Therapeutic cancer vaccination against telomerase: clinical developments in melanoma.

PURPOSE OF REVIEW: Checkpoint inhibitors (CPIs) have revolutionized treatment outcomes for patients with malignant melanoma. Long-term follow-up shows that a substantial subset of patients who exhibit clinical responses achieve extended overall survival. Nevertheless, most patients do not achieve durable benefit from CPIs, and improvements are urgently needed. The clinical efficacy of CPIs depends on highly variable preexisting spontaneous T-cell immune responses. Cancer vaccines represent an independent treatment modality uniquely capable of expanding the repertoire of tumor-specific T cells in cancer patients and thus have the capacity to compensate for the variability in spontaneous T-cell responses. Vaccines are, therefore, considered attractive components in a CPI-combination strategy. RECENT FINDINGS: Here we discuss recent results obtained through therapeutic vaccination against telomerase human telomerase reverse transcriptase (hTERT). Recent publications on translational research and clinical results from phase I trials indicate that vaccination against telomerase in combination with CPIs provides relevant immune responses, negligible added toxicity, and signals of clinical efficacy. CONCLUSION: In the near future, randomized data from clinical trials involving therapeutic cancer vaccines and checkpoint inhibitors will be available. Positive readout may spark broad development and allow cancer vaccines to find their place in the clinic as an important component in multiple future CPI combinations.

Technology to accelerate pangenomic scanning for unknown point mutations in exonic sequences: cycling temperature capillary electrophoresis (CTCE).

BACKGROUND: Rapid means to discover and enumerate unknown mutations in the exons of human genes on a pangenomic scale are needed to discover the genes carrying inherited risk for common diseases or the genes in which somatic mutations are required for clonal diseases such as atherosclerosis and cancers. The method of constant denaturing capillary electrophoresis (CDCE) permitted sensitive detection and enumeration of unknown point mutations but labor-intensive optimization procedures for each exonic sequence made it impractical for application at a pangenomic scale. RESULTS: A variant denaturing capillary electrophoresis protocol, cycling temperature capillary electrophoresis (CTCE), has eliminated the need for the laboratory optimization of separation conditions for each target sequence. Here are reported the separation of wild type mutant homoduplexes from wild type/mutant heteroduplexes for 27 randomly chosen target sequences without any laboratory optimization steps. Calculation of the equilibrium melting map of each target sequence attached to a high melting domain (clamp) was sufficient to design the analyte sequence and predict the expected degree of resolution. CONCLUSION: CTCE provides practical means for economical pangenomic detection and enumeration of point mutations in large-scale human case/control cohort studies. We estimate that the combined reagent, instrumentation and labor costs for scanning the approximately 250,000 exons and splice sites of the approximately 25,000 human protein-coding genes using automated CTCE instruments in 100 case cohorts of 10,000 individuals each are now less than U.S. $500 million, less than U.S. $500 per person.

[Anticancer drug dosing--pharmacogenomic biomarkers or body surface area?]. / Cytostatikadosering etter farmakogenomiske markører eller kroppsoverflate?

BACKGROUND: Chemotherapeutic treatment regimes are established for most cancer forms. In general, these substances have extremely narrow therapeutic windows, which render cancer patients vulnerable to over- and underdosing. Individual drug dosing is currently based on the patients' body surface area. This practice is an extrapolation from animal studies. Recent advances in molecular medicine raise the question of whether the present dosing strategy should be adjusted to individual functional DNA variants affecting the metabolism, transport and efficacy of anticancer drugs. MATERIAL AND METHODS: This review is based on selected references retrieved from PubMed and the authors' experience in drug treatment of cancer patients. RESULTS: Several single nucleotide polymorphisms and other DNA variants that contribute to varying clinical response to chemotherapeutic agents were identified. For some drugs it has been shown that unfavourable DNA variants can lead to life-threatening side effects and/or suboptimal treatment. INTERPRETATION: There is a compelling need for prospective, randomized studies to establish the prognostic values of pharmacogenomic markers. With few exceptions the current knowledge is insufficient to include genotype analyses in routine planning of anticancer drug treatment. In most clinical situations, individual drug dosing according to body surface area in addition to therapeutic drug monitoring and close clinical surveillance is still the preferred approach to treat cancer patients.

