Search

Antibiotic-induced microbiome dysbiosis is widespread in oncology, adversely affecting outcomes and side effects of various cancer treatments, including immune checkpoint inhibitors and chimeric antigen receptor T (CAR-T) cell therapies. In this study, we observed that prior exposure to broad-spectrum ABX with extended anaerobic coverage like piperacillin-tazobactam and meropenem was associated with worsened anti-CD19 CAR-T therapy survival outcomes in large B-cell lymphoma patients (n=422), compared to other ABX classes. In a discovery subset of these patients (n=67), we found that the use of these ABX was in turn associated with substantial dysbiosis of gut microbiome function, resulting in significant alterations of the gut and blood metabolome, including microbial effectors such as short-chain fatty acids (SCFAs) and other anionic metabolites, findings that were largely reproduced in an external validation cohort (n=58). Broader evaluation of circulating microbial metabolites revealed reductions in indole and cresol derivatives, as well as trimethylamine N-oxide, in patients who received ABX treatment (discovery n=40, validation n=28). These findings were recapitulated in an immune-competent CAR-T mouse model, where meropenem-induced dysbiosis led to a systemic dysmetabolome and decreased murine anti-CD19 CAR-T efficacy. Furthermore, we demonstrate that SCFAs can enhance the metabolic fitness of CAR-T cells, leading to improved tumor killing capacity. Together, these results suggest that broad-spectrum ABX deplete metabolically active commensals whose metabolites are essential for enhancing CAR-T efficacy, shedding light on the intricate relationship between ABX exposure, microbiome function and their impact on CAR-T cell efficacy. This highlights the potential for modulating the microbiome to augment CAR-T immunotherapy., (Copyright © 2024 American Society of Hematology.)

Assessment of prognostic biomarkers of nonrelapse mortality (NRM) after allogeneic hematopoietic cell transplantation (HCT) in the pediatric age group is lacking. To address this need, we conducted a prospective cohort study with 415 patients at 6 centers: 170 were children age 10 years or younger and 245 were patients older than age 10 years (both children and adults were accrued from 2013 to 2018). The following 4 plasma biomarkers were assessed pre-HCT and at days +7, +14, and +21 post-HCT: stimulation-2 (ST2), tumor necrosis factor receptor 1 (TNFR1), regenerating islet-derived protein 3α (REG3α), and interleukin-6 (IL-6). We performed landmark analyses for NRM, dichotomizing the cohort at age 10 years or younger and using each biomarker median as a cutoff for high- and low-risk groups. Post-HCT biomarker analysis showed that ST2 (>26 ng/mL), TNFR1 (>3441 pg/mL), and REG3α (>25 ng/mL) are associated with NRM in children age 10 years or younger (ST2: hazard ratio [HR], 9.13; 95% confidence interval [CI], 2.74-30.38; P = .0003; TNFR1: HR, 4.29; 95% CI, 1.48-12.48; P = .0073; REG3α: HR, 7.28; 95% CI, 2.05-25.93; P = .0022); and in children and adults older than age 10 years (ST2: HR, 2.60; 95% CI, 1.15-5.86; P = .021; TNFR1: HR, 2.09; 95% CI, 0.96-4.58; P = .06; and REG3α: HR, 2.57; 95% CI, 1.19-5.55; P = .016). When pre-HCT biomarkers were included, only ST2 remained significant in both cohorts. After adjustment for significant covariates (race/ethnicity, malignant disease, graft, and graft-versus-host-disease prophylaxis), ST2 remained associated with NRM only in recipients age 10 years or younger (HR, 4.82; 95% CI, 1.89-14.66; P = .0056). Assays of ST2, TNFR1, and REG3α in the first 3 weeks after HCT have prognostic value for NRM in both children and adults. The presence of ST2 before HCT is a prognostic biomarker for NRM in children age 10 years or younger allowing for additional stratification. This trial was registered at www.clinicaltrials.gov as #NCT02194439., (© 2020 by The American Society of Hematology.)

