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Results for “molecular profiling”

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PubMed2005

Australian sporadic CJD analysis supports endogenous determinants of molecular-clinical profiles.

OBJECTIVE: To define the protease-resistant prion protein (PrPres) types and associated clinical profiles in Australian patients with sporadic Creutzfeldt-Jakob disease (CJD) to allow comparison with those reported from other continents and concomitantly reaffirm absence of variant CJD (vCJD). METHODS: Reassessment of available clinical and neuropathologic data on patients referred to the Australian National Creutzfeldt-Jakob Disease Registry (ANCJDR) who died between January 1, 1992, and June 30, 2003, was conducted. Molecular classification of PrPres was determined by immunoblot analysis of available frozen brain tissue. Brain homogenate pH and codon 129 genotype on the prion protein gene (PRNP) were established. RESULTS: PrPres patterns in 35 of 37 patients with sporadic CJD conformed to one of three common reported types. Of a range of clinical features assessed, illness duration was the only clinical feature significantly associated with PrPres type. Two patients displayed coexistence of more than one PrP type, with one displaying a novel pattern of three PrPres types in a single brain region. The absence of vCJD was reconfirmed, supported by the lack of the typical PrPres glycoform pattern. CONCLUSIONS: Given Australia's geographic isolation and environmental uniqueness, the general congruity of these results with those reported from other continents suggests that endogenous factors predominantly determine sporadic Creutzfeldt-Jakob disease (CJD) phenotypic subtypes or "strains." These results support a clinicopathologic classification system whereby both PrPres type and codon 129 genotype are utilized to most accurately depict phenotypic subtypes or strains of sporadic CJD.

Adult↗

PubMed2001

[Genomic profiling: from molecular cytogenetics to DNA arrays].

Genetic instability results, in a large majority of solid tumors, in deep chromosomal rearrangements. However, because chromosomal instability produces highly complex caryotypes, rarely showing stereotypic aberrations, it has not been possible to characterize solid cancers according to specific patterns of chromosomal rearrangements. This contrasts with the situation in hematological malignancies, where cytogenetics has allowed to lay out the basis of a renewed classification. New insights have been brought by the development of comparative genomic hybridization (CGH). This molecular cytogenetics approach was originally devised to detect regions in the genome of tumor cells undergoing quantitative changes, i.e. gains or losses of copy numbers. The large body of studies based on CGH show that solid tumors undergo frequent gains and losses and that every chromosomes show at least one region of anomaly. Furthermore, different tumor types present distinct CGH patterns of gains and losses. These observations favor the idea that it may be possible to type human solid cancers according to their patterns of genomic aberrations. However, despite the fact that a number of CGH based studies present data suggesting that different tumor types or cancers at different stages of evolution show distinct patterns of gains and losses, it has proven difficult to be conclusive. This can be mainly attributed to the lack of spatial resolution of CGH. Indeed, CGH uses metaphase chromosomes as hybridization targets and therefore its resolution is at the level of chromosomal banding. The recent adaptation of DNA array technology to CGH will allow to pass this limitation. In DNA array based CGH (array-CGH) metaphase chromosomes have been replaced by spots of cloned DNA. These DNA clones may either be genomic (BACs, YACs or cosmids) or coding (cDNAs). The resolution of array-CGH is therefore determined by the size of the cloned DNA insert (100 Kb for BACs, 1-2 kb for cDNAs). Data corresponding to each of these clones is or will be in a near future linked to DNA sequence data. Hence, in a near future, array-CGH will allow to increase the resolution from a cytogenetic level to a molecular level. Finally, because array technology is highly adaptable to automation, going from classical CGH to array-CGH will produce a quantum leap in throughput.

Chromosome Aberrations↗

PubMed2026

Personalizing endometrial cancer care beyond histology: clinical applications and limits of molecular classification.

