The aim of this study was to determine the expression of transforming growth factor-beta (TGFbeta)-1 and type I TGFbeta-receptor on sequential biopsies from renal transplants with and without chronic allograft nephropathy. Twenty-four renal transplant recipients entered the study. They underwent sequential biopsies performed before (T1: 1.44 +/- 1.2 months) and 6 months after (T2: 15.96 +/- 7.2 months) transplantation. Lesions were graded according to the criteria of the Banff classification. C4d was detected by fluorescence microscopy. Immunohistochemistry was performed in order to identify cells expressing TGFbeta-1 and type I TGFbeta-receptor. In normal renal tissue (n = 4), TGFbeta-1 is expressed by tubular epithelial cells and endothelial cells lining glomerular and peritubular capillaries, whereas type 1 TGFbeta-receptor is expressed by tubular epithelial cells and smooth muscle cells in the media of arteries. In recipients with chronic allograft nephropathy (group 1, n = 14), diffuse epithelial expression of both molecules was found in more patients at T2 than at T1 (42.8% vs 21.4%). In contrast, this pattern of expression remained stable or decreased over time in recipients with long-term normal transplants (group 2, n = 10). Furthermore, type 1 TGFbeta-receptor was detected on the smooth muscle cells of arteries in 12/14 (85.7%) of recipients in group 1 and only in 4/9 (44.4%) of recipients in group 2. No relationship was noticed with regard to C4d deposits. These data suggest that the synthesis of TGFbeta-1 and type I TGFbeta-receptor increases over time in recipients developing chronic allograft nephropathy. Further studies are in progress in order to quantify mRNA of both molecules with real-time polymerase chain reaction.

The aim of this study was to assess the feasibility of detecting anti-HLA antibodies in eluates from needle core biopsies of renal transplants with chronic allograft nephropathy. Two methods of screening, the enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FlowPRA) were compared. Twenty renal transplants with CAN were removed after irreversible graft failure. To assess the feasibility of detecting anti-HLA antibodies in small samples, needle core biopsies were sampled at the same place as surgical samples and at a second cortical area. Antibodies were eluted with an acid elution kit and anti-class I and class II IgG HLA antibodies detected using ELISA and flow cytometry. Flow cytometry was found to be more sensitive than ELISA for detecting anti-HLA antibodies in eluates from renal transplants with CAN (95% vs. 75% of positive cases). Detection of anti-HLA antibodies showed good agreement between surgical samples and needle core biopsies performed at the same place for anti-class I (80% vs. 65%, r=0.724 P<0.01) and anti-class II HLA antibodies (70% vs. 55%, r=0.827 P<0.01). In addition, differences in the detection of anti-class I HLA antibodies in needle core biopsies sampled at different sites suggests that immunization to class I donor antigen could be underestimated in needle core biopsy samples. These data indicate that anti-HLA antibodies can be detected in needle core biopsies from renal transplants. Provided further evaluation is done, elution might be a complementary method to detect anti-HLA antibodies when they are bound to the transplant.

La néphropathie chronique de l'allogreffe (NCA) qui se traduit cliniquement par un dysfonctionnement chronique du transplant rénal, reste la principale cause de perte tardive du transplant rénal. Ses mécanismes sont multiples et encore mal compris. Le but de ce travail est d'étudier l'expression du TGFβ1 et de son récepteur de type 1 en fonction de la pathologie initiale et de rechercher un lien avec la NCA. Trente quatre transplantés rénaux ont été inclus dans cette étude. Les biopsies du transplant rénal, réalisées lors de dysfonctionnement ou à titre systématique ont été classées, selon le délai écoulé après la transplantation, en biopsie précoces (délai 6 mois). Les lésions du transplant ont été évaluées selon la classification de Banff 1997. L'expression du TGFβ1 et son récepteur de type 1 ont été déterminés par immunohistochimie et les dépôts de C4d ont été recherchés sur coupes congelées par une technique d'immunofluorescence indirecte. Une technique sensible de cytométrie en flux (FlowPRA) a permis de détecter les anticorps anti-HLA dans les sérums prélevés au moment des biopsies. Les pathologies observées sur les biopsies précoces ont été classées en 4 groupes : rejet aigu humoral (n = 11), rejet aigu cellulaire (n = 7), toxicité médicamenteuse (n = 6) et lésions non spécifiques (n = 10). Des lésions de NCA ont été observées sur les biopsies intermédiaires de 14 patients alors qu'un dysfonctionnement chronique du transplant est apparu chez 17 receveurs. Le profil d'expression du TGFβ1 et de son récepteur n'était pas statistiquement différent dans chaque groupe lésionnel et au cours de la NCA. Néanmoins, nous avons observé au cours du développement de la NCA, une augmentation des types cellulaires exprimant cette cytokine et son récepteur suggérant que leur synthèse est augmentée au cours de cette pathologie. À l'inverse, les RA cellulaires, associés dans cette étude à une durée d'ischémie froide réduite (p = 0,03) et à une faible incidence de dysfonctionnement chronique, ont été suivi par une diminution de l'expression du TGFβ1 par les cellules épithéliales tubulaires et du récepteur de type 1 par les cellules musculaires lisses des parois artérielles. Dans cette étude, le RA humoral, la toxicité médicamenteuse et les lésions de conservation reste les principaux facteurs de risque de la NCA. Une augmentation, au cours du temps, des types cellulaires marqués par le TGFβ1 et son récepteur de type 1 a été observée durant l'apparition NCA sans qu'il soit toutefois possible d'établir un profil d'expression à risque de cette cytokine en fonction de la pathologie initiale.

