We report a rare germline missense variant in the RET protooncogene found in a 55-year-old woman with medullary thyroid carcinoma (MTC). Calcitonin before surgery was 400 pg/ml (N = 2-6 pg/ml). Postoperative calcitonin was 0.45 pg/ml. Pheochromocytoma and hyperparathyroidism were excluded. Sanger sequencing of eight exons (5, 8, 10, 11, 13-16) (peripheral blood-derived genomic DNA) of the RET gene identified the heterozygous germline missense variant p.Arg886Gln (exon 15). But its clinical significance has not yet been determined. The proband`s parents were not available. In the control group we only found wild-type alleles.

MEN 2B syndrome is a subtype of the multiple endocrine neoplasia type 2. It is characterized by the development of aggressive forms of medullary thyroid cancer at an early age, pheochromocytoma and hyperparathyroid syndrome. This article provides an own clinical observation of the patient with MEN 2B syndrome associated with a mutation in the proto-oncogene RET.

MEN 2B syndrome is a subtype of the multiple endocrine neoplasia type 2. It is characterized by the development of aggressive forms of medullary thyroid cancer at an early age, pheochromocytoma and hyperparathyroid syndrome. This article provides an own clinical observation of the patient with MEN 2B syndrome associated with a mutation in the proto-oncogene RET.

Two approaches to somatic point mutations in 12 and 13 codones of K-ras gene were analyzed: PCR/SSCP/ACRS/sequencing and allele-specific PCR in the real-life regimen (Russian set "KRAS-7M"). The comparison was carried out on 62 examples of genomic DNA extracted from frozen colon carcinomas, which underwent manual dissection. The results obtained in two attempts were consistent in 95,2% (N=59). Specificity and sensitivity of K-ras mutations detection using "KRAS-7M" set were 100 and 96,4% respectively, and 94,1 and 100% respectievly using PCR/SSCP/ACRS/automatic sequencing. False positive results were absent when detecting with "KRAS-7M" and accounted for 2 cases (5,9%) when using PCR/SSCP/ ACRS/automatic sequencing. The only false negative response (3,6%) was obtained analyzing mutations using "KRAS-7M".

Genetic analysis of hereditary mutations of gene RET shows a way to effective diagnosis and prevention of medullary thyroid cancer. DNA diagnosis targets at point mutations in RET exons 10, 11, 13, 14, 15, 16. Examination of 68 families of medullary thyroid cancer patients and 382 their blood relatives discovered genetic medullary thyroid cancer in 24.9% of cases including multiple endocrine neoplasia syndrome types 2A (7.4%) and B (11.7%), familial medullary thyroid cancer syndrome (4.4%) and medullary thyroid cancer as a component of neurofibromatosis type 1 (1.4%). In patients with multiple endocrine neoplasia syndrome type 2A RET mutation in codon 634 was found to be associated with pheochromocytoma and hyperparathyroiditis, and mutation in codon 918 was characteristic of multiple endocrine neoplasia syndrome type 2B and associated with mucosal neurinomas and skeleton malformations. This discovery helps to make a more accurate prognosis of disease course in patients with multiple endocrine neoplasia syndrome type 2. Direct RET testing in relatives of patients with multiple endocrine neoplasia syndrome type 2A identified 8 carriers of germinal mutations of this gene. Prophylactic surgery was made in 6 of them. This surgery appeared preventive in 2 (3.5 and 9 years old) cases, and was performed at an early disease stage in 4 (aged 14 years or older). Mean postoperative disease-free follow-up was 7 years. Germinal de novo RET mutation was found in 1 of 13 patients with sporadic medullary thyroid cancer. This proves reasonable DNA testing in cases with sporadic medullary thyroid cancer. A comprehensive program was developed to identify individuals predisposed to medullary thyroid cancer.

