Analytical Validation of SOD2 Genotyping.

Manganese superoxide dismutase-2 (SOD2) plays a crucial role in cells' protection against mitochondrial oxidative damage. A genetic polymorphism in the mitochondrial targeting sequence of the SOD2 gene has been implicated in various diseases, including prostate cancer. Paller et al. have shown an increase in prostate-specific antigen (PSA) doubling time in patients with the Ala/Ala (wildtype) genotype when treated with pomegranate/grape extract antioxidants. We developed and validated a pyrosequencing assay that detects the common germline SOD2 SNP (rs_4880) with the aim of identifying men with castrate-resistant prostate cancer eligible for an antioxidant therapy clinical trial. We first selected 37 samples from the 1000 genomes study with known genotypes determined using Illumina-based sequencing and confirmed them by Sanger sequencing. In a blinded design, we then performed the new pyrosequencing assay on these samples and assigned genotypes. Genotypes for all 37 samples (13 homozygous Ala, 12 heterozygous Ala/Val, and 12 homozygous Val) were all concordant by pyrosequencing. The pyrosequencing assay has been live since May 2018 and has proven to be robust and accurate.

Val16A SOD2 Polymorphism Promotes Epithelial-Mesenchymal Transition Antagonized by Muscadine Grape Skin Extract in Prostate Cancer Cells.

Epithelial-mesenchymal transition (EMT), a key event in cancer metastasis, allows polarized epithelial cells to assume mesenchymal morphologies, enhancing invasiveness and migration, and can be induced by reactive oxygen species (ROS). Val16A (Ala) SOD2 polymorphism has been associated with increased prostate cancer (PCa) risk. We hypothesized that SOD2 Ala single nucleotide polymorphism (SNP) may promote EMT. We analyzed SOD2 expression and genotype in various prostate cell lines. Stable overexpression of Ala-SOD2 or Val-SOD2 allele was performed in Lymph Node Carcinoma of the Prostate (LNCaP) cells followed by analysis of intracellular ROS and EMT marker protein expression. Treatments were performed with muscadine grape skin extract (MSKE) antioxidant, with or without addition of H2O2 to provide further oxidative stress. Furthermore, MTS cell proliferation, cell migration, and apoptosis assays were completed. The results showed that SOD2 expression did not correlate with tumor aggressiveness nor SOD2 genotype. We demonstrated that the Ala-SOD2 allele was associated with marked induction of EMT indicated by higher Snail and vimentin, lower E-cadherin, and increased cell migration, when compared to Val-SOD2 allele or Neo control cells. Ala-SOD2 SNP cells exhibited increased levels of total ROS and superoxide and were more sensitive to co-treatment with H2O2 and MSKE, which led to reduced cell growth and increased apoptosis. Additionally, MSKE inhibited Ala-SOD2 SNP-mediated EMT. Our data indicates that treatment with a combination of H2O2-generative drugs, such as certain chemotherapeutics and antioxidants such as MSKE that targets superoxide, hold promising therapeutic potential to halt PCa progression in the future.

Successful gene therapy requires targeting the vast majority of cancer cells.

Suicide gene therapy using gene-directed enzyme prodrug therapy (GDEPT) is based on delivering a gene-encoded enzyme to cells that converts a nontoxic prodrug into its toxic metabolite. The bystander effect is thought to compensate for inefficiencies in delivery and expression because the produced toxic metabolite can spread to adjacent non-expressing cells.The purpose of this study was to assess the significance of bystander effect in GDEPT over the long term in vivo.We performed experiments using mixtures of yeast cytosine deaminase (yCD) expressing and empty vector (EV) containing cells. First, the bystander effect was assessed in various ratios of colon cancer cell lines RKO with yCD/EV in 2D and 3D culture. Next, tumors raised from RKO with yCD/EV in mice were treated with the prodrug 5-fluorocytosine (5-FC) for 42 days to assess bystander effect in vivo. Cell types constituting relapsed tumors were determined by 5-FC treatment and PCR.We were able to demonstrate bystander effect in both 2D and 3D. In mice, tumors initially regressed, but they all eventually recurred including those produced from 80% yCD expressing cells. Cells explanted from the recurrent tumors demonstrated that suicide gene expressing cells had been selected against during in vivo treatment with 5-FC.We conclude that gene therapy of malignant tumors in patients using the yCD/5-FC system will require targeting well over 80% of the malignant cells, and therefore will likely require improved bystander effect or repeated treatment.

