SP174 Antibody Lacks Specificity for NRAS Q61R and Cross-Reacts With HRAS and KRAS Q61R Mutant Proteins in Malignant Melanoma.

HRAS, KRAS, and NRAS, highly homologous proteins, are often mutationally activated in cancer. Usually, mutations cluster in codons 12, 13, and 61 and are detected by molecular genetic testing of tumor DNA. Recently, immunohistochemistry with SP174 antibody has been introduced to detect NRAS Q61R-mutant protein. Studies on malignant melanomas showed that such an approach could be a viable alternative to molecular genetic testing. This investigation was undertaken to evaluate the value of SP174 immunohistochemistry for detection of NRAS Q61R-mutant isoform. Two hundred ninety-two malignant melanomas were evaluated using Leica Bond-Max automated immunostainer. Twenty-nine tumors (10%) showed positive immunoreactivity. NRAS codon 61 was polymerase chain reaction amplified and sequenced in 24 positive and 92 negative cases using Sanger sequencing, quantitative polymerase chain reaction, and next-generation sequencing approaches. A c.182A>G substitution leading to NRAS Q61R mutation was identified in 22 tumors. Two NRAS wild-type tumors revealed c.182A>G substitutions in HRAS and KRAS codon 61, respectively. Both mutations were detected by next-generation sequencing and independently confirmed by Sanger sequencing. None of 85 NRAS codon 61 wild-type tumors and 7 NRAS mutants other than Q61R showed immunoreactivity with SP174 antibody. Thus, SP174 antibody was 100% sensitive in detecting NRAS Q61R-mutant isoform in malignant melanoma, but not fully specific as it cross-reacted with HRAS and KRAS Q61R-mutant proteins. Therefore, molecular testing is needed to determine which RAS gene is mutated. The rarity of HRAS and KRAS Q61R mutants in malignant melanoma let previous investigations erroneously conclude that SP174 is specific for NRAS Q61R-mutant protein.

Evaluation of molecular diagnostic approaches for the detection of BRAF p.V600E mutations in papillary thyroid cancer: Clinical implications.

Differentiated papillary thyroid cancer (PTC) is the most common cancer of the endocrine system. PTC has a very good prognosis and a high 5 year survival rate; however, some patients are unresponsive to treatment, and their diagnosis eventually results in death. Recent efforts have focused on searching for prognostic and predictive factors that may enable treatment personalization and monitoring across the course of the disease. The presence of the BRAF mutation is considered to contribute to the risk of poor clinical course, according to American Thyroid Association (ATA) recommendations. The method used for genotyping can impact the predicted mutation frequency; however, ATA recommendations do not address this issue. We evaluated the molecular diagnostic (BRAF p.V600E mutation) results of 410 patients treated for PTC. We thoroughly analyzed the impact of three different BRAF mutation detection methods, Sanger Sequencing (Seq), allele-specific amplification PCR (ASA-PCR), and quantitative PCR (qPCR), on the frequency of mutation detection in 399 patients. Using Seq, we detected the BRAF mutation in 37% of patients; however, we were able to detect BRAF mutations in 57% and 60% of patients using the more sensitive ASA-PCR and qPCR technologies, respectively. Differences between methods were particularly marked in the thyroid papillary microcarcinoma group; BRAF p.V600E mutations were found in 37% of patients using Seq and 63% and 66% of patients using ASA-PCR and qPCR, respectively. We also evaluated how these different diagnostic methods were impacted by DNA quality. Applying methods with different sensitivities to the detection of BRAF p.V600E mutations may result in different results for the same patient; such data can influence stratification of patients into different risk groups, leading to alteration of treatment and follow-up schemes.

SP174, NRAS Q61R Mutant-Specific Antibody, Cross-Reacts With KRAS Q61R Mutant Protein in Colorectal Carcinoma.