Endometrial cancer is a biologically heterogeneous disease whose management has been reshaped by molecular classification. This review summarizes the current evidence supporting the integration of molecular subgroups into prognostic assessment and treatment personalization across stages of disease. The Cancer Genome Atlas classification and its clinically applicable surrogates identify four major molecular categories: POLE-mutated, mismatch repair-deficient, p53-abnormal, and no specific molecular profile tumors. These groups differ substantially in biology, prognosis, treatment sensitivity, and areas of unmet need. POLE-mutated tumors have an excellent prognosis and represent the clearest candidates for adjuvant treatment de-escalation, particularly in early-stage disease. Mismatch repair-deficient tumors show intermediate prognosis but strong sensitivity to immune checkpoint inhibition, which has transformed the management of advanced and recurrent disease and is now being tested in earlier settings. p53-abnormal tumors represent the highest-risk subgroup, requiring multimodal treatment and offering opportunities for biomarker-driven strategies including HER2-directed therapy and DNA damage repair targeting. No specific molecular profile tumors remain the most heterogeneous category, increasingly refined by estrogen receptor status, grade, L1 cell adhesion molecule overexpression, and other biomarkers. Mismatch repair-proficient advanced/recurrent disease should be interpreted as a composite clinical trial population rather than a molecular class. Molecular classification should be integrated with traditional clinicopathologic factors, emerging biomarkers, and local implementation strategies to support equitable, biologically informed treatment selection in endometrial cancer.

Humans↗

PubMed2001

Gene expression profiling and molecular aspects in peripheral nerve regeneration.

Regeneration of the peripheral nervous system after injury depends on a complex sequence of histopathological reactions that comprise a highly reproducible sequence of degenerative reactions, termed Wallerian degeneration. During this period a remodelling of the distal nerve stump prepares a microenvironment that permits successful regrowth of nerve fibers from the proximal nerve fragment. This stereotypical sequence of reactions is reflected by a differential and coordinate expression of genes with specific functions in the process of regeneration. This review will summarize cellular and molecular reactions that contribute to peripheral nerve regeneration including data of a pilot study in which membrane based cDNA array expression technology was applied. We examined the expression of 588 annotated genes in response to a crush lesion of rat sciatic nerves. Approximately 40 % of the genes spotted onto the array filters showed expression significantly above background and 55 of these detected genes represented differential expression profiles after nerve lesion. This approach revealed to be suitable for systematic screening of regeneration associated genes.

Animals↗

PubMed2003

Antimicrobial susceptibility profile of molecular typed cystic fibrosis Stenotrophomonas maltophilia isolates and differences with noncystic fibrosis isolates.

Multiresistance in Stenotrophomonas maltophilia limits the effectiveness of antimicrobial therapy for infections due to this organism. It can be of special concern in cystic fibrosis (CF) patients due to frequent antimicrobial administration. The in vitro activity of 41 antimicrobial agents against 76 epidemiologically defined CF S. maltophilia isolates by pulsed-field-gel electrophoresis (PFGE) technique under XbaI and SpeI restriction was compared with that obtained with 51 non-CF strains recovered from respiratory sources. Minimal inhibitory concentrations (MICs) were determined with the standard National Committee for Clinical Laboratory Standards agar dilution technique, but with 24-hr incubation. Forty-seven different PFGE profiles were observed within 76 S. maltophilia CF isolates. Minocycline (resistance rate, 0%; MIC(90), 1 microg/ml), doxycycline (6.4%; 8 microg/ml), trovafloxacin (4.2%; 2 microg/ml), moxifloxacin (6.3%; 2 microg/ml), clinafloxacin (6.3%; 2 g/ml), and moxalactam (17.0%; 64 g/ml) displayed low resistance rates. On the contrary, resistance rates were higher with ceftazidime (70.0%; 256 microg/ml), cefepime (83.0%; 128 microg/ml), piperacillin (87.2%; >1,024 microg/ml), ticarcillin (87.2%; >512 microg/ml), and aztreonam (95.7%; >1,024 microg/ml). Clavulanate reverted resistance to ticarcillin and aztreonam in 40.4% and 31.7% of strains, respectively. Aminoglycosides displayed reduced activities with susceptibility rates lower than 20% and MIC(90) higher than 128 microg/ml. With the exception of trimethoprim-sulfamethoxazole (25.4 vs. 31.3%), CF isolates were more resistant than non-CF isolates. Remarkably, resistance was enhanced in S. maltophilia isolates persistently recovered in chronically colonized patients. Susceptibility analysis demonstrated higher resistance rates among CF S. maltophilia isolates when compared with respiratory isolates from non-CF patients. Moreover, persistently recovered CF S. maltophilia isolates were more resistant than sporadic non-CF isolates.