Genomic characterization of malignant progression in neoplastic pancreatic cysts.

Intraductal papillary mucinous neoplasms (IPMNs) and mucinous cystic neoplasms (MCNs) are non-invasive neoplasms that are often observed in association with invasive pancreatic cancers, but their origins and evolutionary relationships are poorly understood. In this study, we analyze 148 samples from IPMNs, MCNs, and small associated invasive carcinomas from 18 patients using whole exome or targeted sequencing. Using evolutionary analyses, we establish that both IPMNs and MCNs are direct precursors to pancreatic cancer. Mutations in SMAD4 and TGFBR2 are frequently restricted to invasive carcinoma, while RNF43 alterations are largely in non-invasive lesions. Genomic analyses suggest an average window of over three years between the development of high-grade dysplasia and pancreatic cancer. Taken together, these data establish non-invasive IPMNs and MCNs as origins of invasive pancreatic cancer, identifying potential drivers of invasion, highlighting the complex clonal dynamics prior to malignant transformation, and providing opportunities for early detection and intervention.

Metanephric Adenoma-Epithelial Wilms Tumor Overlap Lesions: An Analysis of BRAF Status.

Metanephric adenoma (MA) has historically been considered to represent a differentiated form of epithelial Wilms tumor (WT), based in part upon cases that morphologically overlap these 2 neoplasms. More recently, BRAF V600E mutations have been demonstrated in the majority of MAs but not in unselected or even epithelial-predominant WTs, suggesting 2 genetically distinct entities. However, no prior study has examined BRAF status in neoplasms with overlapping histologic features of epithelial WT and MA. We studied a cohort of 11 such overlapping lesions, 2 of which we considered morphologically to be otherwise typical MAs with unusually prominent mitotic activity and 9 of which we classified as epithelial WTs with areas resembling MA. Both mitotically active MAs demonstrated the BRAF V600E mutation. While the majority (5/9) of epithelial WTs with areas resembling MA were negative for BRAF V600E mutation, 4 such cases were positive. Two BRAF V600E mutation-positive WTs occurred in children. One case in a 6-year-old male was morphologically similar to the BRAF V600E mutation-positive adult cases and subsequently metastasized to the lungs; remarkably, the metastases then completely resolved on Braf targeted therapy. A second occurred in a 3-year-old girl whose posttherapy nephrectomy specimen's tumor was encapsulated and mitotically active like a typical WT, but also had more differentiated areas resembling MA. Immunohistochemistry for Braf V600E paralleled the molecular findings, demonstrating immunoreactivity in both the WT and MA-like areas of all 4 of these neoplasms. In summary, we demonstrate that BRAF V600E mutations are not entirely restricted to typical MA, as they may be seen in MAs showing mitotic activity along with a subset of epithelial-predominant WTs in adults and children that have foci which overlap morphologically with MA.

Circulating Tumor DNA as a Clinical Test in Resected Pancreatic Cancer.

PURPOSE: In research settings, circulating tumor DNA (ctDNA) shows promise as a tumor-specific biomarker for pancreatic ductal adenocarcinoma (PDAC). This study aims to perform analytical and clinical validation of a KRAS ctDNA assay in a Clinical Laboratory Improvement Amendments (CLIA) and College of American Pathology-certified clinical laboratory. EXPERIMENTAL DESIGN: Digital-droplet PCR was used to detect the major PDAC-associated somatic KRAS mutations (G12D, G12V, G12R, and Q61H) in liquid biopsies. For clinical validation, 290 preoperative and longitudinal postoperative plasma samples were collected from 59 patients with PDAC. The utility of ctDNA status to predict PDAC recurrence during follow-up was assessed. RESULTS: ctDNA was detected preoperatively in 29 (49%) patients and was an independent predictor of decreased recurrence-free survival (RFS) and overall survival (OS). Patients who had neoadjuvant chemotherapy were less likely to have preoperative ctDNA than were chemo-naïve patients (21% vs. 69%; P < 0.001). ctDNA levels dropped significantly after tumor resection. Persistence of ctDNA in the immediate postoperative period was associated with a high rate of recurrence and poor median RFS (5 months). ctDNA detected during follow-up predicted clinical recurrence [sensitivity 90% (95% confidence interval (CI), 74%-98%), specificity 88% (95% CI, 62%-98%)] with a median lead time of 84 days (interquartile range, 25-146). Detection of ctDNA during postpancreatectomy follow-up was associated with a median OS of 17 months, while median OS was not yet reached at 30 months for patients without ctDNA (P = 0.011). CONCLUSIONS: Measurement of KRAS ctDNA in a CLIA laboratory setting can be used to predict recurrence and survival in patients with PDAC.

