Heterogeneity of colorectal cancer (CRC) in reference to KRAS proto-oncogene utilizing WAVE technology.

BACKGROUND: New drugs targeting specific genes required for unregulated growth and metastases have improved survival rates for patients with metastatic colorectal cancer. Resistance to monoclonal antibodies specific for the epidermal growth factor receptor (EGFR) has been attributed to the presence of activating point mutations in the proto-oncogene KRAS. The use of EGFR inhibitor monotherapy in patients that have KRAS wild type has produced response rates of only 10-20%. The molecular basis for clinical resistance remains poorly understood. We propose two possible explanations to explain these low response rates; 1) levels of resistant CRC cells carrying mutated KRAS are below the sensitivity of standard direct sequencing modalities (<5%) or 2) the standard practice of analyzing a single area within a heterogeneous tumor is a practice that can overlook areas with mutated KRAS. METHODS: In a collaborative effort with the surgical and molecular pathology departments, 3 formalin fixed paraffin embedded tissue blocks of human CRC were obtained from the human tissue bank maintained by the Lifespan Pathology Department and/or the human tissue bank maintained by the Molecular Pathology Core of the COBRE for Cancer Research Development. The three specimens previously demonstrated KRAS mutations detected by the Applied Biosystems Kit. The Wave system 4500 (high performance ion-pairing liquid chromatography (IP-HPLC)) was utilized to evaluate tissue for the presence of KRAS proto-oncogene mutations at codons 12 and 13. RESULTS: Initially, the sensitivity of WAVE technology was compared with direct sequencing by evaluating a dilutional series. WAVE detected mutant alleles at levels of 2.5% compared to 20% performed with standard direct sequencing. Samples from three patients were evaluated by WAVE technology. Eight samples from patient 1 were analyzed. In two of eight samples, no mutations were detected at concentrations as low as 5%. In one sample a mutation was noted by WAVE and not by direct sequencing. All four samples from patient 2 tested positive for Exon 12/13 mutations. Of the seven samples from patient 3, five were positive for Exon 12/13 mutations and two were negative for Exon 12/13 mutations. CONCLUSION: In these studies the analysis of three patients' colorectal cancer tissues were analyzed utilizing the WAVE technology. Results demonstrated a greater degree of sensitivity in mutation detection when compared to standard sequencing. These studies also demonstrated heterogeneity of expression of KRAS mutations between areas of the tissue samples at a genomic level. The low clinical response rates to EGFR inhibition might be explained by the variation in mutation presence, which was dependent upon the region examined. The heterogeneity demonstrated in these studies provides another phenotypic variant that will impact clinical care.

The efficacy of five techniques for removing root filling material: microscopic versus radiographic evaluation.

AIM: To test and compare the efficacy of five methods for the removal of root filling material and to test the hypothesis that radiographs fail to represent the real extent of remaining material on canal walls. METHODOLOGY: Fifty maxillary anterior single-rooted teeth with straight root canals were selected. The coronal third of each root canal was prepared with Gates-Glidden drills to number 3, whilst the apical two-thirds were prepared with manual K-files to size 40. Root fillings were performed using lateral compaction with gutta-percha and AH-26. After full setting, the coronal third of the root filling was removed with Gates-Glidden drills and the teeth divided into five groups (n=10). The remaining root filling material was then removed with either Hedström files and chloroform (25 µL), using size 40 as the last file, SafeSider files, using a NiTi Pleezer reamer with a 0.06 taper followed by size 40 reciprocating file, with or without chloroform, or ProTaper Universal retreatment files (D2, D3) with or without chloroform. Reaching working length with no more gutta-percha on the last file was defined as the endpoint for all procedures. The presence of remaining filling material was first evaluated radiographically and then by the microscopic evaluation of split roots. The time required to accomplish the procedure was also recorded. anova and anova with repeated measures were used for statistical analysis of the results. RESULTS: Overall, 11-26% of the canal wall remained covered with filling material; no significant difference was found between the groups. The mechanized methods were faster than manual removal of filling material (P < 0.01); the use of solvent did not speed up the mechanized procedures. Radiographic evaluation failed to adequately and reliably detect the extent of filling material remaining on the canal walls, which was later observed by microscopic evaluation. CONCLUSIONS: All methods left root canal filling material on the canal walls. Radiographic evaluation failed to detect the extent of remaining root filling material, which could only be detected using microscopy.

A proteomic analysis reveals the loss of expression of the cell death regulatory gene GRIM-19 in human renal cell carcinomas.

