DNA signature and occult blood in stool led to diagnosis of an early-onset metastatic rectal cancer in a young pregnant woman: a case report.

Background: Colorectal cancer (CRC) is the third most common form of cancer worldwide in terms of incidence and the second in terms of mortality with 1.9 million new cases and 930,000 deaths reported in 2020. Corresponding numbers in the U.S. are 150,000 and 53,000, respectively. Although the majority of CRCs in the U.S. and other high-income countries are in adults aged 50 and older, there has recently been a considerable rise in early-onset CRC, so that 17,930 cases in the U.S. (12% of total cases) are diagnosed in individuals younger than age 50, representing the equivalent of 49 new cases per day. Early diagnosis is essential to improve the prognosis and reduce the number of cancer-related deaths. Here we report the case of a young pregnant woman, who was diagnosed with CRC with the help of the ColoAlert™ multitargeted stool test. Case Description: In this case study, a young pregnant woman presented with obstipation, rectal bleeding, and pelvic pain, symptoms that were ascribed to her pregnancy. On her own, she performed a multitarget stool test (ColoAlert™) that showed occult blood as well as a very high level of human DNA, both known to be associated with presence of CRC. After testing, she was referred for rectoscopy (during her 21st week of pregnancy), which showed an exophytic, semicircular tumor 10 cm from anus in the rectosigmoid junction. Histology confirmed adenocarcinoma in rectum. Further examination showed perirectal infiltration as well as metastases to both liver and adrenal gland. Conclusions: This case report shows the importance of considering CRC as a possible diagnosis in young people. It also demonstrates the usefulness of multitarget stool testing that in this case led to the endoscopic confirmation of the diagnosis followed by an immediate start of potential life-saving treatment.

DNA Marker in Stool Led to a Second High-Quality Colonoscopy Within Three Months and Removal of an Undetected High-Risk Polyp.

BACKGROUND: In this case study, the patient first had a colonoscopy based on an incidental episode of vomiting and abdominal pain. MATERIALS AND METHODS: Two months after recovery, a multitarget stool test (ColoAlert®) was performed and showed a known somatic mutation in the oncogene KRAS, reported to be associated with colorectal cancer. As a result, a second complete colonoscopy was performed at another center. RESULTS: This procedure led to the diagnosis and removal of a later classified high-risk polyp that had been missed during the initial colonoscopy. CONCLUSIONS: This case report shows the use of genetic markers in stool testing has the potential to detect colon cancer in very early stages when treatment is inexpensive and effective.

Development and validation of a rapid and reliable real-time PCR method for CYP3A5 genotyping.

BACKGROUND: A prominent example for inter-individual differences in drug-metabolizing enzymes is the cytochrome P450 family. These monooxygenases comprise enzymes responsible for metabolism of about 90% of common medications. CYP3A5 and CYP3A4 account for 50% of hepatic cytochrome P450 and conversion of about half of all their substrates. CYP3A5 is the predominant extra-hepatic CYP enzyme and shows varying inter-individual expression attributable to genetic variations in the corresponding gene. CYP3A5*2 and *3 are the most common among Caucasian populations. Among CYP3A5 substrates are cyclosporine and tacrolimus prescribed after organ transplantations. A high incidence of nephrotoxic events after administering these drugs is related to low expression of the CYP3A5 enzyme. A fast and reliable genotyping method for the CYP3A5 gene would help avoid unwanted adverse drug reactions. METHODS: Blood samples from 143 Caucasian subjects were genotyped by means of a real-time PCR multiplex assay testing the two CYP3A5 variations, CYP3A5S*2 and *3. This assay was validated against RFLP-PCR. RESULTS: Both mutations examined could be found in the study. No sample was homozygous for CYP3A5*2, but 2 out of 143 showed heterozygosity (allele frequency: 0.7%). For the CYP3A5*3 variant 17 samples were heterozygous and 115 were homozygous (allele frequency 86.4%). The multiplex real-time PCR yields shorter hands-on time and reduced cost compared to RFLP-PCR. CONCLUSIONS: Establishment of a multiplex real-time PCR has been successful as could be proven by correctly identifying the desired mutations CYP3A5*2 and CYP3A5*3 against a standard reference method.