A New Fast Phasing Method Based On Haplotype Subtraction.

We developed a novel phasing approach, based solely on molecules and genotype frequency, that does not rely on inference of new alleles. We initiated the project because of errors that were detected in the phased 1000 Genomes Project data. The algorithm first combined identical genotypes into clusters and ranked them by descending frequency. Using alleles defined in homozygotes, it combined them to produce expected genotypes that were dismissed and subtracted them from remaining genotypes to define additional new putative alleles. Putative alleles had to be confirmed by identifying them in independent genotypes, and the process was iterated until all alleles were identified. The new approach was validated using single-molecule sequencing of eight loci, 145 (8 to 35 per locus) alleles were identified, and an average 98.2% (range, 95.0% to 99.9%) of 1000 genome individuals at these loci were explained. The accuracy of the new method was compared with that from PHASE and SHAPEIT2 to the experimentally determined genotypes based on single-molecule sequencing. Our method was comparable to PHASE and SHAPEIT2 in accuracy but was, on average, 14.6- and 10.8-fold faster, respectively.

Validation Strategy for Ultrasensitive Mutation Detection.

BACKGROUND: Ultrasensitive detection of low-abundance DNA point mutations is a challenging molecular biology problem, because nearly identical mutant and wild-type molecules exhibit crosstalk. Reliable ultrasensitive point mutation detection will facilitate early detection of cancer and therapeutic monitoring of cancer patients. OBJECTIVE: The objective of this study was to develop a method to correct errors in low-level cell line mixes. MATERIALS AND METHODS: We tested sample mixes with digital-droplet PCR (ddPCR) and next-generation sequencing. RESULTS: We introduced two corrections: baseline variant allele frequency (VAF) in the parental cell line was used to correct for copy number variation; and haplotype counting was used to correct errors in cell counting and pipetting. We found ddPCR to have better correlation for detecting low-level mutations without applying any correction (R2 = 0.80) and be more linear after introducing both corrections (R2 = 0.99). CONCLUSIONS: The VAF correction was found to be more significant than haplotype correction. It is imperative that various technologies be evaluated against each other and laboratories be provided with defined quality control samples for proficiency testing.

Haplotype Counting for Sensitive Chimerism Testing: Potential for Early Leukemia Relapse Detection.

Fields of forensics, transplantation, and paternity rely on human identity testing. Currently, this is accomplished through amplification of microsatellites followed by capillary electrophoresis. An alternative and theoretically better approach uses multiple single-nucleotide polymorphisms located within a small region of DNA, a method we initially developed using HLA-A and called haplotype counting. Herein, we validated seven additional polymorphic loci, sequenced a total of 45 individuals from three of the 1000 Genomes populations (15 from each), and determined the number of haplotypes, heterozygosity, and polymorphic information content for each locus. In addition, we developed a multiplex PCR that amplifies five of these loci simultaneously. Using this strategy with a small cohort of leukemic patients who underwent allogeneic bone marrow transplantation, we first attempted to define a threshold (0.26% recipient) by examining seven patients who tested all donor and did not relapse. Although this initial threshold will need to be confirmed in a larger cohort, we detected increased recipient DNA above this threshold 90 to 145 days earlier than microsatellite positivity, and 127 to 142 days before clinical relapse in four of eight patients (50%). Haplotype counting using these novel loci may be useful for ultrasensitive detection in fields such as bone marrow transplantation, solid organ transplant rejection, patient identification, and forensics.

Patients with McCune-Albright syndrome have a broad spectrum of abnormalities in the gastrointestinal tract and pancreas.