Background: Detection of circulating tumor DNA can be limited due to their relative scarcity in circulation, particularly while patients are actively undergoing therapy. Exosomes provide a vehicle through which cancer-specific material can be enriched from the compendium of circulating non-neoplastic tissue-derived nucleic acids. We carried out a comprehensive profiling of the pancreatic ductal adenocarcinoma (PDAC) exosomal 'surfaceome' in order to identify surface proteins that will render liquid biopsies amenable to cancer-derived exosome enrichment for downstream molecular profiling., Patients and Methods: Surface exosomal proteins were profiled in 13 human PDAC and 2 non-neoplastic cell lines by liquid chromatography-mass spectrometry. A total of 173 prospectively collected blood samples from 103 PDAC patients underwent exosome isolation. Droplet digital PCR was used on 74 patients (136 total exosome samples) to determine baseline KRAS mutation call rates while patients were on therapy. PDAC-specific exosome capture was then carried out on additional 29 patients (37 samples) using an antibody cocktail directed against selected proteins, followed by droplet digital PCR analysis. Exosomal DNA in a PDAC patient resistant to therapy were profiled using a molecular barcoded, targeted sequencing panel to determine the utility of enriched nucleic acid material for comprehensive molecular analysis., Results: Proteomic analysis of the exosome 'surfaceome' revealed multiple PDAC-specific biomarker candidates: CLDN4, EPCAM, CD151, LGALS3BP, HIST2H2BE, and HIST2H2BF. KRAS mutations in total exosomes were detected in 44.1% of patients undergoing active therapy compared with 73.0% following exosome capture using the selected biomarkers. Enrichment of exosomal cargo was amenable to molecular profiling, elucidating a putative mechanism of resistance to PARP inhibitor therapy in a patient harboring a BRCA2 mutation., Conclusion: Exosomes provide unique opportunities in the context of liquid biopsies for enrichment of tumor-specific material in circulation. We present a comprehensive surfaceome characterization of PDAC exosomes which allows for capture and molecular profiling of tumor-derived DNA., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Background: Exosomes arise from viable cancer cells and may reflect a different biology than circulating cell-free DNA (cfDNA) shed from dying tissues. We compare exosome-derived DNA (exoDNA) to cfDNA in liquid biopsies of patients with pancreatic ductal adenocarcinoma (PDAC)., Patients and Methods: Patient samples were obtained between 2003 and 2010, with clinically annotated follow up to 2015. Droplet digital PCR was performed on exoDNA and cfDNA for sensitive detection of KRAS mutants at codons 12/13. A cumulative series of 263 individuals were studied, including a discovery cohort of 142 individuals: 68 PDAC patients of all stages; 20 PDAC patients initially staged with localized disease, with blood drawn after resection for curative intent; and 54 age-matched healthy controls. A validation cohort of 121 individuals (39 cancer patients and 82 healthy controls) was studied to validate KRAS detection rates in early-stage PDAC patients. Primary outcome was circulating KRAS status as detected by droplet digital PCR. Secondary outcomes were disease-free and overall survival., Results: KRAS mutations in exoDNA, were identified in 7.4%, 66.7%, 80%, and 85% of age-matched controls, localized, locally advanced, and metastatic PDAC patients, respectively. Comparatively, mutant KRAS cfDNA was detected in 14.8%, 45.5%, 30.8%, and 57.9% of these individuals. Higher exoKRAS MAFs were associated with decreased disease-free survival in patients with localized disease. In the validation cohort, mutant KRAS exoDNA was detected in 43.6% of early-stage PDAC patients and 20% of healthy controls., Conclusions: Exosomes are a distinct source of tumor DNA that may be complementary to other liquid biopsy DNA sources. A higher percentage of patients with localized PDAC exhibited detectable KRAS mutations in exoDNA than previously reported for cfDNA. A substantial minority of healthy samples demonstrated mutant KRAS in circulation, dictating careful consideration and application of liquid biopsy findings, which may limit its utility as a broad cancer-screening method., (© The Author 2017. Published by Oxford University Press on behalf of the European Society for Medical Oncology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Introduction: CDKN2A (p16) inactivation is common in lung cancer and occurs via homozygous deletions, methylation of promoter region, or point mutations. Although p16 promoter methylation has been linked to KRAS mutation and smoking, the associations between p16 inactivation mechanisms and other common genetic mutations and smoking status are still controversial or unknown., Methods: We determined all three p16 inactivation mechanisms with the use of multiple methodologies for genomic status, methylation, RNA, and protein expression, and correlated them with EGFR, KRAS, STK11 mutations and smoking status in 40 cell lines and 45 tumor samples of primary non-small-cell lung carcinoma. We also performed meta-analyses to investigate the impact of smoke exposure on p16 inactivation., Results: p16 inactivation was the major mechanism of RB pathway perturbation in non-small-cell lung carcinoma, with homozygous deletion being the most frequent method, followed by methylation and the rarer point mutations. Inactivating mechanisms were tightly correlated with loss of mRNA and protein expression. p16 inactivation occurred at comparable frequencies regardless of mutational status of EGFR, KRAS, and STK11, however, the major inactivation mechanism of p16 varied. p16 methylation was linked to KRAS mutation but was mutually exclusive with EGFR mutation. Cell lines and tumor samples demonstrated similar results. Our meta-analyses confirmed a modest positive association between p16 promoter methylation and smoking., Conclusion: Our results confirm that all the inactivation mechanisms are truly associated with loss of gene product and identify specific associations between p16 inactivation mechanisms and other genetic changes and smoking status.