Pharmacogenetic prediction of individual variability in drug response based on CYP2D6, CYP2C9 and CYP2C19 genetic polymorphisms.

Interindividual variability in drug response depends on a number of genetic and environmental factors. The metabolic enzymes are well known for their contribution to this variability due to drug-drug interactions and genetic polymorphisms. The phase I drug metabolism is highly dependent upon the cytochrome P450 mono-oxygenases (CYP) and their genetic polymorphism leads to the variable internal drug exposures. The highly polymorphic CYP2C9, CYP2C19 and CYP2D6 isozymes are responsible for metabolizing a large portion of routinely prescribed drugs and contribute significantly to adverse drug reactions and therapeutic failures. In this review, two attractive and easily implementable approaches are highlighted to recommend drug doses ensuring similar internal exposures in the face of these polymorphisms. The first approach relies on subpopulation-based dose recommendations that consider the original population dose as an average of the doses recommended in genetically polymorphic subpopulations. By using bioequivalence principles and assuming linear gene-dose effect, dose recommendations can be made for different metabolic phenotypes. The second approach relates area under the curve to two characteristic parameters; the contribution ratio (CR), computes for the contribution of the metabolic enzyme and the fractional activity (FA), considers the impact of the genetic polymorphism. This approach provides valid and error free internal drug exposure predictions and can take into consideration genetic polymorphisms and drug interactions and/ or both simultaneously. Despite certain advantages and limitations, both approaches provide a good initial frame-work for devising models to predict internal exposure and individualize drug therapy, one of the promises from human genome project.

Development and validation of a rapid and reliable method for TPMT genotyping using real-time PCR.

BACKGROUND: It is widely accepted that many medications exhibit inter-individual variability in their efficacy and toxicity due to polymorphisms in genes encoding drug-metabolising enzymes. One of the most often cited examples in this context is thiopurine S-methyltransferase (TPMT) polymorphism. TPMT is a phase 2 detoxification enzyme that catalyzes the S-methylation of thiopurine drugs such as thioguanine and 6-mercaptopurine. Approximately 11% of the Caucasian population carry a heterozygous deficiency of this enzyme causing intermediate enzyme activity, whereas 0.3% show a homozygous deficiency. In both cases, severe myelosuppression can develop upon treatment with thiopurines. These are commonly used in the treatment of leukemia. Therefore, genotyping of patients before treatment is absolutely necessary. Development of a fast and reliable real-time PCR application for TPMT genotyping would greatly improve thiopurine treatment regimens and allow the avoidance of adverse drug reactions. METHODS: Blood was obtained from a Caucasian cohort of 143 individuals. After extraction of DNA, all samples were genotyped for TPMT polymorphisms *2, *3A, *3B, and *3C by real-time PCR as well as by PCR-RFLP as the reference method, in order to validate the new method. RESULTS: Four different genotypes were found in the population studied. Of the 143 individuals investigated, 1 was heterozygous for TPMT*2 (0.70%), 2 were heterozygous for TPMT*3B (1.40%), and 8 heterozygous for TPMT-*3C (5.60%). No homozygous genotype could be identified. In total, 7.7% of the individuals carried mutations. Results from the newly developed real-time PCR were 100% concordant with those obtained using standard PCR-RFLP analysis, leading to 100% sensitivity and specificity. The hands-on time is approximately one third of the time needed for standard PCR-RFLP methods. CONCLUSIONS: A new high-throughput genotyping method could be successfully established and optimised for the commonly found mutant alleles TPMT*2 (G238C), TPMT*3A (G460A and A719G), TPMT*3B (G460A), and TPMT*3C (A719G) via real-time PCR on the LightCycler (Roche) instrument and using the standard PCR-RFLP as reference method.