McCune-Albright Syndrome (MAS) is a rare sporadic syndrome caused by post-zygotic mutations in the GNAS oncogene, leading to constitutional mosaicism for these alterations. Somatic activating GNAS mutations also commonly occur in several gastrointestinal and pancreatic neoplasms, but the spectrum of abnormalities in these organs in patients with MAS has yet to be systematically described. We report comprehensive characterization of the upper gastrointestinal tract in seven patients with MAS and identify several different types of polyps, including gastric heterotopia/metaplasia (7/7), gastric hyperplastic polyps (5/7), fundic gland polyps (2/7), and a hamartomatous polyp (1/7). In addition, one patient had an unusual adenomatous lesion at the gastroesophageal junction with high-grade dysplasia. In the pancreas, all patients had endoscopic ultrasound findings suggestive of intraductal papillary mucinous neoplasm (IPMN), but only two patients met the criteria for surgical intervention. Both of these patients had IPMNs at resection, one with low-grade dysplasia and one with high-grade dysplasia. GNAS mutations were identified in the majority of lesions analyzed, including both IPMNs and the adenomatous lesion from the gastroesophageal junction. These studies suggest that there is a broad spectrum of abnormalities in the gastrointestinal tract and pancreas in patients with MAS and that patients with MAS should be evaluated for gastrointestinal pathology, some of which may warrant clinical intervention due to advanced dysplasia.

Morphology and genetics of pyloric gland adenomas in familial adenomatous polyposis.

AIMS: Gastric pyloric gland adenomas (PGAs) are rare epithelial polyps that are found more commonly in autoimmune atrophic gastritis and familial adenomatous polyposis (FAP). Little is known about the morphology and genetics of PGAs in FAP. PGAs in FAP are studied morphologically and genetically. Findings in FAP-associated PGAs are compared to sporadic PGAs and related lesions such as oxyntic gland adenoma (OGA) to increase our understanding of these rare polyps. METHODS AND RESULTS: Seven PGAs and 18 fundic gland polyps (FGPs) from FAP patients were collected. KRAS and GNAS mutations were determined in six PGAs and 18 FGPs. Immunohistochemistry was applied on five PGAs to provide further confirmation of the histological subtypes and genetic alterations. Morphology of all PGAs was studied and compared to literature on sporadic PGAs and related lesions. All successfully sequenced PGAs (six of six) carried GNAS mutations and half of the successfully sequenced PGAs carried a KRAS mutation (three of six). Nuclear ß-catenin was seen only in one PGA with focal high-grade dysplasia. Morphologically, PGAs in FAP showed overlapping features with OGA. CONCLUSION: Familial adenomatous polyposis-associated PGAs have a similar genetic background as sporadic PGAs, i.e. KRAS and GNAS mutation. Based on morphological findings in FAP associated PGAs, it is hypothesized that PGAs and OGAs are closely related lesions.

KRAS and guanine nucleotide-binding protein mutations in pancreatic juice collected from the duodenum of patients at high risk for neoplasia undergoing endoscopic ultrasound.

BACKGROUND & AIMS: Pancreatic imaging can identify neoplastic cysts but not microscopic neoplasms. Mutation analysis of pancreatic fluid after secretin stimulation might identify microscopic neoplasias in the pancreatic duct system. We determined the prevalence of mutations in KRAS and guanine nucleotide-binding protein α-stimulating genes in pancreatic juice from subjects undergoing endoscopic ultrasound for suspected pancreatic intraepithelial neoplasia, intraductal papillary mucinous neoplasms, or pancreatic adenocarcinoma. METHODS: Secretin-stimulated juice samples were collected from the duodenum of 272 subjects enrolled in Cancer of the Pancreas Screening studies; 194 subjects were screened because of a family history of, or genetic predisposition to, pancreatic cancer, and 78 subjects were evaluated for pancreatic cancer (n = 30) or other disorders (controls: pancreatic cysts, pancreatitis, or normal pancreata, n = 48). Mutations were detected by digital high-resolution melt-curve analysis and pyrosequencing. The number of replicates containing a mutation determined the mutation score. RESULTS: KRAS mutations were detected in pancreatic juice from larger percentages of subjects with pancreatic cancer (73%) or undergoing cancer screening (50%) than controls (19%) (P = .0005). A greater proportion of patients with pancreatic cancer had at least 1 KRAS mutation detected 3 or more times (47%) than screened subjects (21%) or controls (6%, P = .002). Among screened subjects, mutations in KRAS (but not guanine nucleotide-binding protein α-stimulating) were found in similar percentages of patients with or without pancreatic cysts. However, a greater proportion of patients older than age 50 years had KRAS mutations (54.6%) than younger patients (36.3%) (P = .032); the older subjects also had more mutations in KRAS (P = .02). CONCLUSIONS: Mutations in KRAS are detected in pancreatic juice from the duodenum of 73% of patients with pancreatic cancer, and 50% of asymptomatic individuals with a high risk for pancreatic cancer. However, KRAS mutations were detected in pancreatic juice from 19% of controls. Mutations detected in individuals without pancreatic abnormalities, based on imaging analyses, likely arise from small pancreatic intraepithelial neoplasia lesions. ClinicalTrials.gov no: NCT00438906 and NCT00714701.