Gene associated with retinoid interferon-induced mortality (GRIM)-19, an inhibitor of transcription factor STAT3, was originally identified as a critical regulatory protein in a genetic screen that was designed to identify the gene products necessary for Interferon (IFN)-beta- and retinoic acid-induced cell death. Over expression of GRIM-19 activates cell death. Conversely, inactivation of its expression promotes cell growth. STAT3 is a transcription factor that regulates gene expression in response to multiple extra cellular growth factors. In contrast to its normal feedback inhibition, a constitutive activation of STAT3 has been documented in several tumors. Although many STAT3-inhibitors are described, their relevance to human cancer is unclear. In an attempt to define the molecular alterations associated with human renal cell carcinoma (RCC) using mass spectrometry, we have discovered that expression of GRIM-19 is lost or severely depressed in a number of primary RCC and in some urinogenital tumors. Using an RCC cell line, we show that down regulation of GRIM-19 promotes tumor growth via an augmentation of STAT3-dependent gene expression. These studies for the first time show a tumor-suppressor like activity of GRIM-19.

Lipopolysaccharide endotoxemia reduces cell proliferation and decreases enterocyte apopotosis during intestinal adaptation in a rat model of short-bowel syndrome.

Sepsis is frequently associated with or complicates short-bowel syndrome (SBS). To investigate the effects of lipopolysaccharide (LPS) endotoxemia on enterocyte proliferation and death via apoptosis in a rat model of SBS, adult male Sprague-Dawley rats were divided into three experimental groups: sham rats underwent bowel transection and reanastomosis; SBS rats underwent 75% small-bowel resection; and SBS-LPS rats underwent 75% bowel resection and were given intraperitoneal injections of LPS 10 mg/kg. Parameters of intestinal adaptation (bowel and mucosal weights, mucosal DNA and protein, villus height, and crypt depth), enterocyte proliferation, and death via apoptosis were determined on day 15 after the operation. Statistical analysis was determined by Student's and ANOVA tests with a P less than 0.05 considered significant. SBS-LPS animals demonstrated a significant decrease (vs SBS rats) in duodenal (20%), jejunal (30%), and ileal (15%) overall weight, duodenal (20%), jejunal (27%), and ileal (18%) mucosal weight, jejunal (20%) and ileal (30%) mucosal DNA, jejunal (29%) and ileal (31%) villus height, and jejunal (14%) and ileal (29%) crypt depth. LPS endotoxemia led to reduced cell proliferation and enterocyte apoptosis compared to untreated SBS animals. Thus, in a rat model of SBS, LPS endotoxemia inhibits intestinal adaptation. A possible mechanism may be decreased cell proliferation. Decreased enterocyte loss via apoptosis may reflect a reduced number of enterocytes. Other mechanisms (necrosis) may be mainly responsible for cell death following LPS injection.

Cytotoxic phenotype of intra-epithelial lymphocytes in normal and cryptorchid human testicular excurrent ducts.

BACKGROUND: Most testicular and epididymal lymphocytes express T-cell markers, but their cytotoxic potential and activation status have not been reported. In this study, distribution of the cytotoxic cells was compared between normal and cryptorchid testes stratified into two groups: the first with complete absence of germ cells [Sertoli cell-only (SCO)] and the second with arrested spermatogenesis (SCA). METHODS: Immunohistochemistry for the T-lymphocyte marker CD3 and cytotoxic markers CD8, TIA-1 and granzyme B was performed on paraffin-embedded sections. RESULTS: The number of CD8+ and CD3+ intra-epithelial lymphocytes (IELs) increased distally throughout the normal epididymis. TIA-1 immunostaining revealed that a significant proportion of IELs exhibited cytotoxic potential, whereas granzyme B staining disclosed a subpopulation of activated cytotoxic lymphocytes (CTLs). TIA-1/CD8 and granzyme B/CD8 double immunostaining revealed that the vast majority of TIA-1+ and granzyme B+ cells were CD8+. The proportion of activated granzyme B+ lymphocytes increased distally throughout the normal epididymis. The number of TIA-1+ and granzyme B+ intra-epithelial and stromal lymphocytes was significantly increased in the normal as opposed to the SCO cryptorchid epididymis and proximal vas deferens. CONCLUSIONS: These results suggest that exposure of the testicular excurrent ducts to spermatozoa or immature germ cells triggers the activation and recruitment of CTLs. Cytotoxic granule effector mechanisms may contribute to the immunological barrier preventing the immune response to spermatozoa in testicular ducts.

Apoptosis, proliferation, and Fas (APO-1, CD95)/Fas ligand expression in medullary carcinoma of the breast.