Adolescent↗

PubMed2002

Gene expression profiling defines molecular subtypes of classical Hodgkin's disease.

Although the prognosis of Hodgkin's disease is relatively good, around 20% of patients do not benefit from current therapies and succumb to their disease. A large-scale molecular characterization of disease might help improve HD management. Using cDNA arrays, we studied the mRNA expression levels of approximately 1000 selected genes in 34 benign and malignant lymphoid samples including 21 classical Hodgkin's disease (HD) tissue samples. Hierarchical clustering identified three main molecular groups of HD tumours relevant with respect to histology and clinical outcome (response to therapy and survival). Samples from all bad outcome HD (BOHD) patients clustered in one group whereas the two other groups contained most good outcome HD (GOHD) cases. The nodular sclerosis GOHD samples overexpressed genes involved in apoptotic induction and cell signalling, including cytokines, while the BOHD samples were characterized by the upregulation of genes involved in fibroblast activation, angiogenesis, extracellular matrix remodelling, cell proliferation, and the downregulation of tumour suppressor genes. Our results establish a molecular taxonomy of HD correlating with response to therapy and clinical outcome, thereby suggesting the possibility of improving the current prognostic classification.

Apoptosis↗

PubMed2000

Molecular characterization of HHV-8 positive primary effusion lymphoma reveals pathogenetic and histogenetic features of the disease.

BACKGROUND: Primary effusion lymphoma (PEL) associates with HHV-8 infection, preferentially develops in immunodeficient patients and grows in the serous body cavities. PEL derives from post-germinal center, pre-terminally differentiated B-cells. The pathogenesis of PEL is unclear and the sole identified genetic lesions are human herpesvirus type-8 (HHV-8) infection in all cases and EBV infection in 70% of cases. Epstein-Barr virus (EBV) infection in PEL displays a latency I phenotype. OBJECTIVES: To clarify the pathogenesis and histogenesis of PEL by investigating (1) the lymphoma karyotype; (2) the expression status of the Met tyrosine kinase receptor and of its ligand hepatocyte growth factor (HGF); (3) the molecular profile of EBV, with particular focus on mutations of EBNA-1 genes, which are thought to affect viral tumorigenicity in EBV-infected neoplasms displaying the latency I phenotype. STUDY DESIGN: Twenty-four PEL (nine cell lines and 15 primary specimens) formed the basis of the study. Karyotypes were investigated by conventional cytogenetics and fluorescent in situ hybridization (FISH) in selected cases. The expression status of Met and HGF was defined by multiple techniques, including RT-PCR, FACS analysis, immunocytochemistry, Western blot studies and ELISA. The molecular profile of EBNA-1 genes of EBV were investigated by DNA direct sequencing. RESULTS: Trisomy 7, trisomy 12 and breaks at 1q21-q25 are recurrently associated with PEL. PEL consistently co-express Met and HGF both at the mRNA and protein level. Among aggressive B-cell lymphomas, Met/HGF co-expression appears to be relatively specific for PEL. The EBNA-1 gene of EBV displays a high degree of genetic heterogeneity in PEL, with no preferential association with one specific variant. CONCLUSIONS: PEL associates with recurrent chromosomal alterations, suggesting that viral infection is not sufficient for tumor development and that lesions of cellular genes may be required. The expression of Met/HGF by PEL cells may bear implications for the lymphoma proliferation and growth pattern, since Met/HGF interactions influence cell mitogenesis and motogenesis. EBV infection in PEL displays a latency I phenotype and fails to associate with specific EBNA-1 variants, suggesting that the role of EBV in PEL is not mediated by the major transforming pathways currently known in EBV positive lymphomas.