Haplotype counting by next-generation sequencing for ultrasensitive human DNA detection.

Human identity testing is critical to the fields of forensics, paternity, and hematopoietic stem cell transplantation. Most bone marrow (BM) engraftment testing currently uses microsatellites or short tandem repeats that are resolved by capillary electrophoresis. Single-nucleotide polymorphisms (SNPs) are theoretically a better choice among polymorphic DNA; however, ultrasensitive detection of SNPs using next-generation sequencing is currently not possible because of its inherently high error rate. We circumvent this problem by analyzing blocks of closely spaced SNPs, or haplotypes. As proof-of-principle, we chose the HLA-A locus because it is highly polymorphic and is already genotyped to select proper donors for BM transplant recipients. We aligned common HLA-A alleles and identified a region containing 18 closely spaced SNPs, flanked by nonpolymorphic DNA for primer placement. Analysis of cell line mixtures shows that the assay is accurate and precise, and has a lower limit of detection of approximately 0.01%. The BM from a series of hematopoietic stem cell transplantation patients who tested as all donor by short tandem repeat analysis demonstrated 0% to 1.5% patient DNA. Comprehensive analysis of the human genome using the 1000 Genomes database identified many additional loci that could be used for this purpose. This assay may prove useful to identify hematopoietic stem cell transplantation patients destined to relapse, microchimerism associated with solid organ transplantation, forensic applications, and possibly patient identification.

False positives in multiplex PCR-based next-generation sequencing have unique signatures.

Next-generation sequencing shows great promise by allowing rapid mutational analysis of multiple genes in human cancers. Recently, we implemented the multiplex PCR-based Ion AmpliSeq Cancer Hotspot Panel (>200 amplicons in 50 genes) to evaluate EGFR, KRAS, and BRAF in lung and colorectal adenocarcinomas. In 10% of samples, automated analysis identified a novel G873R substitution mutation in EGFR. By examining reads individually, we found this mutation in >5% of reads in 50 of 291 samples and also found similar events in 18 additional amplicons. These apparent mutations are present only in short reads and within 10 bases of either end of the read. We therefore hypothesized that these were from panel primers promiscuously binding to nearly complementary sequences of nontargeted amplicons. Sequences around the mutations matched primer binding sites in the panel in 18 of 19 cases, thus likely corresponding to panel primers. Furthermore, because most primers did not show this effect, we demonstrated that next-generation sequencing may be used to better design multiplex PCR primers through iterative elimination of offending primers to minimize mispriming. Our results indicate the need for careful sequence analysis to avoid false-positive mutations that can arise in multiplex PCR panels. The AmpliSeq Cancer panel is a valuable tool for clinical diagnostics, provided awareness of potential artifacts.

Clinical validation of KRAS, BRAF, and EGFR mutation detection using next-generation sequencing.

OBJECTIVES: To validate next-generation sequencing (NGS) technology for clinical diagnosis and to determine appropriate read depth. METHODS: We validated the KRAS, BRAF, and EGFR genes within the Ion AmpliSeq Cancer Hotspot Panel using the Ion Torrent Personal Genome Machine (Life Technologies, Carlsbad, CA). RESULTS: We developed a statistical model to determine the read depth needed for a given percent tumor cellularity and number of functional genomes. Bottlenecking can result from too few input genomes. By using 16 formalin-fixed, paraffin-embedded (FFPE) cancer-free specimens and 118 cancer specimens with known mutation status, we validated the six traditional analytic performance characteristics recommended by the Next-Generation Sequencing: Standardization of Clinical Testing Working Group. Baseline noise is consistent with spontaneous and FFPE-induced C:G→T:A deamination mutations. CONCLUSIONS: Redundant bioinformatic pipelines are essential, since a single analysis pipeline gave false-negative and false-positive results. NGS is sufficiently robust for the clinical detection of gene mutations, with attention to potential artifacts.

Clinicopathological correlates of activating GNAS mutations in intraductal papillary mucinous neoplasm (IPMN) of the pancreas.