Medullary carcinoma (MC) of the breast is a unique subtype of infiltrating ductal carcinoma that is characterized by a prominent lymphoid infiltrate and improved prognosis. Activated granzyme B(+)/CD8(+) cytotoxic T-lymphocytes (CTLs) infiltrating tumour cell nests constitute a major subset within the lymphoid infiltrate. As CTLs destroy target tumour cells by triggering apoptosis, it would be of interest to determine whether the apoptotic rate in MC is increased. This study evaluates the extent of apoptosis in relation to Fas (APO-1, CD95)/Fas ligand (FasL) expression in MC. Fourteen cases of typical MC (TMC) and 15 cases of atypical MC (AMC) classified according to the Ridolfi criteria, as well as 19 cases of poorly differentiated infiltrating ductal carcinoma (PDC) were evaluated. The apoptotic index (AI) was assessed by the TUNEL method on paraffin-embedded tissue. Cell proliferation was evaluated immunohistochemically by PCNA staining. The level of Fas/FasL expression was determined semiquantitatively by immunohistochemistry using a four-grade scoring system. The AI was significantly increased in TMC and AMC as opposed to the PDC subgroup (2.2+/-0.8, 2.1+/-0.8, and 1.3+/-0.6, respectively; p<0.05). A significant proportion (31.8+/-7.9% in TMC and 25.8+/-9.7% in AMC) of the apoptotic tumour cells within tumour nests were in close contact with CD3(+) lymphocytes. Increased apoptosis was not accompanied by increased proliferation of tumour cells. The extent of Fas expression did not differ between the three subgroups. FasL was expressed both by tumour infiltrating lymphocytes in MC and by tumour epithelium in all three subgroups. The observation that the majority of MCs express Fas and are infiltrated by lymphocytes expressing FasL suggests that increased apoptosis in MC is mediated by Fas/FasL. However, our observation that the majority of MCs also express FasL and the fact that tumours co-expressing Fas and FasL did not show increased apoptosis suggest that there may be additional cytotoxic pathways that lead to tumour apoptosis in MC.

Cloning of a glucose phosphate isomerase/neuroleukin-like sperm antigen involved in sperm agglutination.

The mouse monoclonal antibody (mAb) A36 produced by us and shown to induce extensive, "tangled" sperm agglutination was used to isolate cDNAs encoding its cognate antigen. Three overlapping cDNA clones specifically recognized by the mAb were isolated from a human testis cDNA expression library in lambdagt11. Sequencing of these cDNAs yielded the complete nucleotide sequence of a 3-kilobase cDNA that encodes the mAb-related polypeptide, designated sperm antigen-36 (SA-36), composed of 558 deduced amino acids. SA-36 cDNA contained a 5' untranslated region of 234 nucleotides (nt), an open reading frame of 1674 nt, and a 3' untranslated region of 1138 nt. SA-36 cDNA displayed > 99% homology to glucose phosphate isomerase (GPI)/neuroleukin (NLK) mRNA. This surprising homology was confirmed in Western blots demonstrating that mAb A36 reacted specifically with GPI obtained from rabbit muscle and from baker's yeast. Moreover, polyclonal, monospecific antibodies produced against beta-galactosidase/SA-36-3 fusion protein stained human spermatozoa and caused intensive agglutination of these cells in a manner similar to that with the mAb. Taken together, the data presented here demonstrated that mAb A36 cognate sperm surface antigen, encoded by SA-36 cDNA, is a GPI/NLK-like protein involved in sperm agglutination.

Cytotoxic phenotype of tumor infiltrating lymphocytes in medullary carcinoma of the breast.