Evaluation of sieving matrices used to separate alleles by cycling temperature capillary electrophoresis.

Denaturing CE (DCE) is a powerful tool for analysis of DNA variation. The development of commercial multi-CE instruments allows large-scale studies of DNA variation (many samples and many fragments). However, the cost of consumables like capillary arrays and sieving matrix might limit the use of DCE in such studies. Thus, we have tested 72 different in-house formulated sieving matrices' ability to suppress EOF and separate PCR-amplified alleles with the DCE variant, cycling temperature CE (CTCE). The data herein demonstrate that alleles can be baseline-separated by use of PVP and poly(N,N-dimethyl acrylamide) polymers at various percentages and pH. Allele separation by CTCE is matrix-independent and consequently applicable to any capillary instrument used for DNA separation. Formulation of sieving matrix for CTCE was done by dissolving appropriate amount of polymer powder into the running buffers. Allele separation was observed at different pH (7.5-8.5), concentrations and molecular size of the polymer, without compromising the separation and reproducibility. Finally, the cost reduction of homemade matrices is more than 1000-fold as compared to commercial sieving matrices.

[Tidsskriftet, peer review and medical publishing]. / Tidsskriftet, ekstern fagvurdering og medisinsk publisering.

The Journal of the Norwegian Medical Association is a peer review-based general medical journal in Norwegian. Peer review is a hallmark of scientific medical journals. The purpose of a peer review system is to evaluate and secure that only high-quality, up-dated and relevant articles are offered to readers. Peer review has been criticised as being subjective, time-consuming and hindering new hypotheses and thinking. During the last years, peer review has been evaluated scientifically. Increased attention is being given to editorial work and to scientific writing and reading.

Review of denaturant capillary electrophoresis in DNA variation analysis.

Analyses of germline and somatic single-nucleotide DNA variations are important in both population genetics research and clinical practice. Reliable and inexpensive methods that are flexible and designed for automation are required for these analyses. Present day DNA sequencing technology is too expensive for testing all 22-25 000 human genes in populations genetics studies or in scanning large numbers of tumors for novel mutations. Denaturant capillary electrophoresis (DCE) has the potential to meet the need for large-scale analysis of DNA variants. Several different analyses can be performed by DCE, including mutation analysis, single-nucleotide polymorphism (SNP) discovery in individual and pooled samples, detection of allelic imbalance, and determination of microhaplotypes. Here we review the theoretical background of the method, its sensitivity, specificity, detection limit, throughput, and repeatability in the light of current literature in the field.

DNA variants in the ATM gene are not associated with sporadic rectal cancer in a Norwegian population-based study.

BACKGROUND AND AIMS: A large number of DNA single-nucleotide polymorphisms (SNPs) have been discovered following the Human Genome Project. Several projects have been launched to find associations between SNPs and various disease cohorts. This study examined the possible association between the reported SNPs and sporadic rectal cancer. It has been proposed that SNPs in the ataxi-telangiectasia mutated (ATM) gene modulate the penetrance of some cancers. The investigated target sequence harbors three polymorphisms (IVS38-8 T/C in intron 38, 5557 G/A and 5558 A/T in exon 39), resulting in eight possible microhaplotypes at the DNA level. Furthermore, the two exonic SNPs are sited next to each other, allowing four possible amino acids in the same codon. METHODS: We report on a new method analyzing SNPs and microhaplotypes based on theoretical thermodynamics and migration of variant fragments by cycling temperature capillary electrophoresis. Fluorophore-labeled PCR products were analyzed without any post-PCR steps on a standard 96 capillary-sequencing instrument under denaturing conditions. RESULTS: More than 7000 alleles were microhaplotyped based on peak migration patterns of individual samples and sequencing results. The ATM polymorphisms and microhaplotypes examined did not significantly differ between sporadic rectal cancer and normal population. CONCLUSION: No associations were found between the IVS38-8 T/C, 5557 G/A and 5558 A/T polymorphisms and microhaplotypes in the ATM gene with respect to sporadic rectal cancer.