There are no plasma biomarkers specific for GVHD of the gastrointestinal (GI) tract, the GVHD target organ most associated with nonrelapse mortality (NRM) following hematopoietic cell transplantation (HCT). Using an unbiased, large-scale, quantitative proteomic discovery approach to identify candidate biomarkers that were increased in plasma from HCT patients with GI GVHD, 74 proteins were increased at least 2-fold; 5 were of GI origin. We validated the lead candidate, REG3α, by ELISA in samples from 1014 HCT patients from 3 transplantation centers. Plasma REG3α concentrations were 3-fold higher in patients at GI GVHD onset than in all other patients and correlated most closely with lower GI GVHD. REG3α concentrations at GVHD onset predicted response to therapy at 4 weeks, 1-year NRM, and 1-year survival (P ≤ .001). In a multivariate analysis, advanced clinical stage, severe histologic damage, and high REG3α concentrations at GVHD diagnosis independently predicted 1-year NRM, which progressively increased with higher numbers of onset risk factors present: 25% for patients with 0 risk factors to 86% with 3 risk factors present (P < .001). REG3α is a plasma biomarker of GI GVHD that can be combined with clinical stage and histologic grade to improve risk stratification of patients.

Protein glycosylation represents one of the major post-translational modifications and can have significant effects on protein function. Moreover, changes in the carbohydrate structure are increasingly being recognized as an important modification associated with cancer etiology. In this report, we describe the development of a proteomics approach to identify breast cancer related changes in either concentration and/or the carbohydrate structures of glycoprotein(s) present in blood samples. Diseased and healthy serum samples were processed by an optimized sample preparation protocol using multiple lectin affinity chromatography (M-LAC) that partitions serum proteins based on glycan characteristics. Subsequently, three separate procedures, 1D SDS-PAGE, isoelectric focusing and an antibody microarray, were applied to identify potential candidate markers for future study. The combination of these three platforms is illustrated in this report with the analysis of control and cancer glycoproteomic fractions. Firstly, a molecular weight based separation of glycoproteins by 1D SDS-PAGE was performed, followed by protein, glycoprotein staining, lectin blotting and LC-MS analysis. To refine or confirm the list of interesting glycoproteins, isoelectric focusing (targeting sialic acid changes) and an antibody microarray (used to detect neutral glycan shifts) were selected as the orthogonal methods. As a result, several glycoproteins including alpha-1B-glycoprotein, complement C3, alpha-1-antitrypsin and transferrin were identified as potential candidates for further study., (Copyright (c) 2009 Elsevier B.V. All rights reserved.)