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.

The assessment of the microsatellite instability (MSI) status in colorectal cancers is presently warranted for three reasons: 1) as a screening tool for hereditary nonpolyposis colorectal cancer, 2) as a prognostic marker, and 3) as a potential predictive factor of chemotherapy response. The aim of this study was to evaluate, on a large scale with tissue samples coming from a number of different sources, the difficulties met with routine use of immunohistochemistry (IHC) and to determine if it really does offer an accurate alternative to PCR genotyping. Colorectal carcinomas from 462 consecutive patients resected in public or private hospitals were assessed for MSI status by two methods: MSI testing (with BAT-26 microsatellite) and IHC detection of hMLH1, hMSH2, and hMSH6 proteins. Of the 398 cancers tested, immunohistochemistry was noncontributory in 42 (10.5%), focal in 9 (2.3%), and discordant with the PCR results in 36 (9%). For these 87 cases, complementary analyses were performed to explain discrepancy. After additional IHC assay with modified processing protocols, 8 cases remained noncontributory, 2 focal, and 28 discordant: 18 microsatellite stability IHC/MSI PCR and 10 MSI IHC/microsatellite stability PCR. For these discordant cases, we performed a multiplex PCR assay on DNA extracted from the frozen sample and BAT-26 was amplified from DNA extracted from the paraffin blocks used for IHC. Four discordant cases were reclassified after PCR multiplex assay (3 as MSI and 1 as microsatellite stability). Five other cases displayed intratumoral heterogeneity and 19 remained discordant. The discrepancy could be partly explained by variable technical protocols of fixation in the different laboratories, leading to variations in staining quality and difficulties in IHC interpretation. This population-based study is the first one to show that IHC is not sensitive and specific enough to be used routinely. Immunohistochemistry analysis of MMR proteins must be performed in standardized conditions and interpreted by confirmed pathologists. It cannot replace PCR as long as protocols are not optimized and harmonized.

1 Review. Immunohistochemistry in the diagnosis of soft tissue tumours. J M Coindre 17 p57 is useful in the classification and differential diagnosis of complete and partial hydatidiform moles. S-Y Jun, J Y Ro and K-R Kim 26 Expression of differentiation and proliferation related proteins in epithelium of prophylactically removed ovaries from women with a hereditary female adnexal cancer predisposition. J M J Piek, R H M Verheijen, F H Menko, A P M Jongsma, J Weegenaar, J J P Gille, G Pals, P Kenemans and P J van Diest 33 The angiogenic receptor KDR is widely distributed in human tissues and tumours and relocates intracellularly on phosphorylation. An immunohistochemical study. M Stewart, H Turley, N Cook, F Pezzella, G Pillai, D Ogilvie, S Cartlidge, D Paterson, C Copley, J Kendrew, C Barnes, A L Harris and K C Gatter 40 Sporadic colorectal cancers with defective mismatch repair display a number of specific morphological characteristics: relationship between the expression of hMLH1 and hMSH2 proteins and clinicopathological features of 273 adenocarcinomas. C Chapusot, L Martin, N Mungra, D Rageot, A M Bouvier, C Bonithon Kopp, T Ponnelle, J Faivre and F Piard 48 Subepithelial myofibroblasts and tenascin expression in microscopic colitis. A Salas, F Fernández-Bañares, J Casalots, C González, X Tarroch, P Forcada and G González 55 A comparative histological and morphometric study of vascular changes in idiopathic portal hypertension and alcoholic fibrosis/cirrhosis. K Tsuneyama, K Ohba, Y Zen, Y Sato, H Niwa, H Minato and Y Nakanuma 62 Lack of histologically suspicious features, proliferative activity, and p53 expression suggests benign diagnosis in phaeochromocytomas. K Salmenkivi, P Heikkilä, C Haglund, J Louhimo and J Arola 72 Synovial sarcoma in older patients: clinicopathological analysis of 32 cases with emphasis on unusual histological features. J A Chan, M E McMenamin and C D M Fletcher 84 Expert opinion. Reporting needle core biopsies of breast carcinomas. S A Hoda, M Harigopal, G C Harris, S E Pinder, A H S Lee and I O Ellis 91 Lesson of the month. Necrotizing sarcoid granulomatosis with skin involvement. C C Shirodaria, A G Nicholson, D M Hansell, A U Wells and R Wilson 94 Correspondence

