Improved polymerase chain reaction-based method to detect early-stage epitheliotropic T-cell lymphoma (mycosis fungoides) in formalin-fixed, paraffin-embedded skin biopsy specimens of the dog.

In the dog, early-stage epitheliotropic T-cell lymphoma (ETCL) can clinically and histologically mimic a large range of inflammatory dermatoses and often progresses rapidly to a more aggressive tumor stage. Early diagnosis of ETCL is essential to proceed with a specific oncologic therapy that is favorable for the prognosis. In the present study, an improved method for the detection of T-cell receptor gamma (TCRgamma) rearrangement was developed by designing a new set of consensus primers to amplify the different forms of rearranged canine TCRgamma gene sequences by polymerase chain reaction. The amplicons were analyzed by conventional polyacrylamide gel electrophoresis, which requires minimal specific equipment and may be performed in almost every pathology laboratory at low costs. The method proved to be highly specific and sensitive to detect early ETCL in formalin-fixed, paraffin-embedded biopsy specimens, providing an efficient tool for veterinary pathologists to distinguish early neoplastic from reactive cutaneous T-cell infiltrates (tumor-specific marker) or to discriminate T-cell lymphoma from B-cell lymphomas or nonlymphoid neoplasms (T-cell lineage marker). By direct sequencing analysis of amplified TCRgamma gene sequences, ETCL was found to rearrange exclusively the joining (J) 4 region, which suggests specific biology for primary cutaneous T-cell lymphomas. Also, a novel (seventh) functional J region in the TCRgamma gene, localized approximately 2.3 kb upstream of J5, was identified.

MBD2-mediated transcriptional repression of the p14ARF tumor suppressor gene in human colon cancer cells.

OBJECTIVE: The p14(ARF) and p16(INK4A) tumor suppressor genes are commonly inactivated by aberrant methylation of their promoter regions in human colon cancer. The methyl-CpG-binding domain protein MBD2 is physically associated with the methylated promoters of the p14(ARF) and p16(INK4A) genes in specific tumor cell lines. Moreover, deficiency of MBD2 strongly inhibits intestinal tumorigenesis in the Min mouse, raising the possibility that the protein might be involved in transcriptional repression of methylated tumor suppressor genes. The aim of this study was to evaluate the role of MBD2 in the silencing of p14(ARF) and p16(INK4A) in cancer. METHODS: The MBD2 protein was stably knocked down by RNA interference in RKO, a colon cancer cell line in which both p14(ARF) and p16(INK4A) are silenced by methylation. RESULTS: We demonstrate here that MBD2 associates with the methylated promoter of the p14(ARF) gene in the RKO colon cancer cell line. Depletion of MBD2 by RNAi leads to selective upregulation of the p14(ARF) but not the p16(INK4A) gene transcript. In addition, p14(ARF) repression can be restored by expressing mouse MBD2 protein in MBD2-deficient RKO cells. CONCLUSION: These findings implicate MBD2 in transcriptional repression of the methylated p14(ARF) tumor suppressor gene and suggest that repression by MBD2 selectively affects a subset of methylated promoters.

Engineering a high-affinity methyl-CpG-binding protein.

Core members of the MBD protein family (MeCP2, MBD1, MBD2 and MBD4) share a methyl-CpG-binding domain that has a specific affinity for methylated CpG sites in double-stranded DNA. By multimerizing the MDB domain of Mbd1, we engineered a poly-MBD protein that displays methyl-CpG-specific binding in vitro with a dissociation constant that is >50-fold higher than that of a monomeric MBD. Poly-MBD proteins also localize to methylated foci in cells and can deliver a functional domain to reporter constructs in vivo. We propose that poly-MBD proteins are sensitive reagents for the detection of DNA methylation levels in isolated native DNA and for cytological detection of chromosomal CpG methylation.

A cost-effective algorithm for hereditary nonpolyposis colorectal cancer detection.