Hox genes encode transcription factors that regulate the morphogenesis of developing embryos. In mammals, knowledge of the genetic pathways, including the possible direct or indirect targets, regulated by HOX proteins is extremely limited. To identify the downstream genes regulated by posterior HOX proteins, we expressed HOXA13 in mouse embryonic fibroblasts lacking paralog group 13 expression using a bicistronic HOXA13/EGFP retroviral vector. Microarray analysis identified 68 genes with significant, reproducible RNA expression changes (50 activated; 18 repressed) in stable HOXA13-expressing cells. Genes with the GO annotation terms "extracellular matrix" and "basement membrane" were greatly overrepresented, and several were shown to be regulated by HOX proteins in other studies. Among the genes strongly activated by HOXA13 were Enpp2, a bifunctional enzyme known to modulate tumor and normal cell motility and which is expressed in precartilaginous condensations; Fhl1, a transcription factor implicated in muscle cell differentiation and development; and M32486, a putative integral membrane molecule expressed in the female reproductive tract. Expression differences in the HOXA13-expressing cells were confirmed for selected downstream genes using semi-quantitative RT-PCR, and in vivo coexpression with Hoxa13 in the limb interdigital mesenchyme was demonstrated for many. For two candidates, Igfbp4 and Fstl, interdigital limb bud expression was reduced in Hoxa13 mutants. To explore whether paralogous and nonparalogous HOX proteins could regulate the same genes, we created new HOX cell lines and examined the expression of selected genes identified by the HOXA13 screen. HOXD13 similarly activated/repressed 6 tested candidates, demonstrating that multiple downstream genetic pathways may be regulated by paralog HOX proteins. In contrast, HOXA9 was only able to repress expression of some gene targets. A HOXD13 mutant, HOXD13(IQN >)(AAA), incapable of monomeric DNA-binding, activated the expression of 5 HOXA13-upregulated genes; but was incapable of repressing the expression of Ngef and Casp8ap2. Our results suggest that HOX protein-protein interactions without direct HOX DNA-binding may play a larger role in HOX transcriptional regulation than generally assumed, and DNA-binding appears critical for repression.

Recent studies suggest accurate prediction of tissue of origin for human cancers can be achieved by applying sophisticated statistical learning procedures to gene expression data obtained from DNA microarrays. We have pursued the hypothesis that a more straightforward and equally accurate strategy for classifying human tumors is to use a simple algorithm that considers gene expression levels within a tree-based framework that encodes limited information about pathology and tissue ontogeny. By considering gene expression data within this framework, we found only a small number of genes were required to achieve a relatively high accuracy level in tumor classification. Using as few as 45 genes we were able to classify 157 of 190 human malignant tumors correctly, which is comparable to previous results obtained with sophisticated classifiers using thousands of genes. Our simple classifier accurately predicted the origin of metastatic tumors even when the classifier was trained using only primary tumors, and the classifier produced accurate predictions when trained and tested on expression data from different labs, and from different microarray platforms. Our findings suggest that accurate and robust cancer diagnosis from gene expression profiles can be achieved by mimicking the classification strategies routinely used by surgical pathologists.

Gene amplification is an important mechanism of oncogene activation in various human cancers, including ovarian carcinomas (OvCas). We used restriction landmark genomic scanning (RLGS) to detect amplified DNA fragments in the genomes of 47 primary OvCas. Visual analysis of the RLGS gel images revealed several OvCa samples with spots of greater intensity than corresponding spots from normal tissues, indicating possible DNA amplification in specific tumors. Two primary tumors (E1 and S12) shared four high-intensity spots. A recently developed informatics tool termed Virtual Genome Scans was used to compare the RLGS patterns in these tumors with patterns predicted from the human genome sequence. Virtual Genome Scans determined that three of the four fragments localized to chromosome 1p34-35, a region containing the proto-oncogene L-MYC. Sixty-eight primary OvCas, including 40 analyzed by RLGS, were screened by quantitative polymerase chain reaction (PCR) for possible amplification of L-MYC. Ten tumors with increased L-MYC copy number were identified, including tumor E1, which showed an approximately 24-fold increase in copy number compared to normal DNA. Southern analysis of several tumors confirmed the quantitative PCR results. Using sequence tagged site (STS) markers flanking L-MYC, increased DNA copy number in tumor E1 was found to span the region flanking L-MYC between D1S432 and D1S463 ( approximately 3.1 Mb). Other tumors showed amplification only at the L-MYC locus. Using oligonucleotide microarrays, L-MYC was found to be more frequently overexpressed in OvCas than either c-MYC or N-MYC relative to ovarian surface epithelium. Quantitative reverse transcriptase-PCR analysis confirmed elevated L-MYC expression in a substantial fraction of OvCas, including nine of nine tumors with increased L-MYC copy number. The data implicate L-MYC gene amplification and/or overexpression in human OvCa pathogenesis.

