Report from the 17th Annual Western Canadian Gastrointestinal Cancer Consensus Conference; Edmonton, Alberta; 11-12 September 2015.

The 17th annual Western Canadian Gastrointestinal Cancer Consensus Conference (wcgccc) was held in Edmonton, Alberta, 11-12 September 2015. The wcgccc is an interactive multidisciplinary conference attended by health care professionals from across Western Canada (British Columbia, Alberta, Saskatchewan, and Manitoba) who are involved in the care of patients with gastrointestinal cancer. Surgical, medical, and radiation oncologists; pathologists; radiologists; and allied health care professionals participated in presentation and discussion sessions for the purposes of developing the recommendations presented here. This consensus statement addresses current issues in the management of gastric cancer.

Generation of T cells with lytic specificity for atypical antigens. III. Priming F1 animals with antigen-bearing cells also having reactivity for host alloantigens allows for potent lytic T cell responses.

Here, we explore the conditions required for generating two different highly potent F1 antiparental killer cell populations to unusual antigens in rats. The first, L/DA anti-DA, has lytic specificity for two antigen systems: MTA, a mitochondrial antigen expressed on DA and DA Lewis (L) target cells restricted by RT1A class I molecules; and H, an antigen that maps to the class I-like RT1C region and is present only on parental target cells from donors homozygous at the major histocompatibility complex. The second killer population is generated in the reciprocal DA/L anti-DA combination and has lytic specificity only for the H antigen system. We show that the killer cells are T cells, and that generation of these F1 cytotoxic T lymphocytes (CTL) requires an in vivo priming step in which it is essential that the inoculated parental cells bear the relevant target antigens and possess alloreactivity for F1 host antigens. The requirement for alloreactivity and antigen on the same priming cell population suggests that these potent lytic responses depend on a situation akin to a hapten-carrier effect that bypasses otherwise ineffective helper responses by the host to these unusual antigens. Restimulation of F1 lymphocytes in culture is also necessary, requiring the presence of antigen on irradiated lymphoblast stimulator cells, but alloreactivity to responder cell antigens is not necessary; normal, nonactivated lymph node cells are completely ineffective as stimulators. For effective lysis, the target cells need not possess the potential for alloreactivity to responder F1 CTL. We also demonstrate in a preliminary way additional antigen systems defined by killer populations raised with other F1 antiparental strain combinations.

Generation of T cells with lytic specificity for atypical antigens. II. A novel antigen system in the rat dependent on homozygous expression of major histocompatibility complex genes of the class I-like RT1C region.

Lymphocytes from parental strain DA rats can induce potent killer cell responses to atypical antigen systems in F1 Lewis (L)/DA and DA/L recipients. Here, we describe an antigen system, H, present on homozygous parental target cells, but not on F1 cells. This antigen system is unusual in several respects: it does not involve class I RT1A gene products usually used by killer cell responses in the rat, it maps to the major histocompatibility complex (MHC) class I-like RT1C region, and it requires homozygous expression of RT1Cav1 alleles. This may be another example, this time involving the RT1C region, of an MHC gene product antigenically altered by an MHC-linked trans-activating modifier gene.

Generation of T cells with lytic specificity for atypical antigens. I. A mitochondrial antigen in the rat.

F1 rats primed with normal parental strain lymphocyte populations and restimulated in culture with parental lymphoblasts generate potent cytotoxic T cell responses to unusual antigen systems. Here we describe in the Lewis (L)/DA anti-DA combination an antigen system most likely of mitochondrial origin with the following properties: it is transmitted maternally from DA strain females, inherited in an extra-chromosomal manner, restricted by class I RT1Aa major histocompatibility complex gene products, extinguished on target cells treated with chloramphenicol, and its pattern of expression in different rat strains correlates with restriction fragment-length polymorphisms of mitochondrial DNA. Sequence analysis of the rat ND1 gene indicates that the maternally transferred factor in the rat is not a homologue of the maternally transmitted factor responsible for the mitochondrial antigen in mice. In keeping with its inheritance from DA females, this antigen is present on target cells from (DA female x L male)F1 donors and all other F1 combinations derived from DA female parents, but absent from target cells from some F1 combinations (L/DA and Wistar-Furth [WF]/DA) derived from DA strain males. The presence of this antigen in other F1 combinations (Brown Norway [BN]/DA, August 2880 [AUG]/DA, and PVG/DA) indicates that this mitochondrial antigen system is shared by the DA, BN, and PVG strains, but not by the L and WF strains.

