Jacob Kagan

Localization of chromosome 8p regions involved in early tumorigenesis of oral and laryngeal squamous carcinoma

We analysed 30 primary invasive oral and laryngeal squamous carcinomas (SC), with concurrent dysplastic lesions, for genetic alterations at 15 microsatellite loci on the short arm of chromosome 8. Overall, loss of heterozygosity (LOH) was observed, in at least one informative locus, in 27% of the dysplastic lesions and in 67% of the invasive carcinomas. The highest frequency of allele losses in dysplasia (20% and 17%), and invasive carcinoma (40% and 48%) were detected in the same D8S298 and LPL-tet loci located on chromosomes 8p21 and 8p22 respectively. The minimal region with LOH was limited to 4.6 megaBases (mBs) at 8p22 and 7.1 mBs at 8p21. In addition, allelic losses in both dysplastic and corresponding invasive specimens were noted at the same loci in some tumors suggesting their emergence from a common preneoplastic clone. Allele losses correlated significantly with male gender, oral and laryngeal sites and high proliferative index. The data suggest that inactivation of tumor suppressor gene(s), within these loci, may constitute an early event in the evolution of oral and laryngeal SC.

Towards Clinical Application of Methylated DNA Sequences as Cancer Biomarkers: A Joint NCI's EDRN and NIST Workshop on Standards, Methods, Assays, Reagents and Tools

The workshop report, entitled Towards Clinical Application of Methylated DNA Sequences as Cancer Biomarkers: A Joint National Cancer Institutes Early Detection Research Network and National Institute of Standards and Technology Workshop, presents a summary of the main issues, current challenges, outcomes, and recommendations toward application of methylated DNA sequences as cancer biomarkers.

Limiting the location of putative human prostate cancer tumor suppressor genes on chromosome 18q.

We studied loss of heterozygosity (LOH) on the long arm of human chromosome 18 in prostate cancer to determine the location of a putative tumor suppressor gene (TSG) and to correlate these losses with the pathological grade and stage of the cancer. Of 48 specimens analysed 17 (35.4%) lost at least one allele on chromosome 18q. All the specimens with allelic losses lost at least one allele within chromosomal region 18q21. Allelic losses picked at D18S51 (19%) and D18S858 (17%). A 0.58 cM DNA segment that includes the D18S858 locus and is flanked by the microsatellite loci D18S41 and D18S381, was lost in eight (47%) of 17 specimens with allelic losses. This segment was designated as a LOH cluster region 1 (LCR 1). Although Smad2 resides within LCR 1, it was not mutated in any of the six prostate cell lines (five prostate cancer cell lines and one immortalized prostate epithelial cell line) analysed, suggesting that it is not a candidate TSG in prostate cancer. A second LCR at 18q21, LCR 2, includes the D18S51 locus and is flanked by the D18S1109 and D18S68 loci, which are separated by 7.64 cM. LCR 2 was lost in six (35%) of the 17 specimens with chromosome 18q losses. These results suggest that chromosome 18q21 may harbor two candidate prostate cancer TSGs. The candidate TSGs DCC and Smad4 are located centromeric to the LCRs. No alleles were lost within or in close proximity to these genes, suggesting that they are not targets for inactivation by allelic losses in prostate cancer. Although there was no obvious correlation between chromosome 18q LOH and the pathological grade or stage, three (37.5%) of eight low-grade cancers and nine (32.1%) of 28 organ-confined cancers lost alleles at 18q21, suggesting that allelic losses are relatively early events in the development of invasive prostate cancer.

Cluster of allele losses within a 2.5 cM region of chromosome 10 in high-grade invasive bladder cancer

A limited number of previous studies have indicated a low frequency of chromosome 10 allele losses and deletions in bladder cancers. We investigated the involvement of chromosome 10 in advanced bladder cancers. Loss of heterozygosity (LOH) was analysed in 19 microsatellite loci in 20 grade III invasive transitional cell carcinomas. Nine (45%) of the 20 tumors had at least one allele loss on the long arm of chromosome 10. The short arm of chromosome 10 was not affected. The most frequent LOH occurred at D10S215, where four (29%) of 14 of the informative cases had an allele loss. The minimal region with allele losses was located between the centromeric marker D10S1644 and the telomeric marker D10S541, which are separated by 2.52 cM. The results strongly suggest the existence within that region of a tumor suppressor gene or genes for advanced bladder cancer.