CONTEXT: - NRAS is a member of the RAS family oncoproteins implicated in cancer. Gain-of-function NRAS mutations were reported in a subset of colorectal cancers. These mutations occur at codons 12, 13, and 61 and are detected by molecular genetic testing. Recently, an antibody (clone SP174) became available to immunohistochemically pinpoint NRAS Q61R mutant protein. In malignant melanoma, NRAS Q61R mutant-specific immunohistochemistry was shown to be a valuable supplement to traditional genetic testing. OBJECTIVE: - To evaluate the significance of NRAS Q61R mutant-specific immunohistochemistry in a cohort of colorectal carcinomas. DESIGN: - A total of 1185 colorectal carcinomas were immunohistochemically evaluated with SP174 antibody. NRAS Q61R mutant-specific immunohistochemistry was validated by molecular genetic testing including Sanger sequencing, quantitative polymerase chain reaction (qPCR), and next-generation sequencing. RESULTS: - Twelve tumors showed strong SP174 immunoreactivity. Sanger sequencing detected an identical c.182A>G substitution, causing NRAS Q61R mutation at the protein level, only in 8 SP174-positive cases. These results were confirmed by qPCR study. Subsequently, NRAS wild-type tumors with strong SP174 staining were evaluated by next-generation sequencing and revealed KRAS c.182A>G substitutions predicted to cause KRAS Q61R mutation. Review of colorectal carcinomas with known KRAS and NRAS genotype revealed that none of 62 wild-type tumors or 47 mutants other than Q61R were SP174 positive. CONCLUSION: - SP174 immunohistochemistry allows sensitive detection of NRAS and KRAS Q61R mutants. However, molecular genetic testing is necessary to determine specifically which RAS gene is mutated.

Increase in Papillary Thyroid Cancer Incidence Is Accompanied by Changes in the Frequency of the BRAF V600E Mutation: A Single-Institution Study.

BACKGROUND: Thyroid cancer (TC) has one of the fastest increasing incidences worldwide and primarily involves papillary thyroid cancer (PTC). The BRAF(V600E) mutation is the most common genetic alteration identified in PTC. There are few data concerning an association between the rising incidence of PTC and the increasing prevalence of BRAF-positive cases. Environmental factors such as iodine intake may be responsible for the changing molecular features of PTC. The aim of this study was to evaluate probable variations in the frequency of the BRAF(V600E) mutation in PTC that were diagnosed at a single institution over 14 years in Poland, a country with a demonstrated improvement in iodine supplementation in the early 21st century. METHODS: Time-dependent trends in the prevalence of the BRAF(V600E) mutation during three time periods (2000-2004, 2005-2009, and 2010-2013) were analyzed. The BRAF mutation was genotyped using direct sequencing, allele-specific polymerase chain reaction (PCR), and real-time PCR in 723 unselected cases of PTC that were diagnosed in 2000-2013. Trends in the clinicopathologic characteristics of all PTCs and BRAF(V600E)-positive PTCs were also analyzed. RESULTS: The proportion of PTCs with mutations significantly increased over the study period (54.8% vs. 70.6%; p = 0.001). The median tumor size of all and BRAF-positive tumors decreased (p = 0.008 and p = 0.001, respectively) and correlated with an increase in the proportion of all and mutated microcarcinomas (p = 0.003 and p = 0.003, respectively). A decrease in all and mutated tumors between 2 and 4 cm was also observed (p = 0.002 and p = 0.006, respectively). A significant decrease in tumors ≥ 4 cm in size was only observed in BRAF-positive cases (p = 0.017). The proportion of classic PTC with BRAF(V600E) mutation was observed to increase (57.6% vs. 74.4%; p = 0.001) and was stable for the follicular variant of PTC (p = 0.336). CONCLUSIONS: The prevalence of the BRAF(V600E)mutation increased significantly in PTCs diagnosed in the authors' institution. Improved detection and several causative factors, most likely environmental and changes in iodine intake, may contribute to the increasing occurrence of TC.

