Nada H. Khattar

Profiling Tumor-Associated Antibodies for Early Detection of Non-small Cell Lung Cancer

Background: A blood test for non-small cell lung cancer (NSCLC) may be a valuable tool for use in a comprehensive lung cancer screening strategy. Here we report the potential of autoantibody profiling to detect early-stage and occult NSCLC. Methods: T7-phage NSCLC cDNA libraries were screened with patient plasma to identify phage-expressed proteins recognized by tumor-associated antibodies. Two hundred twelve immunogenic phage-expressed proteins, identified from 4000 clones, were statistically ranked for their individual reactivity with 23 stage I cancer patient and 23 risk-matched control samples. All 46 samples were used as a training set to define a combination of markers that were best able to distinguish patient from control samples; this set of classifiers was then examined using leave-one-out cross-validation. Markers were then used to predict probability of disease in 102 samples from the Mayo Clinic CT Screening Trial (six prevalence cancer samples, 40 drawn 1 to 5 years before diagnosis, and 56 risk-matched controls). Results: Measurements of the five most predictive antibody markers in 46 cases and controls were combined in a logistic regression model that yielded area under the receiver operating characteristics curve of 0.99; leave-one-out validation achieved 91.3% sensitivity and 91.3% specificity. In testing this marker set with samples from the Mayo Clinic Lung Screening Trial, we correctly predicted six of six prevalence cancers, 32 of 40 cancers from samples drawn 1 to 5 years before radiographic detection on incidence screening, and 49 of 56 risk-matched controls. Conclusions: Antibody profiling may be a useful tool for early detection of NSCLC.

Genetic and epigenetic modification of mismatch repair genes hMSH2 and hMLH1 in sporadic breast cancer with microsatellite instability.

Breast cancer is the most common cancer in women, but its pathogenesis is still unclear. Microsatellite instability (MSI) has been identified in breast cancer cells, suggesting an association with mismatch repair defects. To test this hypothesis, we investigated MSI, protein expression of hMSH2 and hMLH1, as well as genetic and epigenetic modifications of these two genes in 32 sporadic breast tumors. MSI was identified in 15 cases. Immunohistochemistry analysis revealed that all MSI cases but one had lower than normal expression of hMSH2 (nine cases), hMLH1 (12 cases), or both (seven cases). In tumors with MSI, both genetic and epigenetic modifications of these mismatch repair genes were also identified. Eight cases harbored mutations or polymorphisms in hMSH2 and hMLH1, and 10 exhibited hypermethylation in the promoter region of hMLH1. These results suggest that both genetic and epigenetic alterations of hMSH2 and especially of hMLH1 contribute to genomic instability and tumorigenesis in sporadic breast cancer.

Isolation and partial characterization of an opioid-like 88 kDa hibernation-related protein

Previous studies show that infusion of hibernating woodchuck albumin (HWA) induces hibernation in summer-active ground squirrels and results in profound behavioral and physiological depression in primates. These effects are reversed by the administration of opiate antagonists, suggesting that the putative hibernation induction trigger (HIT) may act through opioid receptors. We have demonstrated that both HIT-containing plasma and the synthetic alpha opioid D-Ala2-D-Leu5-enkephalin (DADLE), which mimics the activity of HIT in hibernators, extend tissue survival time of a multi-organ autoperfusion system by 3-fold. In this study we present the first data showing biological activity with a much more highly purified plasma fraction from hibernating woodchucks, identified as the hibernation-related factor (HRF). Both the HRF and DADLE show opiate-like contractile inhibition in the mouse vas deferens (Mvd) bioassay. We also have preliminary evidence in an isolated rabbit heart preparation indicating that the HRF and DADLE act similarly to restore left ventricular function following global myocardial ischemia. Furthermore, we have partially sequenced an alpha 1-glycoprotein-like 88 kDa hibernation-related protein (p88 HRP) present in this fraction, which may prove to be the blood-borne HIT molecule.