A role for the ETS domain transcription factor PEA3 in myogenic differentiation.

Activation of adult myoblasts called satellite cells during muscle degeneration is an important aspect of muscle regeneration. Satellite cells are believed to be the only myogenic stem cells in adult skeletal muscle and the source of regenerating muscle fibers. Upon activation, satellite cells proliferate, migrate to the site of degeneration, and become competent to fuse and differentiate. We show here that the transcription factor polyomavirus enhancer activator 3 (PEA3) is expressed in adult myoblasts in vitro when they are proliferative and during the early stages of differentiation. Overexpression of PEA3 accelerates differentiation, whereas blocking of PEA3 function delays myoblast fusion. PEA3 activates gene expression following binding to the ets motif most efficiently in conjunction with the transcription factor myocyte enhancer factor 2 (MEF2). In vivo, PEA3 is expressed in satellite cells only after muscle degeneration. Taken together, these results suggest that PEA3 is an important regulator of activated satellite cell function.

CD8 blockade promotes the expansion of antigen-specific CD4+ FOXP3+ regulatory T cells in vivo.

Treatment with a cocktail of CD4 and CD8-specific monoclonal antibodies (mAb) induces long-term transplantation tolerance and regulatory CD4(+) T cells that induce tolerance in non-tolerant T cells. In contrast, treatment with a CD4-specific mAb alone fails to induce long-term tolerance. The current study was designed to test the hypothesis that CD8 blockade plays a role in promoting the development of CD4(+) regulatory T cells. Using the DO11.10 CD4(+) TCR transgenic mouse model we show that treatment with a CD4/CD8-specific mAb cocktail induces antigen-specific tolerance to OVA, measured by a significant decrease in OVA-specific IgG, on challenge with antigen. Although treatment with OVA and the CD4-specific mAb alone also induces a significant decrease in OVA-specific antibody, the number of DO11.10 cells is significantly greater in mice treated with the CD4/CD8-specific mAb cocktail, and this is associated with a significant increase in proliferation of DO11.10 cells in response to specific antigen. DO11.10 cells sorted from mice made tolerant to OVA with the CD4/CD8-specific mAb cocktail promote an OVA-specific IgG1 (Th2-type) response but not an OVA-specific IgG3 (Th1-type) response on transfer into new syngeneic recipients, suggesting their ability to regulate the type of antigen-specific immune response that ensues after priming with antigen. In addition, DO11.10 cells from tolerant mice express markers that are characteristic of CD4(+) regulatory cells, including FOXP3, GITR and CTLA4, but not CD25. Taken as a whole, these data suggest that CD8 blockade promotes CD4(+) FOXP3(+) regulatory CD4(+) T cells by promoting their proliferation in tolerant mice.

Acetylated low-density lipoprotein stimulates human vascular smooth muscle cell calcification by promoting osteoblastic differentiation and inhibiting phagocytosis.

BACKGROUND: Vascular smooth muscle cells (VSMCs) in atherosclerotic lesions display an osteogenic phenotype, and calcification commonly occurs in association with lipid. We therefore tested the hypothesis that lipid components in atherosclerotic lesions influenced VSMC phenotype and calcification using an in vitro model of calcification. METHODS AND RESULTS: In situ hybridization of human atherosclerotic plaques (n=10) collected from patients undergoing carotid endarterectomy demonstrated that subsets of lipid-filled VSMCs adjacent to sites of calcification expressed alkaline phosphatase, bone Gla protein, and bone sialoprotein, suggesting an osteogenic phenotype. Treatment of VSMCs in culture with acetylated low-density lipoprotein (acLDL) or lipoprotein-deficient serum altered the time course of bone-associated protein gene expression and calcification. AcLDL increased nodule calcification 3-fold, whereas lipoprotein-deficient serum significantly inhibited it. Reverse transcriptase-polymerase chain reaction and Western analysis demonstrated the presence of the acLDL receptor, SRA1, exclusively in calcifying nodular VSMCs, and blockade of SRA with polyinosinic acid inhibited acLDL-induced calcification. Because apoptotic bodies can serve as nucleation sites for calcification, we investigated whether acLDL could stimulate apoptosis in nodules. Apoptosis of nodular VSMCs was unaltered, but the number of apoptotic bodies per nodule increased approximately 3-fold, implying a defect in phagocytosis. Consistent with these observations, binding of apoptotic bodies to VSMCs was decreased in the presence of acLDL. CONCLUSIONS: These studies suggest that modified lipoproteins stimulate calcification by enhancing osteogenic differentiation of VSMCs and by a novel mechanism whereby acLDL interacts with SRA on VSMCs and blocks phagocytic removal of apoptotic bodies.