Epstein-Barr Virus Nuclear Antigens↗

PubMed2005

Distinct molecular alterations in complex endometrial hyperplasia (CEH) with and without immature squamous metaplasia (squamous morules).

Several molecular alterations, most commonly PTEN mutations but also K-ras mutations, microsatellite instability, and beta-catenin mutations have been detected in endometrioid endometrial carcinoma (EEC). Specifically, mutations in the beta-catenin gene are seen in 15% to 20% of EECs, whereas immunohistochemical expression of beta-catenin ranges from 13% to 44%, nuclear staining being concentrated in areas of immature squamous metaplasia (squamous morules). Complex endometrial hyperplasia with atypia (CEH-A) is a well-known precursor of EEC, which can also show immature squamous metaplasia. In this study, we compared the immunohistochemical and molecular profiles of 13 CEH-A with and 11 CEH-A without squamous morules (SM) for mutations of beta-catenin, PTEN, K-ras, and microsatellite instability (MSI). In all cases of CEH-A with SM, beta-catenin immunostaining showed strong and diffuse nuclear expression in areas of SM and weak to moderate nuclear expression in the glandular component. Six different beta-catenin mutations were found in 7 of 13 cases (54%) (G34E, G34V, S33C, D32Y, S33F, D32A); however, no mutations of the PTEN or K-ras genes were identified. beta-Catenin immunostaining showed focal nuclear staining in only 2 cases of CEH-A without SM. Only 1 case had a beta-catenin mutation (S45A), which was associated with a K-ras mutation (G12D). Another 3 cases had both PTEN and K-ras mutations (C296Stop Ex 8 and G12V, 244del C Ex 7 and G12D, 251ins TGAT Ex 7 and G13D), and one had a PTEN mutation (G230E Ex 7) only. Of all 24 cases, only 1 case of CEH-A without SM showed MSI. In conclusion, marked differences in the molecular profiles regarding beta-catenin, PTEN, and K-ras mutations were observed between CEH-A with and without SM. beta-catenin mutations might represent a signaling pathway leading to a distinctive morphology in hyperplastic/neoplastic endometrium with SM. Other molecular events such as K-ras or PTEN mutations are likely to occur in CEH-A independently from beta-catenin. Furthermore, morphologic differences between CEH-A with SM and CEH-A without SM seem to correlate, at least to some extent, with the clinical course of the disease. In our series, cases of CEH-A with SM and beta-catenin alterations appeared to have a less aggressive behavior when compared with CEH-A without SM and with K-ras and PTEN mutations.

Adult↗

PubMed2002

Expression of tyrosinase, MIA and MART-1 in sentinel lymph nodes of patients with malignant melanoma.

BACKGROUND: Regional lymph node status is an important predictor of survival in patients with malignant melanoma. Mapping of sentinel lymph nodes using sensitive molecular techniques has recently been introduced. Malignant melanoma is heterogeneous in terms of its biological, immunological and metastatic properties, and melanoma cells exhibit a polymorphous expression of tumour markers. Thus, assays that include multiple markers appear to be more sensitive than single-marker assays. OBJECTIVES: To characterize the molecular profiles of melanoma cells in sentinel lymph nodes employing the mRNA expression of tyrosinase, MIA and MART-1 as markers. METHODS: Samples of sentinel lymph nodes from 17 melanoma patients and 18 control nodes from non-melanoma patients were assayed by reverse transcriptase-polymerase chain reaction, using specific primers for each marker. RESULTS: We found that both tyrosinase and MIA expression were sensitive indicators of micrometastases in sentinel lymph nodes that were negative on routine histopathological examination, and that the finding of micrometastases expressing MART-1 in sentinel lymph nodes was negatively correlated with overall survival. CONCLUSIONS: Characterization of the molecular profiles of melanoma cells constitutes a valid means of detecting metastatic melanoma cells in sentinel lymph nodes, and of predicting the survival of melanoma patients.