Heterogeneity of Ovarian Cancer: Relationships Among Histological Group, Stage of Disease, Tumor Markers, Patient Characteristics, and Survival

Epidemiological studies have established associations between various reproductive factors and risk of ovarian cancer; it has also been observed that some of these risk factors are only associated with specific histological subgroups. To investigate the correlation of genetic alterations with these risk factors, we examined a consecutive series of 158 ovarian cancer cases treated at the University of Kentucky (1990–96). Common molecular genetic alterations (LOH on chromosome 17, P53 alterations, K-RAS mutations), histological and clinical characteristics of the disease, demographic patient information and survival were evaluated. These latter data were from the Kentucky Cancer Registry. Univariate analysis showed higher frequencies of chromosome 17 loss and P53 mutations in tumors of advanced stage and grade, and in older and post-menopausal women. Non-mucinous tumors were more likely to be classified as late stage, high-grade cancers, and to have chromosome 17 loss and P53 mutations. Survival analysis indicated that stage was the only independent significant variable. When stage was the outcome variable in multiple logistic regression analysis, histology and chromosome 17 loss were significantly associated with poor survival. This case-case study provides evidence that ovarian cancers of mucinous and non-mucinous histology are significantly different with respect to clinical characteristics, survival and molecular alterations. It also lends support to the hypothesis that ovarian cancer is a heterogeneous disease with distinct etiological factors and clinical outcomes, which may require different approaches to treatment.

Challenge in the differentiation between attenuated familial adenomatous polyposis and hereditary nonpolyposis colorectal cancer: Case report with review of the literature

The clinical differentiation between hereditary nonpolyposis colorectal cancer (HNPCC) and attenuated familial adenomatous polyposis (AFAP) is very difficult. The 62-yr-old proband presented with duodenal adenocarcinoma. His history of subtotal colectomy for colon cancer, the rarity of duodenal adenocarcinoma in the general population, and his family history of colon cancer made us suspect that he might have FAP. We investigated this family by obtaining medical records and performing gene analysis. The proband had only 10 adenomatous colon polyps when he underwent subtotal colectomy for the cancer, so classic FAP was excluded. His family history included rectal cancer in his brother at 69 yr of age, colon cancer in his mother at 75 yr, and colon cancer in one maternal cousin at 42 yr. Three months after we started to study this family, the probands 32-yr-old son presented with rectal cancer. His family fulfilled the Amsterdam criteria for HNPCC, but AFAP could not be excluded. Upon gene testing, the proband was negative for APC gene germline mutation, which made AFAP highly unlikely. Moreover, high microsatellite instability (MSI) was detected in his adenomas and cancer tissues. The fulfillment of Amsterdam criteria, the exclusion of FAP and AFAP, and the high MSI established the diagnosis of HNPCC in this family. We also summarize the differences between FAP, AFAP, and HNPCC; extend the graphic description of the MSI mechanism; and propose a diagnostic strategy for HNPCC.

Common polymorphisms and somatic mutations in human base excision repair genes in ovarian and endometrial cancers.

The purpose of this study was to determine whether the human APEX and OGG1 genes, encoding proteins important in base excision repair (BER) of DNA, contain nucleotide sequence polymorphisms or are mutated somatically in tumors from women diagnosed with ovarian or endometrial cancer. Based upon the analysis of germline DNA from 83 individuals, 63 with ovarian cancer and 20 with endometrial cancer, we found two missense polymorphisms in APEX (Q51H and D 148E) and two missense (A3P and S326C) and one intronic (Exon 5-15 bp) polymorphism in OGG1. The frequencies of the various alleles (in the ovarian and endometrial cancer patients combined) were 4.8% for 51-His and 56.2% for 148-Glu in APEX, and 1.0% for 3-Pro and 20.0% for 326-Cys in OGG1. Somatic mutations in APEX (P112L, W188X and R237C) were identified in three of 20 endometrial tumors, but no mutations were identified in APEX in 43 ovarian tumors, or in OGG1 at either tumor site. Given the crucial role of the APEX and OGG1 proteins in BER of oxidative DNA damage, the identified polymorphisms are good candidates for genetic epidemiologic studies of cancer susceptibility, while the finding that three of 20 (15%) endometrial tumors have somatic mutations in APEX suggests that inactivation of the BER pathway is important for the development of endometrial cancer in at least a subset of cases.

Characterization of the Human Polymeric Immunoglobulin Receptor (PIGR) 3′UTR and Differential Expression of PIGR mRNA during Colon Tumorigenesis

The human cell lines VACO-235 and VACO-411 constitute a novel in vitro model of colon adenoma to carcinoma progression. By differential display RT-PCR we identified a transcript that is expressed in the parental nontumorigenic adenoma line (VACO-235E), but is not expressed in the tumorigenic daughter (VACO-235L) or granddaughter (VACO-411) lines. This cDNA represents a previously uncharacterized portion of the 3′UTR of human PIGR. Human PIGR mRNA was found to be highly expressed in normal colon epithelium, but was decreased in 6 of 8 colon tumors and was negligible in 8 of 10 colon tumor cell lines. We sequenced the entire 1.8 kb 3′UTR of human PIGR, and found it to contain multiple repetitive elements as well as elements that could affect the processing and stability of PIGR mRNA. We hypothesize that differential regulation of PIGR mRNA stability may contribute to its downregulation in colon cancer.