Otago Diagnostic Laboratories' (ODL) Method for the detection of beta-lactamases in Enterobacteriaceae.

AIM: The rapid evolvement of beta-lactamases in Enterobacteriaceae is an important concern and the clinical microbiology laboratory is required to detect them, where possible, using a rapid, reliable, simple and low cost methodology. MATERIALS AND METHODS: A disc diffusion method using NCCLS breakpoints, Jarlier's principle and cefoxitin test for AmpC was carried out. It incorporated seven antimicrobial discs in one agar plate: cefotaxime, aztreonam, amoxicillin-clavulanate, ceftazidime, cefpodoxime, cefepime and cefoxitin. NCCLS disc confirmation test for extended-spectrum beta-lactamase (ESBL) was carried out simultaneously. RESULTS: AmpC, ESBL, CTX-M, and K1 were detected using these tests. The prevalence of ESBL was <1% in the hospital. CONCLUSION: The method is recommended for the phenotypic detection of beta-lactamases in Enterobacteriaceae or for confirmation after the results are obtained by conventional automated systems.

Distribution of leukemia, lymphoma, and allied disease in parts of Great Britain: analysis by administrative districts and simulations of adjacencies. Leukaemia Research Fund 1984 Data Collection Group.

An analysis is presented of the distribution of cases of leukemia and allied disorders occurring in 151 administrative districts from England, Wales, and Scotland during 1984. The age-adjusted rates for certain conditions present an unusual pattern highlighting excessively high and low rates in parts of the country, some of which share contiguous boundaries. In particular, high rates for non-Hodgkin's lymphoma are found in rural Yorkshire districts, whereas leukemias and primary polycythemias are much more common in the Midland districts.

Consistency of histopathological reporting of breast lesions detected by screening: findings of the U.K. National External Quality Assessment (EQA) Scheme. U. K. National Coordinating Group for Breast Screening Pathology.

The aim of the scheme was to determine consistency of histopathological reporting in the United Kingdom National Breast Screening Programme. This external quality assessment scheme involved 51 sets of 12 slides which were circulated to 186-251 pathologists at intervals of 6 months for 3 years. Participants recorded their diagnoses on standard reporting forms, which were submitted to the U.K. National Cancer Screening Evaluation Unit for analysis. A high level of consistency was achieved in diagnosing major categories of breast disease including invasive carcinoma and the important borderline lesions, radial scar and ductal carcinoma in situ (DCIS), the latter exceeding a national target set prior to the onset of the scheme. Atypical hyperplasia (AH) was reported with much less consistency although, where it was the majority opinion, over 86% of diagnoses were of benign disorders and only 14% were of DCIS. Inconsistency was encountered in subtyping and measuring DCIS, the former apparently due to current uncertainties about classification and the latter to poor circumscription, variation in size in different sections and merging with zones of AH. Reporting prognostic features of invasive carcinomas was variable. Measurement of size was achieved with adequate consistency except in a small number of very poorly circumscribed tumours. Grading and subtyping were inconsistent although the latter was not specifically tested and will be the subject of future study. Members of the National Coordinating Group achieved greater uniformity than the remainder of the participants in all diagnostic categories, but both groups experienced similar types of problem. Our findings suggest that participation in the scheme improves diagnostic consistency. In conclusion, consistency in diagnosing invasive carcinoma and radial scar is excellent, and good in DCIS, but improvements are desirable in diagnosing atypical hyperplasia, classifying DCIS and reporting certain prognostic features of invasive tumours. Such improvements will require further research, the development of improved diagnostic criteria and the dissemination of clearer guidelines.

Amplification of cytokine-specific ELISAs increases the sensitivity of detection to 5-20 picograms per milliliter.