Соматические мутации в гене KRAS являются важными маркерами некоторых видов опухолей, например рака поджелудочной железы, и могут применяться для ранней диагностики этого заболевания. Разработан биологический микрочип, позволяющий выявлять 13 наиболее частых мутаций в кодонах 12, 13 и 61 гена KRAS. Для повышения чувствительности метода и обнаружения минорных фракций опухолевых клеток в клинических образцах амплификацию нуклеотидных последовательностей гена KRAS дикого типа в ходе ПЦР подавляли с помощью LNA-олигонуклеотидов. Продукт LNA-блокирующей ПЦР далее гибридизовали с олигонуклеотидными зондами, иммобилизованными в геле на поверхности биочипа. Биочип проверен на 42 клинических образцах ДНК, полученных от больных с опухолями поджелудочной железы, в основном, протоковыми аденокарциномами. В качестве референс-методов использовали ПДРФ-анализ и секвенирование. Метод с использованием биочипов позволяет с высокой достоверностью обнаруживать мутации в гене KRAS, если доля клеток, несущих мутацию, составляет не менее 1%.

To estimate diagnostic value of K-ras mutations during cancer risk group formation, they were studied in the samples of sporadic carcinomas (n = 58), benign (n = 33), and malignant (n = 13) polyps of large intestine obtained during surgery or polypectomy. Using PCR analysis, restriction analysis, SSCP analysis and automated sequencing, eight various point mutations were revealed. Six of them were located in codon 12 and two, in codon 13 of the K-ras gene. Mutation frequency in carcinomas, benign and malignant polyps was 43, 49, and 69%, respectively. In the normal tissue samples of colorectum, no changes in codons 12 and 13 in the K-ras gene were observed. Mutations in the groups of Russian patients examined partially overlapped. In patients with colorectal carcinoma the mutation frequency in the K-ras gene was not associated with disease onset age, location, and the extent of tumor differentiation while it was associated with the stage of tumor process. In polyps, the maximum mutation frequency was revealed among patients over 70 years of age as well as in the adenomas of villous histology and large size (≥1cm). No correlation between the K-ras mutation frequency and the extent of polyp dysplasia was observed.

The diagnostic significance of molecular markers was assessed for the most common somatic aberrations at the K-ras, TP53, CDKN2A, and MADH4 loci, as well as less common mutations of BRCA1, BRCA2, and CHEK2, arising in preinvasive stages of sporadic adenocarcinoma of the pancreas. The study was performed on paired primary pancreatic adenocarcinoma and normal pancreatic tissue specimens obtained from 37 Russian patients. Surgical adenocarcinoma specimens were subjected to manual microdissection. Mutations of K-ras codon 12 were found in 24 tumor specimens (0.65), but not in normal pancreatic tissue specimens. Mutations of BRCA1 (185delAG, 300T > G, 4153delA, 4158A > G, 5382insC), BRCA2 (695insT, 6174delT), and CHEK2 (1100delC) were not found. The informativeness of allelic losses did not differ significantly among the three tumor suppressor loci and was 60% for TP53 (GDB186817) and CDKN2A (D9S974 + D9S162) and 65.7% for MADH4 (D18S363 + D18S474) (t = 0.48). The CDKN2A locus had the highest LOH frequency of 0.95. For TP53 and MADH4 the LOH frequency was 0.62 and 0.70, respectively. In 80% of adenocarcinomas, at least one locus was characterized with LOH. The overall informativeness of the combined data on K-ras mutations and loss of heterozygosity at 9p, 17p, and 18q was 85.7%. Only 9% of the tumors were characterized with microsatellite instability.

Диагностическую значимость молекулярных маркеров оценили при помощи анализа наиболее частых соматических изменений в генах K-ras, TP53 и CDKN2A и MADH4 и более редких мутаций в генах BRCA1, BRCA2 и CHEK2, которые возникают на преинвазивной стадии развития спорадических аденокарцином поджелудочной железы. Использовали парные образцы первичных аденокарцином и нормальной ткани поджелудочной железы 37 российских больных. Операционный материал карцином подвергали ручной микродиссекции. Мутации в кодоне 12 протоонкогена K-ras найдены в 24 образцах опухолей (0.65) и не найдены в образцах нормальной ткани поджелудочной железы. Мутации в генах BRCA1 (185delAG, 300T > G, 4153delA, 4158A > G, 5382insC), BRCA2 (695insT, 6174delT) и CHEK2 (1100delC) не обнаружены. Информативность потери аллелей трех генов-супрессоров не имела статистически значимых различий: 60% – в случае TP53 (GDB186817) и CDKN2A (D9S974 + D9S162) и 65.7% – в случае MADH4 (D18S363 + D18S474) (t = 0.48). Максимальную частоту потери гетерозиготности проявляет ген CDKN2A – 0.95. У генов TP53 и MADH4 она составила 0.62 и 0.70 соответственно. Потерю гетерозиготности как минимум по одному локусу наблюдали в 80% аденокарцином. “Суммарная” информативность при объединении данных по мутациям K-ras и потере гетерозиготности в локусах 9p, 17p и 18q составила 85.7%. Нестабильность микросателлитных маркеров обнаружена только в 9% опухолей.

