurrence was 32 months. Of the patients in this group, two died from metastatic disease., Conclusions: The prognosis after surgical resection with negative and sometimes positive microscopic margins for patients with DFSP is very good. However, increased age, high mitotic index, and increased cellularity are predictors of poor clinical outcome. The FS-DFSP variant represents a much more aggressive tumor with metastatic potential. Patients who are treated with curative intent for FS-DFSP should undergo aggressive attempts at complete surgical resection. Patients with recurrent classic DFSP without evidence of adverse prognostic features may benefit from conservative management, especially in the setting of potentially unresectable disease., (Copyright 2000 American Cancer Society.)

Self-antigens, in the form of differentiation antigens, are commonly recognized by the immune system on melanoma and other cancers. We have shown previously that active immunization of mice against the melanocyte differentiation antigen, a tyrosinase-related protein (TRP) gp75(TRP-1) (the brown locus protein) expressed by melanomas, could induce tumor immunity and autoimmunity manifested as depigmentation. In this system, tumor immunity and autoimmunity were mediated by autoantibodies. Here, we characterize immunity against another tyrosinase family glycoprotein TRP-2 (the slaty locus protein), using the same mouse model and method of immunization. As observed previously for gp75(TRP-1), immunity was induced by DNA immunization against a xenogeneic form of TRP-2, but not against the syngeneic gene, and depended on CD4(+) cells. Immunization against TRP-2 induced autoantibodies and autoreactive cytotoxic T cells. In contrast to immunization against gp75(TRP-1), both tumor immunity and autoimmunity required CD8(+) T cells, but not antibodies. Only autoimmunity required perforin, whereas tumor immunity proceeded in the absence of perforin. Thus, immunity induced against two closely related autoantigens that are highly conserved throughout vertebrate evolution involved qualitatively different mechanisms, i.e., antibody versus CD8(+) T cell. However, both pathways led to tumor immunity and identical phenotypic manifestations of autoimmunity.

An important issue for effective vaccines is the development of potent adjuvants that can facilitate induction or augmentation of immunity. Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth factor for myeloid progenitors of monocytes and dendritic cells (DC), which upon maturation are antigen-presenting cells (APC). The adjuvant effects of inoculation of DNA encoding GM-CSF into skin were studied. Initial experiments examined whether the GM-CSF gene injected into the skin of mice could affect the density of epidermal DC (Langerhans cells). DNA encoding GM-CSF delivered by particle bombardment into skin resulted in a significant increase of epidermal DC at the inoculation site. Kinetic analysis of epidermal recruitment after GM-CSF inoculation showed an increase in DC that peaked at seven days. This increase was accompanied by recruitment of DC into draining lymph nodes. The adjuvant effects of DNA encoding GM-CSF inoculated into skin were measured by the ability to augment antibody and T-cell responses against poorly immunogenic tumor antigens. Peptide immunization at skin sites containing epidermal DC newly recruited by GM-CSF DNA elicited T-cell responses against mutant p53, whereas peptide immunization of control skin sites did not elicit any detectable T-cell responses. Likewise, generation of antibodies following immunization with DNA encoding human gp75TRP1, a tyrosinase family member expressed by melanomas, was accelerated and protection from tumor challenge augmented by GM-CSF DNA.

In the last decade, the most important factor in the rekindled interest in immune therapy for cancer is the development of new methods to identify tumor antigens that can be recognized by T-cells and other immune effectors. In addition, greater knowledge about tolerance and mechanisms of tumor cell evasion from immune effectors has made the prospect of developing clinically effective immune therapies for cancer seem promising. Research in immune therapies for sarcoma has been limited, mainly because of the previous lack of defined tumor antigens in this disease and the low prevalence of sarcoma in the general population. We will review the fundamental concepts of tumor immunobiology, both cellular and humoral, and highlight the new, powerful methods for identifying novel tumor antigens. Further, we will focus on the unique situation presented by sarcoma as the only solid tumor in which many cytogenetic abnormalities have been characterized which encode for unique, tumor-specific fusion proteins that are ideal targets for immune-based therapy. We will review the specifics of vaccine therapy approach to this disease, with emphasis on strategies to improve the immunogenicity of newly defined tumor antigens in sarcoma.

In tumor transplantation models in mice, cytotoxic T lymphocytes (CTLs) are typically the primary effector cells. CTLs recognize major histocompatibility complex (MHC) class I-associated peptides expressed by tumors, leading to tumor rejection. Peptides presented by cancer cells can originate from viral proteins, normal self-proteins regulated during differentiation, or altered proteins derived from genetic alterations. However, many tumor peptides recognized by CTLs are poor immunogens, unable to induce activation and differentiation of effector CTLs. We used MHC binding motifs and the knowledge of class I:peptide:TCR structure to design heteroclitic CTL vaccines that exploit the expression of poorly immunogenic tumor peptides. The in vivo potency of this approach was demonstrated using viral and self-(differentiation) antigens as models. First, a synthetic variant of a viral antigen was expressed as a tumor antigen, and heteroclitic immunization with peptides and DNA was used to protect against tumor challenge and elicit regression of 3-d tumors. Second, a peptide from a relevant self-antigen of the tyrosinase family expressed by melanoma cells was used to design a heteroclitic peptide vaccine that successfully induced tumor protection. These results establish the in vivo applicability of heteroclitic immunization against tumors, including immunity to poorly immunogenic self-proteins.

Oogenesis of four cases of 47,XX,+21 at gestational ages of 19 and 20 weeks was studied using pachytene cytogenetic methods. We found a variable pattern of pairing behavior of the 21 chromosomes among the cases, which included partially synapsed trivalents, a bivalent plus a univalent, and three univalents. The bivalent/univalent conformation of 21 chromosomes predominated. Pachytene chromomere maps were normal for all autosomal bivalents in 84 oocytes analyzed, except for minor variations in some 21 chromosomes. This complex system of pairing behavior of the 21 chromosomes theoretically affects subsequent disjunctional behavior and therefore may account for the observations of normal and trisomic progeny observed for Down's syndrome mothers. Further study of meiotic behavior during all stages of oogenesis in such patients would provide essential data for predicting chromosomal outcome of pregnancy in this population.

Z-DNA has been detected in several pro- and eukaryotic cells and possible roles in regulating transcriptional activity and meiotic recombination proposed. The present study examined the localization of reaction product to potential Z-DNA sites in human testicular tubule epithelium from three subjects using an avidin-biotin complex (ABC)-immunoperoxidase method with a specific rabbit antibody previously shown to react with rat spermatogonial nuclei. A total of 46,626 cells were scored, of which 5656 were Sertoli cells. Eighty-six percent of spermatogonia in mitotic metaphase were found to be negative. Interphase spermatogonia identified as A dark or pale were positive in 93 and 92%, respectively, of cells, and this positivity persisted through B spermatogonia into meiosis. Of 8083 leptotene/zygotene spermatocytes, 99% were positive. Pachytene spermatocytes were 98% positive in the autosomal bivalents. First meiotic metaphase nuclei were negative (92%), as were almost all cells scored of the spermiogenic series (16,195). Nuclei of Sertoli cells had reaction product over the chromatin in 81% of 5656 cells, with no reaction product on the prominent nucleolus. The presence of potential Z-DNA sites in the genome of human spermatogenesis and Sertoli cells during known active stages of transcription (pachytene) and recombination (zygotene/pachytene) suggests a role for this conformation during these stages.