Medullary carcinoma (MC) of the breast is considered to carry a more favorable prognosis than other subtypes of infiltrating ductal carcinoma This is a biological paradox because its clinical behavior contrasts with its anaplastic morphology. MC is characterized by a dense lymphocytic infiltrate. In this study, we determined the cytotoxic potential and activity of tumor infiltrating lymphocytes (TILs) in MC by CD3, CD8, TIA-1, and granzyme B immunostaining on paraffin-embedded sections. Fourteen cases of typical MC (TMC) and 15 cases of atypical MC (AMC) classified according to Ridolfi criteria, and 19 cases of poorly differentiated infiltrating ductal carcinoma (PDC) were studied. TILs were quantified separately into two groups: cells infiltrating tumor nests and cells within stroma The number of CD8+ and TIA-1+ cells infiltrating tumor cell nests were markedly increased in TMC and AMC, as opposed to the PDC subgroup (159.6+/-132.8; 77.4+/-59.3; 9.4+/-10.5 and 171.2+/-152.4; 72.3+/-55.0; 10.8+/-12.7 per high power field, respectively). The number of tumor infiltrating granzyme B+ cells was significantly greater in TMC and AMC, as compared with the PDC subgroup (82.1+/-64.9, 33.9+/-19.7, and 3.1+/-5.1, respectively). Although no significant difference was found between the number of stromal CD3+ and CD8+ lymphocytes among the three subgroups, stromal granzyme B+ cells were significantly elevated in TMC and AMC as compared with the PDC subgroup. Finally, the relative proportion of granzyme B+ as opposed to CD3+ intraepithelial and stromal lymphocytes was greater in TMC and AMC as compared with the PDC subgroup (0.52+/-0.29; 0.47+/-0.31; 0.19+/-0.18 and 0.18+/-0.11; 0.13+/-0.11; 0.06+/-0.05, respectively). The presence of increased numbers of activated cytotoxic lymphocytes in MC of the breast may be a key mechanism active in the host versus tumor response leading to improved prognosis.

Assessment and diagnostic utility of the cytotoxic T-lymphocyte phenotype using the specific markers granzyme-B and TIA-1 in esophageal mucosal biopsies.

Most esophageal intraepithelial lymphocytes (IELs) express T-cell markers. Increased numbers of esophageal IELs have been shown in reflux esophagitis. The cytotoxic potential and activity of esophageal IELs have not as yet been examined. Our objectives were to determine whether esophageal IELs express the recently described cytotoxic T-cell (CTLs) markers, TIA-1 and granzyme-B, and whether the number of CTLs correlates with well-defined endoscopic, clinical, and histological features of esophagitis. In this study, most CD-3+ esophageal IELs exhibit the CD-8+/TIA-1+ T cell with cytotoxic potential phenotype in both histologically normal biopsy specimens and in biopsy specimens with esophagitis. A subpopulation of esophageal IELs that express cytotoxic activity was identified by granzyme-B immunostaining. A significant positive association was found between the number of esophageal IELs seen by light microscopy in biopsy specimens with histological features of reflux (21 IELs/HPF) and Candida esophagitis (31 IELs/HPF) as compared with normal-appearing biopsy specimens (10 IELs/HPF) (P< or =.05). Furthermore, the number of TIA-1 or granzyme-B-positive IELs were significantly increased in biopsy specimens with reflux esophagitis (34 and 15 cells/HPF) and Candida esophagitis (44 and 18 cells/HPF) as compared with normal (11 and 2 cells/HPF) (P< or =.05). Granzyme-B and CD-3-positive IELs were also significantly elevated in biopsy specimens with reflux-associated squamous hyperplasia (P< or =.05). Finally, biopsy specimens of patients with dysphagia and to a lesser extent dyspepsia/heartburn exhibited increased numbers of IELs bearing the cytotoxic phenotype when compared with asymptomatic patients. In conclusion, we provide immunohistochemical evidence that most esophageal IELs exhibit the cytotoxic phenotype and that activated cytotoxic IELs are increased in reflux and Candida esophagitis.

Characterization of a potent sperm-agglutinating monoclonal antibody and its cognate antigens.

OBJECTIVE: To identify sperm antigens that are capable of eliciting infertility-related sperm-agglutinating antibodies. DESIGN: In vitro laboratory experiments. SETTING: University research laboratory. PATIENT(S): Fertile semen donors. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): Sperm agglutination, immunofluorescence localization, and flow cytometric analysis of surface expression of A36 antigens. Antigen analysis by Western immunoblotting. RESULT(S): Monoclonal antibody A36 induced intensive head-to-head, tail-to-tail, and head-to-tail agglutination of motile human spermatozoa. Antigens recognized by A36 were localized on the acrosomal cap and in the principal tail regions of motile, noncapacitated human sperm. Changes in subcellular levels and localization of the A36-recognized epitope occurred after capacitation and acrosomal loss. A36 reacted with a polymorphic series of proteins in Western blots of sperm extracts from humans and various other animal species, including mouse testis extracts. A common 53-kd antigen was recognized by the antibody in the different antigenic preparations. CONCLUSION(S): A mouse antibody to human sperm, monoclonal antibody A36, caused intensive agglutination of noncapacitated human spermatozoa and reacted with antigens on the acrosomal cap and in the principal tail regions. Of the multiple polypeptides that were reactive with the monoclonal antibody in sperm extracts from humans and other animal species, a common 53-kd antigen was recognized.