This review covers recent developments towards the implementation of multi-dimensional (MuD) liquid phase based systems for proteome investigations. Although two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) has been used as a standard approach in proteomics, its drawbacks including the limited dynamic range and molecular mass range, together with lack of on-line integration with biological mass spectrometery (Bio-MS) have limited its widespread use and applications in proteomics. In the meantime, various liquid-phase based multi-dimensional separation techniques have been explored. Especially, with the emergence of the combination of nanoflow capillary high-performance liquid chromatography (cHPLC) and Bio-MS, attention is again refocused on utilizing multi-dimensional liquid-phase based separation of proteins. Some remarkable applications of on-line analysis of intact proteins and on-column digested proteins, and the emergence of approaches such as multiple HPLC-electrospray ionization tandem MS and capillary array electrophoresis-matrix assisted laser desorption ionization MS, have stimulated thinking towards developing a automated multi-dimensional system (MuDSy) that integrates liquid phase based separation, digestion and identification of proteins in complex biological mixtures., (Copyright 2002 Elsevier Science B.V.)

Cytokeratins (CK) are intermediate filaments whose expression is often altered in epithelial cancer. Systematic identification of lung adenocarcinoma proteins using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry has uncovered numerous CK isoforms. In this study, 93 lung adenocarcinomas (64 stage I and 29 stage III) and 10 uninvolved lung samples were quantitatively examined for protein expression. Fourteen of 21 isoforms of CK 7, 8, 18, and 19 occurred at significantly higher levels (P < .05) in tumors compared to uninvolved adjacent tissue. Specific isoforms of the four types of CK identified correlated with either clinical outcome or individual clinical-pathological parameters. All five of the CK7 isoforms associated with patient survival represented cleavage products. Two of five CK7 isoforms (nos. 2165 and 2091), one of eight CK8 isoforms (no. 439), and one of three CK19 isoforms (no. 1955) were associated with survival and significantly correlated to their mRNA levels, suggesting that transcription underlies overexpression of these CK isoforms. Our data indicate substantial heterogeneity among CK in lung adenocarcinomas resulting from posttranslational modifications, some of which correlated with patient survival and other clinical parameters. Therefore, specific isoforms of individual CK may have utility as diagnostic or predictive markers in lung adenocarcinomas.

17q23 is a frequent site of gene amplification in breast cancer. Several lines of evidence suggest the presence of multiple amplicons on 17q23. To characterize distinct amplicons on 17q23 and localize putative oncogenes, we screened genes and expressed sequence tags (ESTs) in existing physical and radiation hybrid maps for amplification and overexpression in breast cancer cell lines by semiquantitative duplex PCR, semiquantitative duplex RT-PCR, Southern blot, and Northern blot analyses. We identified two distinct amplicons on 17q23, one including TBX2 and another proximal region including RPS6KB1 (PS6K) and MUL. In addition to these previously reported overexpressed genes, we also identified amplification and overexpression of additional uncharacterized genes and ESTs, some of which suggest potential oncogenic activity. In conclusion, we have further defined two distinct regions of gene amplification and overexpression on 17q23 with identification of new potential oncogene candidates. Based on the amplification and overexpression patterns of known and as of yet unrecognized genes on 17q23, it is likely that some of these genes mapping to the discrete amplicons function as oncogenes and contribute to tumor progression in breast cancer cells.