Colorectal cancer with microsatellite instability (MSI) may occur sporadically or be inherited in cases of hereditary nonpolyposis colorectal cancer (HNPCC) syndrome. However, there is no consensus as to which patients must be tested and how to test MSI. In this study, MSI was tested by immunohistochemical analysis and by polymerase chain reaction in 148 cases of colorectal cancer, and methylation of the hMLH1 promoter was examined. MSI status was correlated with tumor phenotype. We found that localization, tumor infiltrating lymphocytes, and mucinous differentiation were predictive of high-frequency MSI (MSI-H) colorectal cancer and might be used to select cases for MSI analysis. Immunohistochemical analysis detected most MSI-H colorectal cancer and might constitute the first step in MSI detection. Absence of hMLH1 promoter methylation in MSI-H colorectal cancer could be predictive of hereditary colorectal cancer, and, hence, methylation analysis might constitute the second step in the identification of patients with HNPCC.

Systemic investigation of keratoepithelin deposits in TGFBI/BIGH3-related corneal dystrophy.

PURPOSE: To investigate the location and tissue-specificity of the pathologic keratoepithelin (KE) deposition in a patient with a keratoepithelinopathy (KEP), TGFBI/BIGH3-related corneal dystrophy. METHODS: An autopsy was performed in a patient with lattice type I corneal dystrophy (LCDI) after authorization was obtained from the family. Mutation screening in TGFBI/BIGH3 was done on the patient several years ago. Eighteen different tissues or organs, including brain, heart, lung, kidney, liver, lymph nodes, spleen, aorta, esophagus, bone marrow, urinary bladder (including a papillary urothelial carcinoma), samples of a metastatic squamous cell carcinoma, adrenal gland, parathyroid gland, muscle, prostate, and cornea were investigated, and sections from the tissues were labeled with KE2 rabbit TGFBI/BIGH3 antiserum. RESULTS: The patient, diagnosed with LCDI and Alzheimer's disease, died at 79 years of age from a complicated chronic obstructive lung disease. Mutation analysis showed the classical Arg124Cys mutation in exon 4 of TGFBI/BIGH3, associated with LCDI. Except for the cornea, immunostaining with KE2 antisera did not reveal any deposits in any of the 17 other organs analyzed. CONCLUSIONS: Pathologic deposits caused by KE accumulation were only observed in the cornea and in no other tissue or organ in this patient. These results suggest a cornea-specific mechanism in the aggregation of KE. Further studies need to be done to investigate whether the degradation of mutated KE generates cornea-specific fragments that aggregate or whether the clearing of normal fragments is different in affected corneas, which then leads to aggregation.

De novo concurrent papillary renal cell carcinoma and angiomyolipoma in a kidney allograft: evidence of donor origin.

In the general population, renal cell carcinoma (RCC) is a relatively common neoplasm; however, the papillary RCC subtype is infrequent and represents only 10 to 15% of all RCC. Angiomyolipoma is a well-known common benign tumor. The occurrence of RCC in association with angiomyolipoma is a rare event, with only approximately 50 cases reported in the nontransplantation setting. In transplant recipients, RCC can develop in native kidneys, but its occurrence "de novo" in the renal allograft is very rare with an estimated incidence of less than 0.5%. We report here the case of a 39-year-old woman who underwent cadaveric renal transplantation in 1990. No lesion was observed in the allograft during the pre- and perioperative period or on early postoperative ultrasounds. No graft rejection occurred under a standard triple immunosuppressive therapy. Thirteen years later, during a routine ultrasonography, 2 solid masses were discovered in the allograft, both of them richly vascularized. She underwent allograft nephrectomy and the histologic findings revealed that one of the tumors was a chromophilic (type 1) papillary RCC (2.5 cm in diameter) and the other, an angiomyolipoma (1.5 cm). Microsatellite analysis of the allograft, as compared with the recipient peripheral blood leukocytes, demonstrated that the 2 tumors (1 malignant and 1 benign) were of donor origin. To our knowledge, this is the first report of de novo concurrent papillary RCC and angiomyolipoma in a renal allograft.