Adult↗

PubMed2005

Molecular markers predicting radiotherapy response: report and recommendations from an International Atomic Energy Agency technical meeting.

PURPOSE: There is increasing interest in radiogenomics and the characterization of molecular profiles that predict normal tissue and tumor radioresponse. A meeting in Amsterdam was organized by the International Atomic Energy Agency to discuss this topic on an international basis. METHODS AND MATERIALS: This report is not completely exhaustive, but highlights some of the ongoing studies and new initiatives being carried out worldwide in the banking of tumor and normal tissue samples underpinning the development of molecular marker profiles for predicting patient response to radiotherapy. It is generally considered that these profiles will more accurately define individual or group radiosensitivities compared with the nondefinitive findings from the previous era of cellular-based techniques. However, so far there are only a few robust reports of molecular markers predicting normal tissue or tumor response. RESULTS: Many centers in different countries have initiated tissue and tumor banks to store samples from clinical trials for future molecular profiling analysis, to identify profiles that predict for radiotherapy response. The European Society for Therapeutic Radiology and Oncology GENEtic pathways for the Prediction of the effects of Irradiation (GENEPI) project, to store, document, and analyze sample characteristics vs. response, is the most comprehensive in this regard. CONCLUSIONS: The next 5-10 years are likely to see the results of these and other correlative studies, and promising associations of profiles with response should be validated in larger definitive trials.

Biomarkers, Tumor↗

PubMed2003

Small lymphocytic lymphoma, marginal zone B-cell lymphoma, and mantle cell lymphoma exhibit distinct gene-expression profiles allowing molecular diagnosis.

Non-germinal center small B-cell lymphomas represent a heterogeneous group of non-Hodgkin lymphomas, the most frequent histologic subtypes being small lymphocytic lymphoma (SLL), splenic marginal zone B-cell lymphoma (MZL), and mantle cell lymphoma (MCL). In order to identify genomic signatures specific for each disease, we analyzed 128 primary tumors using high-density microarrays. Several clusters of genes significantly discriminated the 3 histologic subtypes. Genes associated with cell adhesion, angiogenesis, and inhibition of apoptosis were up-regulated in SLL. Genes associated with intracellular signaling via the AKT1 pathway were up-regulated in splenic MZL. Genes associated with cell cycle control and multidrug resistance were up-regulated in MCL. Using 44 genes selected within the gene clusters discriminant for the 3 lymphoma subtypes, we generated a class prediction score that allowed us to classify the 3 entities in 96% of the cases, including borderline cases. Whereas specific transcriptional profiles easily distinguished all MZL samples, SLL samples, and most of the MCL samples into separate groups, few MCL cases exhibited MZL-type transcriptional profiles. This study demonstrates that SLL, splenic MZL, and MCL possess specific transcriptional profiles that may be relevant to the pathogenesis and the diagnosis of these histologic subtypes.

Biopsy↗

PubMed2004

Gene expression profiles and molecular markers to predict recurrence of Dukes' B colon cancer.

PURPOSE: The 5-year survival rate of patients with Dukes' B colon cancer is approximately 75%. Identification of the patients at high risk of recurrence in this group would allow better staging and more informed use of adjuvant chemotherapy. In this study, we used DNA chip technology to systematically identify new prognostic markers for tumor relapse in Dukes' B patients. PATIENTS AND METHODS: Using Affymetrix U133a GeneChip containing approximately 22,000 transcripts (Affymetrix, Santa Clara, CA), RNA samples from 74 patients with Dukes' B colon cancer were analyzed. Thirty-one patients developed tumor relapse in less than 3 years, whereas 43 patients remained disease-free for more than 3 years after surgery. Two supervised class prediction approaches were used to identify gene markers that can best discriminate between patients who would experience relapse and patients who would remain disease-free. A multivariate Cox model was built to predict recurrence. RESULTS: Gene expression profiling identified a 23-gene signature that predicts recurrence in Dukes' B patients. This signature was validated in 36 independent patients. The overall performance accuracy was 78%. Thirteen of 18 relapse patients and 15 of 18 disease-free patients were predicted correctly, giving an odds ratio of 13 (95% CI, 2.6 to 65; P =.003). The log-rank test indicated a significant difference in disease-free time between the predicted relapse and disease-free patients (P =.0001). CONCLUSION: The clinical value of these markers is that the patients at a high predicted risk of relapse (13-fold risk) could be upstaged to receive adjuvant therapy, similar to Dukes' C patients. Our data highlight the feasibility of a prognostic assay that could focus more intensive treatment for localized colon cancer.