Molecular classification of tumors based on their gene expression profiles promises to significantly refine diagnosis and management of cancer patients. The establishment of organ-specific gene expression patterns represents a crucial first step in the clinical application of the molecular approach. Here, we report on the gene expression profiles of 154 primary adenocarcinomas of the lung, colon, and ovary. Using high-density oligonucleotide arrays with 7129 gene probe sets, comprehensive gene expression profiles of 57 lung, 51 colon, and 46 ovary adenocarcinomas were generated and subjected to principle component analysis and to a cross-validated prediction analysis using nearest neighbor classification. These statistical analyses resulted in the classification of 152 of 154 of the adenocarcinomas in an organ-specific manner and identified genes expressed in a putative tissue-specific manner for each tumor type. Furthermore, two tumors were identified, one in the colon group and another in the ovarian group, that did not conform to their respective organ-specific cohorts. Investigation of these outlier tumors by immunohistochemical profiling revealed the ovarian tumor was consistent with a metastatic adenocarcinoma of colonic origin and the colonic tumor was a pleomorphic mesenchymal tumor, probably a leiomyosarcoma, rather than an epithelial tumor. Our results demonstrate the ability of gene expression profiles to classify tumors and suggest that determination of organ-specific gene expression profiles will play a significant role in a wide variety of clinical settings, including molecular diagnosis and classification.

Insulin-like growth factor II (IGF-II) acts as an autocrine growth factor for many in vitro tumor cell lines including neuroblastoma. To examine the role of IGF-II in tumor biology we have analyzed a total of 56 primary neuroblastoma tumor samples for the presence of IGF-II using a combination of mRNA and protein analysis. A group of 21 samples was examined for the presence of IGF-II mRNA by slot blot and a separate group of 37 samples was examined for IGF-II immunoreactivity. IGF-II was detected in 48% of the total tumor specimens analyzed. IGF-II immunoreactivity was observed in cells resembling developing neuroblasts and was confined to the cytoplasm and proximal neurites. The appearance of IGF-II mRNA and protein did not correlate with tumor prognostic features including stage, histology, or N-myc amplification. These data suggest that the expression of IGF-II is not confined to a specific stage of the disease but may have a broader role in the pathogenesis of neuroblastoma.

Op18 is a highly conserved major cytosolic phosphoprotein which has been implicated in signal transduction in a wide variety of cell types. Freshly isolated peripheral blood lymphocytes (PBL) constitutively express low levels of mostly unphosphorylated Op18. Following mitogenic stimulation of PBL, Op18 synthesis is induced at a time when cells are entering S-phase. In this study we have characterized Op18 phosphorylation during progression of freshly isolated PBL through the cell cycle. Transition from G0 to G1 following activation with OKT3 was associated with an increase in a phosphorylated form designated Op18c. Progression of cells through G1 into S resulted in an increase in phosphorylated Op18 forms, designated Op18a and Op18b, which paralleled new Op18 synthesis. Transition of cells into G2 + M resulted in the appearance of the more acidic phosphorylated forms Op18d and Op18e. Calphostin C, a specific inhibitor of protein kinase C, dramatically decreased all forms of phosphorylated Op18 in OKT3 treated Jurkat cells. Our results suggest that Op18 phosphorylation is mediated in part by PKC activation as well as by other kinases yielding different phosphorylated forms at specific stages of the cell cycle.

Op18 is a highly conserved major cytosolic phosphoprotein that is expressed at high levels in acute leukemia and in neuroblastoma. In this study we present evidence pointing to a role for Op18 in cellular proliferation. Blocking of Op18 mRNA translation using antisense oligonucleotides delayed entrance of mitotically stimulated normal peripheral blood lymphocytes into the S phase. Moreover treatment of HL-60 promyelocytic leukemia cells with DMSO or PMA which induced terminal differentiation resulted in a decrease in the level of Op18 RNA and protein. Inhibition of lymphoid proliferation with cyclosporin also resulted in reduced Op18 levels.

We have previously reported lack of expression of a polypeptide designated L3 in infant acute lymphoblastic leukemia (ALL). Expression of L3 occurred predominantly in older children with pre-B ALL. We have recently reported the expression during B cell ontogeny of two other polypeptides, designated L2 and L4 with a similar Mr as L3, which were identified as phosphorylated and non-phosphorylated forms respectively of the low Mr heat shock protein. hsp27. In this study we have characterized L3 and identified it as another phosphorylated form of hsp27. The two phosphorylated forms appear to be differentially expressed in acute leukemia. L3 levels in infants who expressed hsp27 isoforms L2 and L4 were significantly diminished compared to levels in older children with an equivalent amount of hsp27. We conclude that leukemic cells in infant ALL exhibit a unique pattern of phosphorylation of hsp27 expressed at a pre-B cell stage of differentiation.