Genetic mutations in SPINK1, CFTR, CTRC genes in acute pancreatitis.

BACKGROUND: Explanation of the ultimate causes of acute and chronic pancreatitis is challenging. Hence, it is necessary to seek various etiological factors, including genetic mutations that may be of importance in triggering recurrence and progression of acute to chronic pancreatitis. The aim of this study was to determine the frequency of genetic mutations in patients with acute pancreatitis and to investigate their relationship with the etiology and clinical course. METHODS: The study included 221 patients treated for acute pancreatitis and 345 healthy subjects as a control group. Peripheral blood samples were collected from each study participant and genomic DNA was isolated. Genotyping of common mutations in the SPINK1 (p.N34S and p.P55S) and CTRC (p.I259V, p.V235I, p.K247_R254del, p.E225A) genes was performed using the high-resolution melting method. Mutations in the CFTR p.F508del (delF508_CTT) were genotyped using allele-specific amplification polymerase chain reaction. All detected mutations were confirmed with direct capillary DNA sequencing. RESULTS: Mutations in SPINK 1, CFTR and CTRC were detected in 6.3%, 2.3% and 1.8% of patients with acute pancreatitis versus 3.2%, 3.8% and 1.2% of volunteers in the control group. No relationship was found between the detected mutations and severity of acute pancreatitis: mild acute pancreatitis, mutation of CFTR in 4 (2.8%) and CTRC in 2 (1.4%) patients; severe acute pancreatitis, mutation of CFTR and CTRC in 1 (2.6%) case each. The SPINK1 mutation was significantly more frequent in 8 (10.4%) severe cases than in 6 (4.2%) mild cases (P < 0.05), and was observed in 5/70 (7.1%) patients with alcohol-related AP, 5/81 (6.2%) with biliary AP, and 4/63 (6.3%) in those without any established cause of the disease. CONCLUSIONS: Mutation p.N34S in SPINK1 may predispose patients to acute pancreatitis, especially in those abusing alcohol, and may promote a more severe course of the disease.

The BRAF(V600E) mutation in papillary thyroid microcarcinoma: does the mutation have an impact on clinical outcome?

CONTEXT: An activating mutation in the gene BRAF has been correlated with poorer prognosis and more aggressive clinical course in papillary thyroid carcinoma (PTC). We therefore hypothesized that the good prognosis, high 5-year disease-free rate and high survival rate of patients with less aggressive papillary thyroid microcarcinoma (pT1aNo-x) would be associated with a lower incidence of the BRAF(V600E) mutation. OBJECTIVES: To evaluate the frequency of the activating mutation BRAF(V600E) in low-risk papillary thyroid microcarcinoma (pT1aNo-x at the moment of diagnosis) and the association of the mutation with the clinical outcome in a retrospective analysis. STUDY DESIGN: BRAF(V600E) was characterized in 113 PTC patients diagnosed with pT1aNo-x (one PTC focus with a diameter <1 cm, without lymph node or distant metastases according to IUCC/AJCC TNM staging system 2010). Genotyping was performed on DNA extracted from thyroid tumour tissue using direct capillary sequencing, and allele-specific amplification PCR was used to resolve equivocal results. Retrospective analysis of the clinical course of PTC was then correlated with BRAF status in the primary tumour tissue. RESULTS: The BRAF(V600E) mutation was detected in 78 of the 113 pT1aNo-x patients (69·0%). We observed no persistence, locoregional recurrence, lymph node or distant metastases or deaths in the study group during the 12-year study (January 2001 to December 2012). CONCLUSIONS: The presence of the activating BRAF(V) (600E) mutation in a significant percentage of papillary thyroid microcarcinoma indicates that further analyses are required to verify its usefulness as a predictor of clinical outcome in PTC. In this study, there was no correlation between BRAF-positive primary focus of papillary microcarcinoma and more aggressive or recurrent disease.