Tissue engineered fetal skin constructs for paediatric burns.

Autologous skin-grafting is the gold standard for treatment of deep second and third degree burns. Available bioengineered skin products also necessitate this two-step surgical procedure. Therefore, we developed fetal skin constructs to improve healing of such degree burns. A bank of fetal skin cells was developed from one organ donation (4 cm2 of skin allowing the preparation of several million three-dimensional skin constructs, 9x12 cm, on native horse collagen). Successive fetal constructs were applied to eight patients at every change of dressing during 1-3 weeks in an outpatient setting. Complete closure was rapid (mean 15.3 days [SD 5.5]) with little hypertrophy of new skin and no retraction seen. This simple technique provided complete treatment without auto-grafting, showing that fetal skin cells might have great potential to treat burns and eventually acute and chronic wounds of other types.

Naturally acquired MAGE-A10- and SSX-2-specific CD8+ T cell responses in patients with hepatocellular carcinoma.

Immunotherapy is being proposed to treat patients with hepatocellular carcinoma (HCC). However, more detailed knowledge on tumor Ag expression and specific immune cells is required for the preparation of highly targeted vaccines. HCC express a variety of tumor-specific Ags, raising the question whether CTL specific for such Ags exist in HCC patients. Indeed, a recent study revealed CTLs specific for two cancer-testis (CT) Ags (MAGE-A1 and MAGE-A3) in tumor infiltrating lymphocytes of HCC patients. Here we assessed the presence of T cells specific for additional CT Ags: MAGE-A10, SSX-2, NY-ESO-1, and LAGE-1, which are naturally immunogenic as demonstrated in HLA-A2(+) melanoma patients. In two of six HLA-A2(+) HCC patients, we found that MAGE-A10- and/or SSX-2-specific CD8(+) T cells naturally responded to the disease, because they were enriched in tumor lesions but not in nontumoral liver. Isolated T cells specifically and strongly killed tumor cells in vitro, providing evidence that these CTL were selected in vivo for high avidity Ag recognition. Therefore, besides melanoma, HCC is the second solid human tumor with clear evidence for in vivo tumor recognition by T cells, providing the rational for specific immunotherapy, based on immunization with CT Ags such as MAGE-A10 and SSX-2.

Expression of NDRG1, a differentiation-related gene, in human tissues.

NDRG1 is a member of the new N-myc downregulated gene (NDRG) family which belongs to the alpha/beta hydrolase superfamily, but without presenting a hydrolytic catalytic site. Diverse physiological and pathological conditions (hypoxia, cellular differentiation, heavy metal, N-myc, neoplasia) modulate NDRG1 transcription, mRNA stability, and translation. In this report we present the immunohistochemical localization of NDRG1 in a large set of normal human tissues at light and electron microscopic levels. The immunoreactivity of NDRG1 is mostly found in epithelial cells with different aspects. We observed NDRG1 primarily in the cytoplasm, but it is also associated with the cellular membrane and adherens junctions. Given the strong upregulation of NDRG1 under hypoxia and its nuclear localization, we propose a role for NDRG1 in protection from ischemic cell damage. The multiple localizations of this protein also suggest pleiotropic functions amongst which a functional involvement in the E-cadherin/catenin complex.

Role of metallothioneins in irradiated human rectal carcinoma.