EPIDEMIOLOGIC DETERMINANTS OF CLINICALLY RELEVANT PROSTATE CANCER

While tumor volume and Gleason scores are the best available prognostic indicators for prostate cancer, contemporary predictive methods are unable to identify which men with Gleason scores of 7 have clinically insignificant tumors that will not progress and which men will develop highly aggressive prostate cancer. Our objective was to evaluate potential environmental determinants of significant prostate cancer. Subjects were patients identified from a university‐based hospital and tertiary cancer center who had undergone radical prostatectomy for prostate cancer. Cases were 103 patients whose tumor volumes were ≤0.5 ml. The comparison group was comprised of 225 men with larger‐volume disease or with histologic evidence of extracapsular extension but without lymph node involvement. The matching criteria were ethnicity, age at diagnosis (±5 years), and date of diagnosis (±1 year). Epidemiologic data, current weight, and height were obtained. The comparison group was significantly more likely than cases to be current smokers (7.6% vs. 3.9%) and to report more pack‐years smoked (30.1 vs. 23.0 years, p = 0.06). Cases tended to weigh less (85.2 vs. 87.1 kg, p = 0.1) and have lower body mass indices (26.8 vs. 27.6, p = 0.07). A similar trend was evident for weight at age 40 (79 vs. 81 kg). Cases reported a mean weight gain of 4.9 kg compared with 6.6 kg in the comparison subjects (p = 0.05) between the ages of 25 and 40. There was no significant difference in weight gain from age 40 to current age. Cases were more likely to report having prostate cancer screening (90% vs. 80%, p = 0.02). Cases with Gleason scores ≤7 (3 + 4, with 3 being the dominant grade) were younger at diagnosis than those with scores of 7 (4 + 3, with 4 being the dominant grade), were more likely (93%) to have had prostate screening, were less likely to be current smokers (4%), reported the fewest pack‐years smoked (21.5 vs. 28.6 years for high‐score cases and 30.1 for comparison subjects), and had the lowest average weight gain from ages 25 to 40 (4.62 vs. 6.31 kg for high‐score cases). Weight gain in early adulthood and smoking thus appear to be important predictors of virulent prostate cancer. Our data also suggest that prior screening is associated with diagnosis of lower‐volume and lower‐score disease. Int. J. Cancer 89:259–264, 2000.

Genotypic alterations in benign and malignant salivary gland tumors: histogenetic and clinical implications.

Loss of heterozygosity (LOH) and microsatellite instability (MI) were examined at 24 microsatellite loci in 46 primary benign and malignant salivary gland tumors. Among the 27 benign tumors, 11 (40.7%), manifested microsatellite alterations in at least one locus; of these, five (18.5%) showed LOH and four (14.8%) had microsatellite instability at two or more loci. Four of 11 pleomorphic adenomas (36.4%) had allele loss on the long arm of chromosome 8. Among the 19 malignant neoplasms examined, 10 (52.6%) and one (5.2%) had allele losses and MI, respectively, at multiple loci; three tumors showed MI at only one locus. Frequent LOH was detected at D8S166 (8q11-12), D17S799, and D17S122 (17p-17p11-2) loci, with an incidence of 40%, 37.5%, and 43%, respectively. In general, malignant neoplasms with LOH exhibited aggressive tumor characteristics. Statistically significant correlations were found between LOH and pathologic classification (chi 2, p = 0.05), higher grade (p = 0.02), DNA aneuploidy (p = 0.005), and a proliferative index of > 6% (p = 0.005) of the malignant tumors. Carcinomas with 17p loci alterations, including two carcinomas expleomorphic adenoma with concurrent 8q LOH, showed more aggressive features. The results suggested that (a) loci on chromosome 8q may harbor a tumor suppressor gene or genes associated with the development or progression of some salivary neoplasms; (b) alterations on the short arm of chromosome 17 may represent an event related to tumor progression; and (c) tumors with LOH at multiple loci have aggressive biologic characteristics.

Inactivation of MMAC1 in bladder transitional-cell carcinoma cell lines and specimens.