The ability to detect a protein is always limited to the sensitivity of the assays available. Progress in improving the sensitivity of protein detection will allow a more complete understanding of biological systems. Of particular interest to the field of immunology is the ability to characterize an immune response based upon the pattern of cytokines that are released in response to antigen. A Th1 response is characterized by the presence of IL-2, IL-12, TNF and IFN-gamma, whereas a Th2 response is characterized by IL-4, IL-5, IL-6 and IL-10. Often, these cytokines are present in in vitro-derived culture supernatants at extremely low concentrations and are therefore very difficult to detect. Although a number of improvements have been made to the sensitivity of the relevant detection assays, the most successful assays involve the presence of the cells being cultured thereby limiting the number of tests per culture to one. Here we describe an enhanced ELISA protocol where the sensitivity is equivalent or better than corresponding cell-based assays. This protocol will permit the sensitive measurement of multiple cytokines per single culture supernatant.

Strain variation in susceptibility to monoclonal antibody-induced transplantation tolerance.

BACKGROUND: We have reproducibly induced specific tolerance to multiple minor histocompatibility antigens with nondepleting anti-CD4 and -CD8 monoclonal antibodies. The tolerance induced is effective for the lifetime of the host. We have tested this therapy in a number of mouse strain combinations to further understand the mechanisms. METHODS: Various mouse strains were grafted with allogeneic tail skin with and without nondepleting CD4- and CD8-specific monoclonal antibody therapy. The grafts were monitored daily for signs of rejection. RESULTS: Whereas the CBA/Ca (H2k) strain can be made tolerant to skin grafts that are mismatched at multiple minor histocompatibility antigens indefinitely, using the same protocol, long-term survival of similarly mismatched grafts on the HW80 (B6 congenic for BALB H1) mouse strain is limited to around 8 weeks. Interestingly, the B10.BR strain, which is also of the H2k haplotype, is also not readily tolerized. In addition, an F1 between the CBA/Ca and the resistant B10.BR strains is B10.BR-like in its susceptibility to tolerance induction. Susceptibility to such antibody-dependent tolerance induction is not related to immunogenicity because grafts mismatched at only a single minor antigen also do not reproducibly survive beyond 8 weeks when grafted onto HW80 mice in the presence of the antibody therapy. CONCLUSIONS: The data strongly suggest that the B6/B10 genetic background confers a level of resistance to CD4- and CD8-specific monoclonal antibody-dependent tolerance induction.

Interleukin-4 secretion by the allograft fails to affect the allograft-specific interleukin-4 response in vitro.

BACKGROUND: The role of the cytokine, interleukin (IL)-4, in allograft rejection and protection is not clear. We have previously shown that IL-4 transgenically expressed in a pancreas allograft does not protect the allograft from rejection. Here, we analyze the effect of the transgenically expressed IL-4 on the cytokine profile of the allograft-specific immune response. METHODS: C57BL/6SCID mice were infused with small numbers of spleen cells from C57BL/6 donors. The former received pancreas grafts from 1- to 2-day-old BALB/c donors which did or did not transgenically express IL-4 in the graft. Three weeks after the cell infusion, the spleens were removed and the splenocytes were restimulated in vitro with BALB/c APC, and third party BALB.K APC. IL-2 and IL-4 levels in the culture supernatants were measured. RESULTS: The presence of a pancreatic allograft induced an increase in the levels of both IL-2 and IL-4 in culture supernatants from splenocytes of mice receiving grafts compared with mice not receiving grafts. The presence of IL-4 transgenically expressed in the pancreas allograft had no effect on the in vitro cytokine profile. CONCLUSIONS: from these results we conclude that the failure of transgenically expressed IL-4 to protect the allograft was not associated with up-regulation of a graft antigen-specific IL-4 response.

Tolerance and suppression in a primed immune system.

The induction of tolerance in a primed immune system would be valuable therapeutically, but has been difficult to achieve. Mice primed to multiple minor histoincompatible antigens (minors) are able to rapidly reject secondary grafts using either their CD4+ or CD8+ T-cell subpopulations. Short courses of treatment with nonlytic anti-CD4 and anti-CD8 antibodies targeted at both T-cell subsets can induce long-term peripheral T-cell tolerance in primed mice. We examine the mechanisms by which peripheral tolerance is maintained, and show that tolerant mice harbor CD4+ T cells capable of specifically suppressing rejection mediated by either subset of primed T cells. Remarkably, elimination of CD4+ T cells from tolerant mice resulted in graft rejection, suggesting that graft-reactive CD8+ T cells had not been eliminated, but had been under continuous regulation by "tolerant" CD4+ T cells. This result demonstrates that it may be possible to establish therapeutic operational tolerance without permanently inactivating all antigen-reactive cells.