At least two distinct epigenetic mechanisms are correlated with high-frequency "switching" for APRT phenotypic expression in mouse embryonal carcinoma stem cells.

A series of clones displaying high frequency “switching” phenotypes for expression of the adenine phosphoribosyltransferase (aprt) gene were previously isolated from the P19 mouse embryonal carcinoma stem cell line. Most clones contained only oneaprt allele. We report here the characterization of each of these clones with regards to enzymatic activity, mRNA steady state levels, DNA methylation, and chromatin conformation. When clones were selected for resistance to the purine analog 2,6-diaminopurine, which requires markedly reduced levels of APRT enzymatic activity, two distinct classes were observed. The first class was associated with reduced or undetectable levels ofaprt mRNA, hypermethylation of the 5′ CpG island, and a closed chromatin conformation within this region. When clones of this class were selected for reacquisition of APRT enzymatic activity they were found to have increased mRNA levels, a hypomethylated CpG island, and an open chromatin conformation. In contrast, the second class of clones displayed wild-type levels of mRNA, CpG island hypomethylation, and an open chromatin conformation regardless of whether they were selected for the presence or absence of APRT enzymatic activity. The implications of these results for general mechanisms of epigenetic change in somatic cells and the possibility that expression of the mouseaprt gene may be developmentally regulated are discussed.

Lung cancer-associated auto-antibodies measured using seven amino acid peptides in a diagnostic blood test for lung cancer

Abstract Autoantibody profiling is a developing approach that incorporates immune recognition of myriad aberrant cancer proteins into a single diagnostic assay. We have previously described methodology to screen T7-phage NSCLC-cDNA libraries for phage-expressed proteins recognized by NSCLC-associated antibodies, and developed a multiplex assay that has excellent ability to discriminate NSCLC from control samples. This follow-up report describes the development and testing of a diagnostic autoantibody assay that uses seven amino-acid peptides as capture proteins. A random-peptide M13-phage library was screened for proteins recognized by cancer-associated antibodies. One hundred twenty-one NSCLC case and control samples were divided into two groups for training and validation, or alternately, evaluated sequentially in a leave-one-out analysis. Candidate antibody-markers were ranked by statistical discrimination between cases and controls. Receiver-Operating-Characteristic (ROC-AUC) suggested the predictive potential of various marker combinations. A five-marker combination (AUC=0.982) afforded 90% sensitivity and 73% specificity in a training-and-testing strategy. Leave-one-out validation provided similar class prediction. Data confirm the potential of antibody profiling to provide high levels of cancer prediction. Random peptide libraries offer a universal source of capture proteins for antibody profiling that obviates the need for tumor-specific library construction and abrogates inherent problems with tumor heterogeneity during biomarker discovery.

A role for certain mouseAprt sequences in resistance to toxic adenine analogs

A mouse embryonal carcinoma cell line hemizygous for the adenine phosphoribosyltransferase gene (aprt) was exposed to ultraviolet light (UV) or to the alkylating agent, ethyl methanesulfonate (EMS). Thirty eight cell lines retaining theaprt gene were isolated by selecting for resistance to 2,6-diaminopurine (DAP), an adenine analogue which selects against aprt activity. Of these, six cell lines distinguished by significant levels of aprt enzymatic activity after selection in DAP, were found to carry mutations in theaprt gene affecting the apparent Km of the enzyme for adenine in every cell line, and the apparent Km for phosphoribosylpyrophosphate in two of the six cell lines. The results indicate that the ability of these cells to survive in the presence of toxic adenine analogues while maintaining significant levels of aprt enzyme activity may be due to a reduced affinity for the adenine analogue, DAP. This biochemical analysis along with results obtained from sequencing theaprt gene from 31 DAP resistant cell lines with no detectable aprt activity were used to implicate certain amino acids within aprt in substrate binding. It was also determined that, in contrast to UV, EMS did not appear to exhibit any strand bias in the distribution of mutations.