Algorithms↗

PubMed1982

Immunochemical and biosynthetic analysis of monoclonal antibody-defined melanoma-associated antigen.

Immunoprecipitation studies with application of monoclonal antibody F11 originally made to a partially purified spent medium antigen of melanoma cells, made it possible to delineate the molecular profiles of both the cell associated and spent medium antigens recognized on melanoma cells intrinsically labeled with glycoprotein precursors. F11 distinguishes a glycoprotein of Mr 100,000 (100 K) in the spent media of melanoma cells while a parallel analysis of detergent lysates of cells reveals a pattern of three glycoproteins of Mr 75, 77, and 100 K. Pulse-chase analysis of the biosynthesis of these antigens indicated that F11 first recognizes the 75 and 77 K antigens in the absence of a 100K component suggesting strongly that these molecules contain an antigenic site recognized by F11. The 100 K antigen appears later in the pulse-chase analysis with kinetics that suggest some of the 75 and 77 K antigens are biosynthetic precursors of the 100 K antigen. This molecule is ultimately secreted into the extracellular media and appears to be a sialoglycoprotein judging from its sensitivity to neuraminidase. A cross-reactive species with an approximate Mr 90 K is also recognized by F11 in indirect immunoprecipitation analysis of spent media from a neuroblastoma cell line indicating that a common antigenic site exists on this secreted but structurally different neuroblastoma antigen. Thus, a combination of immunochemical and biosynthetic analyses of cell-associated and secreted antigens recognized by monoclonal antibody F11 demonstrate such molecules can differ structurally when isolated from the same or different tumor cells. These findings indicate the necessity to establish molecular profiles of melanoma-associated glycoprotein antigens recognized by monoclonal antibodies to characterize and define their potential biological functions within tumor cells.

Antibodies, Monoclonal↗

PubMed2004

Distinct gene expression profiles determine molecular treatment response in childhood acute lymphoblastic leukemia.

Treatment resistance, as indicated by the presence of high levels of minimal residual disease (MRD) after induction therapy and induction consolidation, is associated with a poor prognosis in childhood acute lymphoblastic leukemia (ALL). We hypothesized that treatment resistance is an intrinsic feature of ALL cells reflected in the gene expression pattern and that resistance to chemotherapy can be predicted before treatment. To test these hypotheses, gene expression signatures of ALL samples with high MRD load were compared with those of samples without measurable MRD during treatment. We identified 54 genes that clearly distinguished resistant from sensitive ALL samples. Genes with low expression in resistant samples were predominantly associated with cell-cycle progression and apoptosis, suggesting that impaired cell proliferation and apoptosis are involved in treatment resistance. Prediction analysis using randomly selected samples as a training set and the remaining samples as a test set revealed an accuracy of 84%. We conclude that resistance to chemotherapy seems at least in part to be an intrinsic feature of ALL cells. Because treatment response could be predicted with high accuracy, gene expression profiling could become a clinically relevant tool for treatment stratification in the early course of childhood ALL.