We recently limited the location of a candidate tumor suppressor gene in invasive (T3a/b) bladder transitional‐cell carcinoma (TCC) to a 2.5‐cM region at chromosome 10q23.3. This region harbors the MMAC1/PTEN/TEP1 gene (referred to hereafter as MMAC1), a dual‐phosphatase tumor‐suppressor gene frequently inactivated in variety of malignant tumors. In the present study, we examined whether MMAC1 is a target for inactivation by mutations and deletions in bladder TCC cell lines and specimens. MMAC1 was inactivated by homozygous deletions and mutations in three (27%) of 11 bladder cancer cell lines. One cell line, UC‐3, had homozygous deletions, and two other cell lines, T‐24 and UC‐9, had missense mutations. T‐24 had also a nonsense mutation. However, none of the 33 bladder TCC specimens examined had a mutation or deletion in the coding region. These results suggest that MMAC1 is not the primary target for inactivation in bladder TCC and that another gene, in close proximity to the MMAC1 locus, within this region of frequent allelic losses, may be the target for inactivation. Mol. Carcinog. 29:143–150, 2000.

Immobilization strategies for single-chain antibody microarrays.

Sandwich ELISA microarrays have great potential for validating disease biomarkers. Each ELISA relies on robust‐affinity reagents that retain activity when immobilized on a solid surface or when labeled for detection. Single‐chain antibodies (scFv) are affinity reagents that have greater potential for high‐throughput production than traditional IgG. Unfortunately, scFv are typically less active than IgG following immobilization on a solid surface and not always suitable for use in sandwich ELISAs. We therefore investigated different immobilization strategies and scFv constructs to determine a more robust strategy for using scFv as ELISA reagents. Two promising strategies emerged from these studies: (i) the precapture of epitope‐tagged scFv using an antiepitope antibody and (ii) the direct printing of a thioredoxin (TRX)/scFv fusion protein on glass slides. Both strategies improved the stability of immobilized scFv and increased the sensitivity of the scFv ELISA microarray assays, although the antiepitope precapture method introduced a risk of reagent transfer. Using the direct printing method, we show that scFv against prostate‐specific antigen (PSA) are highly specific when tested against 21 different IgG‐based assays. In addition, the scFv microarray PSA assay gave comparable quantitative results (R2 = 0.95) to a commercial 96‐well ELISA when tested using human serum samples. In addition, we find that TRX‐scFv fusions against epidermal growth factor and toxin X have good LOD. Overall, these results suggest that minor modifications of the scFv construct are sufficient to produce reagents that are suitable for use in multiplex assay systems.

The clinical impact of recent advances in LC-MS for cancer biomarker discovery and verification

Mass spectrometry (MS) -based proteomics has become an indispensable tool with broad applications in systems biology and biomedical research. With recent advances in liquid chromatography (LC) and MS instrumentation, LC–MS is making increasingly significant contributions to clinical applications, especially in the area of cancer biomarker discovery and verification. To overcome challenges associated with analyses of clinical samples (for example, a wide dynamic range of protein concentrations in bodily fluids and the need to perform high throughput and accurate quantification of candidate biomarker proteins), significant efforts have been devoted to improve the overall performance of LC–MS-based clinical proteomics platforms. Reviewed here are the recent advances in LC–MS and its applications in cancer biomarker discovery and quantification, along with the potentials, limitations and future perspectives.

Developing recombinant antibodies for biomarker detection

Monoclonal antibodies (mAbs) have an essential role in biomarker validation and diagnostic assays. A barrier to pursuing these applications is the reliance on immunization and hybridomas to produce mAbs, which is time-consuming and may not yield the desired mAb. We recommend a process flow for affinity reagent production that utilizes combinatorial protein display systems (e.g., yeast surface display or phage display) rather than hybridomas. These systems link a selectable phenotype--binding conferred by an antibody fragment--with a means for recovering the encoding gene. Recombinant libraries obtained from immunizations can produce high-affinity antibodies (<10 nM) more quickly than other methods. Non-immune libraries provide an alternate route when immunizations are not possible, or when suitable mAbs are not recovered from an immune library. Directed molecular evolution (DME) is an integral part of optimizing mAbs obtained from combinatorial protein display, but can also be used on hybridoma-derived mAbs. Variants can easily be obtained and screened to increase the affinity of the parent mAb (affinity maturation). We discuss examples where DME has been used to tailor affinity reagents to specific applications. Combinatorial protein display also provides an accessible method for identifying antibody pairs, which are necessary for sandwich-type diagnostic assays.