Andrei P. Kozlov

The Human T-Box Mesodermal Transcription Factor Brachyury Is a Candidate Target for T-Cell–Mediated Cancer Immunotherapy

Purpose: Identification of tumor antigens is essential in advancing immune-based therapeutic interventions in cancer. Particularly attractive targets are those molecules that are selectively expressed by malignant cells and that are also essential for tumor progression. Experimental Design and Results: We have used a computer-based differential display analysis tool for mining of expressed sequence tag clusters in the human Unigene database and identified Brachyury as a novel tumor antigen. Brachyury, a member of the T-box transcription factor family, is a key player in mesoderm specification during embryonic development. Moreover, transcription factors that control mesoderm have been implicated in the epithelial-mesenchymal transition (EMT), which has been postulated to be a key step during tumor progression to metastasis. Reverse transcription-PCR analysis validated the in silico predictions and showed Brachyury expression in tumors of the small intestine, stomach, kidney, bladder, uterus, ovary, and testis, as well as in cell lines derived from lung, colon, and prostate carcinomas, but not in the vast majority of the normal tissues tested. An HLA-A0201 epitope of human Brachyury was identified that was able to expand T lymphocytes from blood of cancer patients and normal donors with the ability to lyse Brachyury-expressing tumor cells. Conclusions: To our knowledge, this is the first demonstration that (a) a T-box transcription factor and (b) a molecule implicated in mesodermal development, i.e., EMT, can be a potential target for human T-cell–mediated cancer immunotherapy.

In silico screening for tumour-specific expressed sequences in human genome.

A computer‐based differential display tool named HsAnalyst has been developed and successfully used for the comparison of expression patterns in a set of tumours versus a set of normal tissues. A list of EST clusters highly represented in tumours and rarely observed in normal tissues has been developed as a resulting output file of the program. These differentially expressed EST clusters (genes) can be useful for developing new tumour markers and prognostic indicators for a wide set of human malignancies. Tumour‐specific protein‐coding genes may be considered a manifestation of tumour‐specific gene expression.

The feasibility of audio computer-assisted self-interviewing in international settings

Objective:To determine the feasibility of using audio computer-assisted self-interviewing (ACASI) for data collection in developing countries, and to compare responses to questions eliciting sensitive information about sexual behavior using ACASI versus computer-assisted personal interviewing (CAPI) in five developing countries. Design:A feasibility study determined whether ACASI could be used in populations in developing countries. A follow-up, randomized crossover study compared responses to questions eliciting sensitive information about sexual behavior using ACASI versus CAPI. Methods:The NIMH Collaborative HIV/STD Prevention Trial conducted a feasibility study of ACASI in convenience samples in China, India, Peru, and Russia, then a randomized crossover ACASI versus CAPI study among volunteers in these countries plus Zimbabwe. Results:Approximately equal numbers of men and women completed the feasibility study; the results suggested a high comfort level among participants. Married respondents in China and India appeared to give unreliable responses on sexual activity. In the crossover study, the pattern of responses to sensitive questions showed few differences. In China, higher rates of sexual risk were reported on CAPI. In Peru and Russia, differences by mode were found in the number of partners in the past year. Conclusion:Despite variable computer experience and literacy, feasibility study participants reported ease in completing ACASI, and preferred a computer to an interviewer for answering sensitive questions, or had no preference. In the crossover study, most participants gave similar responses on both modes of survey administration. ACASI appears to be feasible in these settings, although low literacy may pose problems if participants cannot clarify questions.

Ethical issues in the NIMH collaborative HIV/STD prevention trial

Objective:To develop decision rules regarding key ethical dimensions in scientific protocols for the National Institute for Mental Health (NIMH) Collaborative HIV/STD Prevention Trial taking place in five countries (China, India, Peru, Russia, and Zimbabwe). Design:Countries had HIV rates from 27 to 0.1%, the standard of care varied from access to antiretroviral drugs to no availability, and the reporting of sexually transmitted diseases (STD) to government agencies was mandatory in some countries and not in others. These variations presented challenges when developing decision rules that could be uniformly adopted across countries and simultaneously follow the ethical principles of beneficence, respect, and justice. Methods:We used several strategies to identify and resolve ethical dilemmas for this international HIV prevention trial. First, we identified key principles, especially those derived for clinical therapeutic, biomedical preventive, or device trials. We convened a ‘workgroup on protecting human participants’ and charged them with identifying and implementing optimal procedures for ensuring the ethical and equitable treatment of participants and making recommendations to minimize physical, psychological, and social harm to the participants. Each site had a community advisory board, essential in identifying local ethical issues and possible resolutions to them. The NIMH established a data safety and monitoring board with ultimate responsibility for adjudicating ethical dilemmas and decisions. The protocols were deliberated thoroughly by the Trial steering committee, and approved by nine United States and five in-country institutional review boards. Results:We summarize the decision rules adopted to resolve the ethical dilemmas identified. Especially important were the translation of clinical trials principles for a behavioral intervention trial, strategies for ensuring confidentiality and informed consent, dilemmas relating to partner notification of sexually transmitted infections including HIV, minimizing the risks of social harm, establishing community partnerships, ensuring equity among United States and in-country principal investigators, and building capacity for additional research. Conclusion:We document our processes and decisions, and their underlying rationales, and hope they contribute to the development of further thinking and practice regarding the ethics of social and behavioral HIV and STD prevention trials in resource-poor settings.

Evolutionarily new sequences expressed in tumors

BackgroundEarlier we suggested the concept of the positive evolutionary role of tumors. According to this concept, tumors provide conditions for the expression of evolutionarily new and/or sleeping genes in their cells. Thus, tumors are considered as evolutionary proving ground or reservoir of expression. To support this concept we have previously characterized in silico and experimentally a new class of human tumor-related transcribed sequences.ResultsIn this article we describe results of further studies of previously described tumor-related sequences. The results of molecular phylogeny studies, Southern hybridization experiments and computational comparison with genomes of other species are presented.ConclusionThese results suggest that these previously described tumor-related human transcripts are also relatively evolutionarily new.

ELFN1-AS1: A Novel Primate Gene with Possible MicroRNA Function Expressed Predominantly in Human Tumors

Human gene LOC100505644 uncharacterized LOC100505644 [Homo sapiens] (Entrez Gene ID 100505644) is abundantly expressed in tumors but weakly expressed in few normal tissues. Till now the function of this gene remains unknown. Here we identified the chromosomal borders of the transcribed region and the major splice form of the LOC100505644-specific transcript. We characterised the major regulatory motifs of the gene and its splice sites. Analysis of the secondary structure of the major transcript variant revealed a hairpin-like structure characteristic for precursor microRNAs. Comparative genomic analysis of the locus showed that it originated in primates de novo. Taken together, our data indicate that human gene LOC100505644 encodes some non-protein coding RNA, likely a microRNA. It was assigned a gene symbol ELFN1-AS1 (ELFN1 antisense RNA 1 (non-protein coding)). This gene combines features of evolutionary novelty and predominant expression in tumors.