Background. Chronic allograft nephropathy (CAN), which remains the main cause of graft loss after kidney transplantation, is still poorly understood. Because anti-HLA antibodies may be involved in the pathogenesis of CAN, this study was performed to look for donor-specific antibodies (DSA) fixed onto renal transplants with CAN. Methods. DSA were identified after elution with flow cytometric assay and/or flow cytometric crossmatches in 20 transplants removed after irreversible graft failure caused by CAN and in control samples from 2 transplants with relapsing glomerulopathy, 2 transplants lost after vascular thrombosis, and 4 normal kidneys. The results were compared with those obtained in the serum samples 1 year after grafting, at the time of transplantectomy, and within 2 months after transplantectomy. Results. IgG anti-class I, anti-class II, or both DSA were identified in 70.6% of eluates versus 73.6% of posttransplantectomy serum samples (NS), 42.1% of 1-year postgrafting serum samples (P <0.05), and 31.6% of serum samples at the time of transplantectomy (P <0.05). Our data show a good correlation between the target of anti-HLA antibodies found in both eluates and posttransplantectomy serum samples, but the precise specificity of anti-HLA antibodies is more often assigned in posttransplantectomy serum samples than in eluates. This problem needs further evaluation. Conclusion. This study shows that testing for anti-HLA DSA in eluates from removed kidney transplants using flow cytometry can be achieved and is highly efficient. It already suggests that both anti-class I and anti-class II HLA antibodies can be involved in CAN. Further studies are now needed to evaluate the possibility of identifying such antibodies in the eluates of transplant biopsy specimens from recipients experiencing CAN.

The Inhibitor of Apoptosis Protein (IAP) family includes several critical cell death inhibitors, the expression of which could be involved in colorectal carcinogenesis. Among them, c-IAP1 and c-IAP2 expression has never been investigated in colorectal cancer. The present study was designed to determine whether expression of both IAPs was related to pathological parameters and survival in sporadic colon carcinomas. Analysis of five human colon cancer cell lines by both western blotting of cell fractions and immunocytochemistry showed that the two IAPs could be expressed both in the nucleus and in the cytoplasm. Immunohistochemical analysis of a series of 46 sporadic colorectal adenocarcinomas demonstrated that c-IAP1 expression was more frequent in the nucleus (85%), and that c-IAP2 was more often expressed in the cytoplasm (82%). A significant association was identified between a strong lymphoid infiltrate in the stroma and the nuclear expression of c-IAP2 (p = 0.02). No other relationship was observed between IAP expression and pathological features. After adjusting by age and stage, the relative risk of death was lower for cytoplasmic c-IAP1, cytoplasmic c-IAP2, and nuclear c-IAP2 expression. It was higher for nuclear c-IAP1 expression. These associations were not statistically significant, but this work underlines the importance of taking into account the subcellular location of the IAP family members in the evaluation of their prognostic significance.

AIMS:The aim of this study was to assess the independent value of pathological criteria in the diagnosis of mismatch repair (MMR)-defective sporadic colorectal cancers.METHODS AND RESULTS:Resected colorectal adenocarcinomas (n = 273) were reviewed in order to identify a number of specific morphological features of MMR-defective carcinomas. Of the 273 cases, 35.2% were right-sided and 5.9% were poorly differentiated. Focal extracellular mucin secretion was seen in 5.1% of cases and a stromal follicular reaction in 4.6%. The expression of the two major MMR proteins hMLH1 and hMSH2 was studied by immunohistochemistry. Carcinomas were considered deficient in the MMR system when a loss of nuclear signal in neoplastic cells was observed for one of the proteins. Such an extinction was seen in 37 of the cases (13.6%). The hMLH1 protein was the one most frequently altered (86.5%). After multivariate analysis, three independent factors were significantly associated with MMR deficiency: proximal location [odds ratio (OR) = 9.30; 95% confidence interval (CI) 2.79, 30.98], the presence of a true stromal follicular reaction (OR = 13.60; 95% CI 2.98, 62.00) and poor differentiation (OR = 8.33; 95% CI 1.63, 40.32).CONCLUSIONS:These results confirm that sporadic colorectal MMR-defective adenocarcinomas display certain specific morphological characteristics. However, these pathological features are not sufficiently predictive and immunohistochemistry is needed to identify such tumours accurately.

Microsatellite instability has been proposed as an alternative pathway of colorectal carcinogenesis. The aim of this study was to evaluate the interest of immunohistochemistry as a new tool for highlighting mismatch repair deficiency and to compare the results with a PCR-based microsatellite assay. A total of 100 sporadic proximal colon adenocarcinomas were analysed. The expression of hMLH1, hMSH2 and hMSH6 proteins evaluated by immunohistochemistry was altered in 39% of the cancers, whereas microsatellite instability assessed by PCR was detected in 43%. There was discordance between the two methods in eight cases. After further analyses performed on other tumoural areas for these eight cases, total concordance between the two techniques was observed (Kappa=100%). Our results demonstrate that immunohistochemistry may be as efficient as microsatellite amplification in the detection of unstable phenotype provided that at least two samples of each carcinoma are screened, because of intratumoural heterogeneity.