Marking planes of surgical excision on breast biopsy specimens: use of artists' pigments suspended in acetone.

The performance of carbon and metallic inks, silver nitrate solution, and artists' pigments mounted in acetone was compared for marking the surface of surgical biopsy specimens. Using India ink is an unsatisfactory procedure because of slow drying, messiness, and spreading of the ink. It is concluded that use of artists' pigments has many advantages over other reagents, because of their rapid drying, resistance to tissue processing, and the ability to mark simultaneously many different planes of excision. Furthermore, the pigments are readily visible, are distinguishable from each other on microscopical examination, and the method entails little extra cost.

Smooth muscle pseudotumours: a potentially confusing artefact of rectal biopsy.

An artefactual smooth muscle lesion was found in seven of 500 consecutive rectal biopsy specimens. The lesions had the deceptive appearance of a genuine tumour although none of the patients with the lesion had presented with a rectal mucosal swelling. The morphology of the lesion and its poor reproducibility under experimental conditions suggested that it was an artefact of the biopsy procedure: it was easily reproduced in resected specimens of large bowel using punch or basket forceps but not when using flat forceps. The presence of the lesion seems to depend on the type of forceps used rather than on differences in deployment and seems to be caused by avulsion of the superficial part of the muscularis propria and its incorporation into the tissues included in rectal biopsy specimens.

Segmental lymph-node infarction after fine-needle aspiration.

Wedge-shaped lymphoid depletion and sinus distention is described in the pole of an intramammary lymph node. The origin of the lesion appears to be traumatic venous thrombosis. Topographically the lesion differs from spontaneous venous infarction of lymph nodes, and resembles segmented infarction due to small arterial lesions. The usually localised trauma of fine-needle aspiration appears to account for its distinctive microanatomical distribution.

Frozen section diagnosis: an audit.

A consecutive series of 1000 operative frozen section diagnoses was reviewed. Correct diagnosis was made at the time in 96.5% of the cases. Clinically relevant errors were found in 1.3% of the cases and unimportant errors in 0.9%. Diagnosis was deferred, to await subsequent paraffin sections, in a further 1.3%. All the errors and provisional diagnoses in the deferred cases were conservative false negative results; no false positive diagnosis of malignancy was made. The cases of incorrect or deferred diagnosis were analysed to ascertain the origin of the difficulties, which comprised: technical imperfection (three cases); the focal nature of the lesion (14); and pathological misinterpretation (28). More than one of these factors played a part in eight cases. Further retrospective assessment indicated that the factors leading to error or deferred diagnosis were avoidable in 57% and potentially avoidable in 43% of cases. Misinterpretation was the single factor responsible for all avoidable misdiagnoses or deferred diagnosis. None the less, unavoidable factors led to erroneous or deferred diagnosis in about 2% of operative frozen section requests. Using present methods this seems to be the irreducible minimum of failures to make the correct diagnosis when frozen sections are assessed.

Food-starch granulomatous peritonitis.

Two cases of peritoneal granulomatous reactions to food starch are described. They followed bowel perforation and clinically mimicked tuberculous and glove-powder starch peritonitis. Their histological differences from corn-starch peritonitis warrant attention in the absence of previous documentation of starch as a component of peritoneal food granulomas. Food-starch granules tend to be larger than those of glove powder, are often oval, and may be extremely resistant to salivary diastase digestion.

Spontaneous infarction of superficial lymph nodes.

Five cases of extensive infarction of lymph nodes were traced in just over 16 years' surgical material. All presented with painful swelling in a superficial lymph node chain. None was diagnosed clinically; two were interpreted as fibroadenoma of the axillary tail of the breast, and two as a femoral hernia. Microscopically the lymph nodes in the first three weeks after infarction were characterized by extensive necrosis of medullary and cortical lymphoid cells, but the central reticulin architecture and a narrow, incomplete rim of viable subcapsular lymphoid tissue were preserved. Reactive perinodal inflammation and the formation of granulation tissue resembled the reaction to myocardial infarction. The late stage of the lesion was characterized by incomplete regeneration of lymphoid tissue in the lymph nodes. The lesions appeared attributable to thrombosis of veins within the substance and the hila of the nodes.