Development of a high throughput single nucleotide polymorphism screening method for the cytochrome P450 1A2 polymorphisms CYP1A2*1C and CYP1A2*1F: are they useful as predictive markers in mental disorders?

BACKGROUND: The cytochrome P450 1A2 (CYP1A2) gene encodes one of the most important enzymes of the Phase I drug metabolism, which is involved in the metabolism of many lipophilic xenobiotics, such as haloperidol, theophylline, phenacetine, and others. The recently discovered single nucleotide polymorphisms CYP1A2*1C (-3860G-->A) in the 5' flanking region of the gene and CYP1A2*1F (-163C-->A) in intron 1 seem to interfere with the expression rate or catalytic function of the enzyme. Polymorphism carriers may either have a risk of reduced drug degradation and side effects, or may present with an increased induction of enzymatic activity resulting in clinical non-response to the prescribed therapy. We investigated two populations, a mental disease group and a healthy control group, to identify whether these two genetic variants are correlated with the general development of a mental disorder and if they could potentially be used as predictive markers for manifestation of the same. METHODS: Using specifically designed primers, we established a high-throughput multiplex screening realtime PCR method for the two polymorphisms on the CYP1A2 gene with the Roche LightCycler instrument. RESULTS: We analysed the two cohorts to identify whether one of the two described genetic variants may be associated with the manifestation of a mental disorder in general. For the CYP1A2*1C variant, we identified an allele frequency of 1.7% for both cohorts. For the CYP1A*1F polymorphism, we found an allele frequency of 74.5% for the mental disease group and 68.6% for the healthy control group. CONCLUSIONS: This new diagnostic method of multiplex detection may be helpful to routinely identify carriers of CYP1A2 variants and to improve the therapeutic effectiveness by selection of the most appropriate therapeutic regimen. As a result of this pilot study, there appeared to be no significant correlation between the existence of one of the investigated genetic variants and the development of a mental disorder.

Development and validation of a high-throughput screening method for two polymorphisms in the serotonin transporter gene.

BACKGROUND AND AIM: The human serotonin (5-hydroxytryptamine) transporter, encoded by the SLC6A4 gene on chromosome 17q11.1-q12, is the cellular reuptake site for serotonin and a site of action for several drugs with central nervous system effects, including both therapeutic agents (e.g. antidepressants) and drugs of abuse (e.g. cocaine). It is known that the serotonin transporter plays an important role in the metabolic cycle of a broad range of antidepressants, antipsychotics, anxiolytics, antiemetics, and antimigraine drugs. The identification and characterization of variations that increase the response to common medications is a challenging and increasingly important task with regard to prediction of drug response. Therefore, the aim of this study was to establish a high-throughput single nucleotide polymorphism (SNP) screening method for two polymorphisms in the serotonin transporter gene, focusing on the SLC6A4 variations rs140701 and rs2066713. METHODS: We developed a classical restriction fragment length polymorphism (RFLP) PCR protocol as a reference, followed by a new protocol established for the LightCycler real-time PCR method. To validate the method, the allele frequencies in 169 individuals (112 women, 57 men) were determined and compared with published data. The population was divided into two groups: one group comprised 87 individuals with various mental disorders and the other consisted of 82 healthy persons. RESULTS: No difference was found in the prevalence of the two SNPs between the two populations. Subsequently, the determined allele frequencies were compared with previously published data. We found that 68% of the whole study population (groups I and II) carried a mutated allele of the rs140701 variation. With regard to the rs2066713 polymorphism, we found an allele frequency of 61% in the population. Both results are consistent with published data. CONCLUSION: The developed protocol for RT-PCR analysis of both variations turned out to be reliable and economical, and thus suitable for routine laboratory use.