BACKGROUND: Metallothioneins (MT) are low-molecular weight, metal-binding proteins that play a role in cellular proliferation and differentiation, as well as in cellular defense mechanisms. They act as scavengers of free radicals produced by irradiation. A number of in vitro and in vivo studies have linked overexpression of cellular MT with tumor cell resistance to radiation. This is the first study that investigates whether MT expression is involved in the radioresistance of rectal carcinoma. METHODS: Using a mouse monoclonal antibody, MT expression was analyzed by immunohistochemistry on surgical samples (n = 85) from 85 patients with locally advanced rectal carcinoma who were treated preoperatively with a hyperfractionated and accelerated radiotherapy schedule and on tumor biopsies (n = 13) obtained before treatment. The potential correlations between MT expression and pathologic variables and survival were examined. RESULTS: MT were expressed strongly in both the cytoplasm and nucleus of tumor cells in 7 biopsy and 42 surgical samples. A comparison of MT expression in biopsy and surgical specimens showed that MT expression did not change after irradiation in most cases. Against all expectations, MT were expressed more frequently in tumors from responders than in those from the nonresponders (P = 0.02). There was no correlation between MT expression and tumor stage, histology after radiotherapy, or survival. CONCLUSION: These findings do not Cansupport the hypothesis that MT overexpression at the end of radiotherapy is a marker for radiation resistance.

Application of molecular genetics to the diagnosis of lymphoid-rich effusions: study of 95 cases with concomitant immunophenotyping.

The cytological differentiation between reactive lymphocytosis and malignant lymphoma in serous effusions is often difficult. The present study was designed to evaluate the potential contribution of molecular genetic clonality analysis to a solution to this problem. We examined the cytological specimens of 95 consecutive patients collected during a 4-yr period, including 74 pleural, 20 peritoneal, and one pericardial fluids. Cytological diagnosis in the 95 lymphocyte-rich effusions was positive for lymphoma in 20 cases, suspicious for lymphoma in 26 cases, and negative in 49 cases. The analysis by ICC was not carried out, inconclusive, or noninterpretable in 25 cases. In five cases molecular genetic analysis was hampered by technical problems. By immunocytochemistry, eight additional cases of lymphoma were detected and lineage classification was achieved in 15 of the 20 cytologically positive effusions. PCR and Southern blot analysis were used to assess B- and T-cell clonality. Monoclonality was found in 40 (42%) of the 95 effusions analyzed. One-third of the effusions with a monoclonal B-cell gene rearrangement were detected by Southern blot analysis but not by the PCR performed in parallel. The results of molecular genetic analysis were corroborated by histological findings and/or clinical evolution in 15 cases. Our results indicate that molecular genetic analysis is a useful tool in the analysis of lymphocyte-rich serous effusions.

Comparison of microsatellite instability and chromosomal instability in predicting survival of patients with T3N0 colorectal cancer.

BACKGROUND: At least 2 apparently independent mechanisms, microsatellite instability (MSI) and chromosomal instability, are implicated in colorectal tumorigenesis. Their respective roles in predicting clinical outcomes of patients with T3N0 colorectal cancer remain unknown. METHODS: Eighty-eight patients with a sporadic T3N0 colon or rectal adenocarcinoma were followed up for a median of 67 months. For chromosomal instability analysis, Ki-ras mutations were determined by single-strand polymerase chain reaction, and p53 protein staining was studied by immunohistochemistry. For MSI analysis, DNA was amplified by polymerase chain reaction at 7 microsatellite targets (BAT25, BAT26, D17S250, D2S123, D5S346, transforming growth factor receptor II, and BAX). RESULTS: Overall 5-year survival rate was 72%. p53 protein nuclear staining was detected in 39 patients (44%), and MSI was detected in 21 patients (24%). MSI correlated with proximal location (P <.001) and mucinous content (P <.001). In a multivariate analysis, p53 protein expression carried a significant risk of death (relative risk = 4.0, 95% CI = 1.6 to 10.1, P =.004). By comparison, MSI was not a statistically significant prognostic factor for survival in this group (relative risk = 2.2, 95% CI = 0.6 to 7.3, P =.21). CONCLUSIONS: p53 protein overexpression provides better prognostic discrimination than MSI in predicting survival of patients with T3N0 colorectal cancer. Although MSI is associated with specific clinicopathologic parameters, it did not predict overall survival in this group. Assessment of p53 protein expression by immunocytochemistry provides a simple means to identify a subset of T3N0 patients with a 4-times increased risk for death.