Proliferating cell nuclear antigen (PCNA) is a 36-Kd nuclear protein, identified as the auxiliary protein of DNA polymerase delta, that is upregulated in activated proliferating cells from a variety of tissues and species, including human lymphocytes. We have examined by two-dimensional polyacrylamide gel electrophoresis the expression of PCNA in various subtypes of childhood acute leukemia and have found differences in its expression according to subtype. These differences were not related to the initial peripheral white blood count, age, or sex, and appeared to reflect differences in proliferative activity between subtypes of acute leukemia.

We analyzed the polypeptide pattern of leukemic cells of infants and older children with acute lymphoblastic leukemia (ALL), using two-dimensional polyacrylamide gel electrophoresis (PAGE). Patterns were analyzed for the occurrence of a previously detected cytosolic polypeptide, designated L3. Quantitative analysis of L3 in 12 infants and 91 older children with non-T ALL indicated lack of expression of polypeptide L3 in leukemic cells of infants which, in most cases, expressed HLA-DR and CD19 and lacked CD10. Quantitative analysis of L3 in relation to cell surface marker expression revealed that L3 was limited in its occurrence to non-T ALL and was not coordinately expressed with any of the surface markers included in the study. Among patients in the HLA-DR-positive, CD19-positive, and CD10-negative group, different levels of polypeptide L3 were observed between infants and older children. These results indicate differences in leukemic cell constituents between infants and older children with ALL and an otherwise similar cell surface marker phenotype.

An anion-exchange high-performance liquid chromatographic procedure was developed for the separation of transferrin in human plasma. The procedure allowed the four molecular forms of transferrin, which differ with respect to bound iron, to be separated from each other and from other plasma proteins. Transferrin variants, including B and D types, could also be identified using anion-exchange high-performance liquid chromatography. The approach followed for optimizing the separation of transferrin included identification of the peaks in the chromatogram by two-dimensional polyacrylamide gel electrophoresis. This approach could be extended to other proteins in plasma or biological fluids in order to optimize their separation.

The use of a recently developed cation-exchange HPLC packing material for the separation of hemoglobin types in human blood has been investigated. Adult and newborn hemolysates from normal individuals and from subjects with hemoglobin disorders were analyzed using a weak cation carboxymethyl-bonded phase on 5-micron-particle-size silica. Elution was accomplished using a Bistris (2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1, 3-propanediol) gradient. Seven well-resolved HbA1 fractions eluted before the major HbA peak. Hbs A1a, A1b, A1c and an HbA1 fraction that increased with aging of the hemolysates were separately eluted. HbF when present or when added to the hemolysates eluted as a distinct peak. HbA was followed by Hbs A2, S, and C when present. An early-eluting peak corresponding to Hb Bart's was identified in newborn hemolysates. It is concluded that cation-exchange HPLC provides a new tool for the reliable separation of minor hemoglobin components.

The detection of neutral amino acid mutants, by isoelectric focusing in immobilized pH gradients (IPGs), is exemplified by the separation of hemoglobin (Hb) Beirut (126 beta Val----Ala) from Hb A in a shallow pH 7.2-7.6 IPG gradient with 2% Ampholine pH 6-8. The mechanism of these separations appears to involve minute alterations in the pK values of ionizable groups bordering the mutation site, which are in turn reflected in tiny alterations in the net surface charge, delta pI. The delta pI values are of the order of 0.01 to 0.001 pH units, outside the resolving limits of conventional isoelectric focusing, and correspond to changes of the order of 0.1-0.01 unit charge (a proton or an electron).

An apparent Mr variant of plasma transthyretin (TTR), previously detected using 2-D PAGE, is the first reported occurrence of this type of human TTR variant. We characterized the variant TTR to determine the nature of this difference. Comparative tryptic peptide maps of variant and normal TTR and sequencing of peptides which differed indicated the variant contained a single amino acid substitution of valine for tyrosine at position 116. Because such a change requires two nucleotide substitutions, we postulate the variant arose through mutation in codon 116 of a heretofore unrecognized polymorphic or rare variant allele of TTR.