BACKGROUND: Intraductal papillary mucinous neoplasms (IPMNs) are the most common cystic precursor lesions of invasive pancreatic cancer. The recent identification of activating GNAS mutations at codon 201 in IPMNs is a promising target for early detection and therapy. The purpose of this study was to explore clinicopathological correlates of GNAS mutational status in resected IPMNs. METHODS: Clinical and pathologic characteristics were retrieved on 54 patients in whom GNAS codon 201 mutational status was previously reported ("historical group", Wu et al. Sci Transl Med 3:92ra66, 2011). In addition, a separate cohort of 32 patients (validation group) was included. After microdissection and DNA extraction, GNAS status was determined in the validation group by pyrosequencing. RESULTS: GNAS activating mutations were found in 64% of the 32 IPMNs included in the validation group, compared with a previously reported prevalence of 57% in the historical group. Overall, 52 of 86 (61%) of IPMNs demonstrated GNAS mutations in the two studies combined. Analysis of both groups confirmed that demographic characteristics, tumor location, ductal system involvement, focality, size, grade of dysplasia, presence of an associated cancer, and overall survival were not correlated with GNAS mutational status. Stratified by histological subtype, 100% of intestinal type IPMNs demonstrated GNAS mutations compared to 51% of gastric IPMN, 71% of pancreatobiliary IPMNs, and 0% of oncocytic IPMNs. CONCLUSIONS: GNAS activating mutations can be reliably detected in IPMNs by pyrosequencing. In terms of clinicopathological parameters, only histological subtype was correlated with mutational frequency, with the intestinal phenotype always associated with GNAS mutations.

GNAS codon 201 mutations are uncommon in intraductal papillary neoplasms of the bile duct.

BACKGROUND: Activating point mutations of GNAS at codon 201 have been detected in approximately two thirds of intraductal papillary mucinous neoplasms (IPMNs) of the pancreas. Intraductal papillary neoplasms of the bile ducts (IPNBs) morphologically resemble pancreatic IPMNs. This study sought to assess the mutational status of GNAS at codon 201 in IPNBs. METHODS: Thirty-four patients were included. DNA from microdissected IPNBs was subjected to a polymerase chain reaction and ligation method for the detection of GNAS mutations at codon 201 and of KRAS mutations at codon 12. Mutational status was compared with clinical and pathologic data. RESULTS: The IPNBs had a median diameter of 3.5 cm and were located intrahepatically (n= 6), extrahepatically (n= 13), both intra- and extrahepatically (n= 4) or in the gallbladder (intracystic papillary neoplasms, n= 11). Most exhibited pancreatobiliary differentiation (n= 20), high-grade dysplasia (n= 26) and an associated adenocarcinoma (n= 20). Analysis of GNAS codon 201 identified only one mutant sample in a multifocal intestinal subtype intrahepatic IPNB with high-grade dysplasia. Six lesions harboured a KRAS codon 12 mutation. CONCLUSIONS: GNAS codon 201 mutations are uncommon in IPNBs, by contrast with pancreatic IPMNs. More comprehensive molecular profiling is needed to uncover the pathways involved in IPNB development.

A virtual pyrogram generator to resolve complex pyrosequencing results.

We report a freely available software program, Pyromaker, which generates simulated traces for pyrosequencing results based on user inputs. Simulated pyrograms can aid in the analysis of complex pyrosequencing results in which various hypothesized mutations can be tested, and the resultant pyrograms can be matched with the actual pyrogram. We validated the software using the actual pyrograms for common KRAS gene mutations as well as several mutations in the BRAF, GNAS, and p53 genes. We demonstrate that all 18 possible single-base mutations within codons 12 and 13 of KRAS generate unique pyrosequencing traces and highlight the distinctions between them. We further show that all reported codon 12 and 13 complex mutations produce unique pyrograms. However, some complex mutations are indistinguishable from single-base mutations. For complicated pyrograms, Pyromaker was used in two modes, one in which hypothesis-based simulated pyrograms were pattern-matched with the actual pyrograms. In a second strategy with only the pyrogram, Pyromaker was used to identify the underlying mutation by iteratively reconstructing the mutant pyrogram. Either strategy was able to successfully identify the complex mutations, which were confirmed by cloning and sequencing. Using two examples of KRAS codon 12 mutations (specifically GGT→TTT, G12F and GGT→GAG, G12E), we report which combinations of five approaches permit unambiguous mutation identification. The most efficient approach was found to be pyrosequencing with Pyromaker.