Рак яичников (РЯ) занимает одно из ведущих мест по смертности от онкологических заболеваний у женщин. Мутации в генах BRCA1/2 обусловливают высокий риск возникновения данного заболевания (от 10 до 60% на протяжении жизни). Показано, что вариант 1100delC в гене CHEK2 ассоциирован с предрасположенностью к раку молочной железы у женщин. Сведения о спектре и частоте мутаций в этих генах в популяции позволяют выявлять “группы риска” развития онкопатологий. В работе исследована частота встречаемости восьми мутаций в генах BRCA1/2 и СНЕК2 (185delAG, 300T>G, 4153delA, 4158A>G и 5382insC в гене BRCA1, 695insT и 6174delT в гене BRCA2, варианта 1100delC в гене СНЕК) у российских женщин с диагнозом РЯ при использовании технологии биочипов. Протестировано 68 пациентов с органоспецифическим РЯ и 19 пациентов с первично-множественными злокачественными новообразованиями с поражением яичников. Выявлены четыре типа мутаций в гене BRCA1 – 185delAG, 300Т>G, 4153delA и 5382insC. У больных органоспецифическим РЯ в гене BRCA1 наиболее часто встречается мутация 5382insC (87.5% от всех мутаций в этом гене); в группе с первично-множественными злокачественными новообразованиями ее доля составляет 50.0%. Мутаций в генах BRCA2 и СНЕК2 в обеих группах не обнаружено.

Ovarian cancer (OC) is among the leading causes of cancer-related mortality in women. A high risk of OC (lifetime estimates ranging 10–60%) is determined by BRCA1/2 mutations. The 1100delC variant of CHEK2 is associated with predisposition to breast cancer (BC) in women. With the known spectrum and frequencies of mutations of these genes, it is possible to identify a risk group in a population. Using biochip technology, the frequencies of eight BRCA1/2 and CHEK2 mutations (185delAG, 300T>G, 4153delA, 4158A>G, and 5382insC of BRCA1; 695insT and 6174delT of BRCA2; and 1100delC of CHEK2) were studied in Russian women with OC, including 68 patients with organ-specific OC and 19 with primary multiple tumors (PMTs) involving the ovaries. Four BRCA1 mutations were observed: 185delAG, 300T>G, 4153delA, and 5382insC. The last one was most common in OC, accounting for 87.5% of all cases with mutant BRCA1, and occurred at a frequency of 50.0% in PMT. BRCA2 and CHEK2 mutations were not found in the two groups.

The spectrum of mutations of the proto-oncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six different mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four types of mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of mutations from three MEN 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.

The spectrum of mutations of the RET proto-oncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six different mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four types of mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MEN 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.

The spectrum of mutations of the RET protooncogene was analyzed in Russian patients with inherited or sporadic medullary thyroid carcinoma (MTC). Four RET exons (11, 13, 15, and 16) were subjected to molecular analysis, and mutations were revealed and identified in 47.4% (9/19) patients with sporadic MTC. In total, six mutations (including three new ones) were observed. The most common mutation affected codon 918 to cause substitution of methionine with threonine and accounted for 31.6% alleles. Analysis of exons 11 and 16 revealed four mutations in patients with inherited multiple endocrine neoplasia type 2 (MEN 2). Mutations were found in each patient. Thyroidectomy was performed in four asymptomatic carriers of RET mutations from three MET 2A families (in two families, affected relatives had bilateral pheochromocytoma). In two patients, analysis of the surgery material revealed MTC microfoci in both lobes of the thyroid gland. The results provide the ground for constructing a bank of genetic information on Russian MTC patients with the clinically verified diagnosis.