Cycling gradient capillary electrophoresis: a low-cost tool for high-throughput analysis of genetic variations.

In the present work, we introduce a new type of DNA variation detection. This method represents a transfer of melting gel technique onto multicapillary electrophoresis DNA sequencing instrument with further improvements to achieve maximum sample throughput while maintaining a high performance. The main improvement comes from application of cycling (revolving) temporal temperature gradient in place of a single-sweep gradient, commonly used in similar gel-based techniques. This improvement enables utilization of multiple-injection technique, in which multiple samples are injected into the same capillary (or sets of capillaries) separated by predefined time intervals of partial electrophoresis. The periodic oscillation of the temperature results in identical separation conditions of all samples injected in such series. Using this novel approach, we demonstrate a dramatic increase in separation throughput by turning a standard commercial 96-capillary array instrument into a semicontinuous flow mutation detection system capable to screen over 15 000 samples in 24 h of operation on a single 96-capillary commercial instrument. This represents a 10-fold increase in sample throughput over the current comparable technology.

Approach to analysis of single nucleotide polymorphisms by automated constant denaturant capillary electrophoresis.

Melting gel techniques have proven to be amenable and powerful tools in point mutation and single nucleotide polymorphism (SNP) analysis. With the introduction of commercially available capillary electrophoresis instruments, a partly automated platform for denaturant capillary electrophoresis with potential for routine screening of selected target sequences has been established. The aim of this article is to demonstrate the use of automated constant denaturant capillary electrophoresis (ACDCE) in single nucleotide polymorphism analysis of various target sequences. Optimal analysis conditions for different single nucleotide polymorphisms on ACDCE are evaluated with the Poland algorithm. Laboratory procedures include only PCR and electrophoresis. For direct genotyping of individual SNPs, the samples are analyzed with an internal standard and the alleles are identified by co-migration of sample and standard peaks. In conclusion, SNPs suitable for melting gel analysis based on theoretical thermodynamics were separated by ACDCE under appropriate conditions. With this instrumentation (ABI 310 Genetic Analyzer), 48 samples could be analyzed without any intervention. Several institutions have capillary instrumentation in-house, thus making this SNP analysis method accessible to large groups of researchers without any need for instrument modification.

Direct identification of all oncogenic mutants in KRAS exon 1 by cycling temperature capillary electrophoresis.

Over the past few decades, advances in genetics and molecular biology have revolutionized our understanding of cancer initiation and progression. Molecular progression models outlining genetic events have been developed for many solid tumors, including colon cancer. Previous reports in the literature have shown a relationship between different KRAS mutations and prognosis and response to medical treatment in colon cancer patients. Furthermore, the presence of a mutated KRAS has been correlated with different clinicopathological variables including age and gender of patients and tumor location. To our knowledge, few institutions screen for KRAS mutations on regular basis in colon cancer patients despite such evidence that knowledge of KRAS exon 1 status is informative. Here, we report on a mutation analysis method adapted to a 96-capillary electrophoresis instrument that allows identification of all 12 oncogenic mutations in KRAS exon 1 under denaturing conditions. To determine the optimal parameters, a series of DNA constructs generated by site-directed mutagenesis was analyzed and the migration times of all mutant peaks were measured. A classification tree was then made based on the differences in migration time between the mutants and an internal standard. A randomized series of 500 samples constructed with mutagenesis as well as 60 blind samples from sporadic colon carcinomas was analyzed to test the method. No wild-type samples were scored as mutants and all mutants were correctly identified. Post polymerase chain reaction (PCR) analysis time of 96 samples was performed within 40 min.

Effect of vaccination with mutant KRAS peptides on rat colon carcinogenesis induced by azoxymethane.