Adolescent↗

PubMed1999

On the importance of a methyl group in beta-lactamase evolution: free energy profiles and molecular modeling.

beta-Lactam antibiotics are generally thought to inhibit their target enzymes, the bacterial cell wall-synthesizing DD-peptidases, because of their resemblance to D-alanyl-D-alanine peptides. Although a favorable conformation of the latter does structurally resemble the beta-lactams with respect to backbone conformation, a significant difference is the presence of a D-methyl substituent on the penultimate alanine residue of the cell wall peptide. A classical beta-lactam antibiotic has a hydrogen in the corresponding position. In the process of evolution of a beta-lactamase from a DD-peptidase, it seems likely that this D-methyl group would be selected against, to ensure that the former enzyme would hydrolyze beta-lactams rather than peptides. In this paper, the effect of the penultimate D-alanine residue (as opposed to a glycine residue) has been examined in peptide substrates of a present-day DD-peptidase and a beta-lactamase. The peptides N-(phenylacetyl)-D-alanyl-D-phenylalanine and N-(phenylacetyl)glycyl-D-phenylalanine were used as a test pair against the DD-peptidase of Streptomyces R61 and the structurally very similar class C beta-lactamase of Enterobacter cloacae P99. The kinetics of turnover of both of these substrates were determined for both enzymes. To quantify the partitioning of the acyl-enzyme intermediate, the aminolysis by D-phenylalanine of a cognate pair of depsipeptides was also studied. Thus, free energy-reaction coordinate diagrams were constructed for turnover of both peptides by both enzymes. Comparison of these profiles showed that the D-methyl group is preferred over hydrogen by the DD-peptidase at all stages of catalysis (acyl-enzyme and acylation and deacylation transition states), whereas the beta-lactamase selects against the D-methyl group only at the peptide acylation transition state. A process of evolution by uniform dissociation of the methyl group by the beta-lactamase has apparently occurred. These results were explored structurally by computational models of the acylation tetrahedral intermediates. A methyl group pocket on the DD-peptidase, less favorable on the beta-lactamase, was identified. The interaction of the leaving group, the terminal D-alanine residue, with the two enzymes was interesting, since it seemed that different positively charged active site residues were directly associated with the carboxylate, Lys 315 in the beta-lactamase and Arg 285 (rather than His 298) in the case of the DD-peptidase. The problems posed by larger substituents on the penultimate residue of the peptide, and in particular by the heterocyclic substituent present in a bicyclic beta-lactam, were analyzed. Qualitative and quantitative analysis of the models support the proposed importance of the penultimate D-alanine in beta-lactamase evolution.

Carboxypeptidases↗

PubMed2026

Single-section multiplex spatial proteomics of immune microenvironments in kidney transplantation.

Characterizing kidney disease is challenged by marked cellular heterogeneity and limited tissue availability from renal biopsies. Conventional diagnostic workflows rely on multiple serial sections for parallel staining, increasing tissue consumption, sampling bias, and loss of spatial information, thereby constraining molecular characterization within intact tissue architecture. High-plex spatial proteomics may overcome these limitations by enabling comprehensive molecular profiling on a single section. Here, we present and evaluate a high-plex cyclic immunofluorescence imaging workflow (MACSima™, Miltenyi Biotec) applied to kidney transplant biopsies, including BK virus nephropathy (BKVN) and focal segmental glomerulosclerosis (FSGS), to characterize spatial immune organization with a focus on complement system components. Feasibility and subcellular resolution were first assessed in a lupus nephritis section, demonstrating compatibility with diagnostic immune panels and preservation of tissue morphology. A 48-marker multiplex panel interrogating immunity, oxidative stress, senescence, and fibrosis was then applied to BKVN samples, including paired pre- and post-treatment biopsies, revealing distinct proteomic patterns and dynamic changes following therapy. In FSGS, a glomerulus-focused panel identified spatially resolved innate and adaptive immune signatures, including complement-related patterns supporting exploratory analysis of glomerular immune architecture. Structural, nuclear, membrane, and phosphorylated signaling markers enabled precise delineation of renal compartments and assessment of cellular states such as proliferation, DNA damage, and pathway activation. The workflow also supported detection of extracellular vesicles in cultured renal cells, highlighting its versatility. Overall, this approach provides a robust, tissue-sparing platform for integrated spatial and molecular profiling of renal biopsies, reducing sampling bias while enabling discovery-level phenotyping from a single section. This unified strategy is particularly suited to kidney transplantation, where diagnosis, therapeutic decision-making, and longitudinal monitoring are closely interconnected.