Rat colon carcinogenesis induced by 2 x 15 mg/kg body weight of azoxymethane (AOM) is a standard model, widely used to evaluate the role of nutritional components and chemopreventive agents at various stages of tumorigenesis. In this model, KRAS mutations have been frequently observed in aberrant crypt foci (ACF), putative preneoplastic lesions, as well as in tumours. Therefore we used this model and vaccinated F344 rats with a mixture of synthetic mutant KRAS peptides (MT KRAS) corresponding to frequent KRAS exon 1 mutations before AOM treatment in order to study the role of KRAS mutations in the development of ACF and subsequently tumours. The controls were sham-vaccinated with KRAS exon 1 wild-type KRAS peptides (WT KRAS). MT KRAS vaccination suppressed the number of ACF by 42% at week 13 (p=0.001). The subpopulation of ACF suppressed by MT KRAS vaccination had higher focal crypt multiplicity than the control ACF population (p=0.001). At week 26, vaccination reduced the KRAS mutation frequency in ACF from 50% in the MT KRAS group to 13% in the WT KRAS (p=0.038). However, at this phase of carcinogenesis, vaccination did not have significant effects on the ACF number and focal crypt multiplicity. Surprisingly, the KRAS mutation frequency was only 5% in the colonic tumours of the controls (1 out of 20 tumours). Although there were no tumours with KRAS mutations in the MT KRAS group, the possible effect of vaccination could not be evaluated. These data indicate that KRAS mutations play a minor role in colonic tumorigenesis and that ACF with KRAS mutations could hardly be the precursors of the AOM-induced tumours in rats. Hence, the cancer protective potential of a KRAS vaccine in the early phase of AOM-induced colon carcinogenesis in the rat appeared minuscule. Additional studies in a model with a high outcome of KRAS mutations in colonic tumours are needed to evaluate the effects of a KRAS vaccine at later stages of tumorigenesis.

Mutation detection in KRAS Exon 1 by constant denaturant capillary electrophoresis in 96 parallel capillaries.

Mutations in KRAS exon 1 oncogene are frequently found in colon carcinomas. A correlation between the mutated KRAS and the prognosis and outcome of treatment of colon cancer patients was reported in the literature. The object of our work was to establish a high-throughput method with high sensitivity to enable screening of tumor mutation status of KRAS exon 1 in large groups of colon cancer patients. KRAS exon 1 sequences from DNA isolated from 191 sporadic colon cancers were PCR amplified using one primer labeled with fluorescein and a second primer extended by a GC-clamp. After PCR amplification samples were subjected to automated 96-array constant denaturant capillary electrophoresis using a modified MegaBACE 1000 sequencing instrument. Mutant samples were identified by characteristic peak patterns. The sensitivity of detection of a mutant allele in a background of the wild-type alleles was 0.3%. Using the 96-array instrument a typical screening of 191 samples for KRAS mutation status could be performed within 2 h. A KRAS exon 1 mutation was found in 66 of 191 (34.6%) of the samples. The 96-array constant denaturant capillary electrophoresis provides an opportunity for the high-sensitivity screening of large cancer populations for KRAS exon 1 mutations.

Population screening of single-nucleotide polymorphisms exemplified by analysis of 8000 alleles.

The authors describe a method in which the population frequency of single-nucleotide polymorphisms (SNPs) can be efficiently detected and their allele frequencies accurately measured. Selected SNPs in TNFbeta, IL-4, and CTLA-4 were used to demonstrate the method. Blood from 4000 individuals was pooled, DNA was extracted, and target sequences were PCR amplified and analyzed by denaturant capillary electrophoresis. Alleles were separated into peaks based on melting properties of the double DNA helix. Frequencies of the different alleles were determined by calculating the area under the peaks. Allele frequencies and Hardy-Weinberg equilibrium estimated from the pooled data were verified by analyzing 7.5% of the samples randomly selected from the blood donor series. The method herein is equally suitable for single-samples and/or pooled-samples analysis of SNPs, in which sample treatment is kept to a minimum. The potential throughput of the method is beyond obtainable numbers of samples.