Kidney Transplantation↗

PubMedDate not supplied

Microsatellite status and cell cycle associated markers in rectal cancer patients undergoing a combined regimen of 5-FU and CPT-11 chemotherapy and radiotherapy.

BACKGROUND: Microsatellite instability (MSI) and expression of cell cycle-related markers may predict a favorable outcome in colorectal cancer patients. The aim of this study was to elucidate the molecular profiles of patients with rectal cancers treated with neoadjuvant chemotherapy (5-Fluorouracil and CPT-11), radiotherapy and surgery that correlate with response to therapy. PATIENTS AND METHODS: Fifty-seven patients with rectal cancer were treated with the same preoperative chemotherapy regimen, radiotherapy (45 to 54 Gy) followed by surgery. The microsatellite status, the expression of the mismatch repair proteins MLH1 and MSH2 and p21WAF1/C1PI, p27, bcl-2, topoisomerase II (topo II) and Ki-67 were assessed in the pretreatment biopsies. The response to adjuvant therapy was categorized in the resected specimens as complete response (CR, no microscopic residual tumor present) and partial response (PR, tumor present). RESULTS: p21WAF1/C1PI, expression characterized the CR with 12 out of 30 tumors (40%) positive for this marker. None of the patients whose tumors did not express p21WAFI/C1PI (10 patients) was a CR (p=0.011). Overall, the tumors with CR also showed higher expression of bcl-2, Ki-67, topo II and p27. However, p53 was more frequently expressed in the PR tumors. Tumors with high microsatellite instability showed CR (3/5, 60%) more often than PR, whereas tumors with stable microsatellites showed PR (26/36, 80%) more often than CR (p=0.099). CONCLUSION: We conclude that a molecular profile characterized by high microsatellite instability with loss of mismatch repair protein expression and p21WAF1/C1PI is predictive of an improved response to neoadjuvant treatment with 5-FU, CPT-11 and radiation therapy.

Adult↗

PubMed2002

Transcriptional profiling develops molecular signatures for ovarian tumors.

Of the cancers unique to women, ovarian cancer has the highest mortality rate. Over 26,000 women are diagnosed with this disease in the U.S. annually, and 60% of those diagnosed will die of the disease. One of the greatest problems with this disease is the lack of strong early warning signs or symptoms resulting in advanced stage at presentation in most women, followed by the outgrowth of chemotherapy-resistant disease in the majority of patients. The 5-year survival for patients with early stage disease ranges from 50-90%, but it is less than 25% for advanced-stage disease. In collaboration with researchers at Millennium Predictive Medicine (Cambridge, MA), the Ovarian Cancer Program of the Mayo Clinic Cancer Center analyzed gene expression in over 50 primary ovarian tumors, as compared with normal ovarian epithelial cells. The technologies utilized included microarray analysis with nitrocellulose filters containing 25,000 arrayed human cDNAs, as well as the construction of subtraction suppression hybridization cDNA libraries and their subsequent sequencing. Our specific focus has been on genes that are underexpressed during the development of ovarian cancer, although this analysis has revealed a large number of consistently up- and down-regulated genes. There were more down-regulated genes in ovarian tumors than up-regulated genes. In addition, the number of genes that had altered expression levels was quite large. For example, we found 409 genes down-regulated at least 5-fold, and 72 genes up-regulated at least 5-fold in 33% of the tumors analyzed. We also observed that most of the expression alterations observed in later stage (Stages III/IV) tumors were also observed in early-stage tumors (Stages I/II). This was corroborated using comparative genomic hybridization analysis on the same tumors that were expression profiled. This analysis revealed that the late-stage tumors had more gene amplification than early-stage tumors, but most regions of change (either increases or decreases) were in common between different stage tumors. We also have verified the altered expression levels of several of these genes using several complementary strategies. Finally, we are taking top candidate genes that are consistently under-expressed in ovarian tumors and attempting to determine their functional role in the development of ovarian cancer.

Female↗