Ras Point Mutations Research Articles

Molecular Profiles of Papillary Thyroid Tumors Have Been Changing in the Last Decades: How Could We Explain It?

The molecular profile of thyroid tumors can be discovered today in at least 70–80% of cases. The most common alterations are point mutations of BRAF and RAS followed by RET/PTC rearrangements. Other less frequent oncogenes involved are PIK3CA, TP53, TSHR, PTEN, GNAS, and CTNNB1 (1), but taken together they are still not able to account for 100% of cases. According to the data reported and collected so far (http://www.sanger.ac.uk/genetics/CGP/), BRAFV600E point mutations are present in about 45% of papillary thyroid cancer (PTC), with a higher rate in classical and tall cell variants and a rather low prevalence in follicular variant (2). There are exceptions with higher prevalence of BRAFV600E in PTC found in areas of China with a very high iodine content in the drinking water (3) and PTC found in the volcanic area of Mount Etna in Sicily where the drinking water is particularly rich in several elements, such as boron, iron, manganese, and vanadium, often present in concentrations exceeding usual maximum limits (4). The observations of Jung et al (5), showing a high prevalence of BRAFV600E mutation in a series of PTC diagnosed in the United States over a 35-year period, from 1974 to 2009, are of great relevance in this regard, associated with a significant increase of its prevalence in the classical variant of PTC over these last decades. The first study that indicated an increase of the prevalence of BRAFV600E mutation was reported in 2005 in a UK series, and the authors hypothesized that some environmental/etiological agent(s) could be responsible for the changing molecular features of PTC over time (6). Similar results were reported 6 years later by Mathur et al (7) in a series of PTC patients reported from California, and the hypothesis in this study was that the increased prevalence of the BRAFV600E mutation could be responsible for the increasing rate of PTC. In 2012, we also reported a significant increasing rate of BRAFV600E mutation in an Italian multicenter study (8), and it was interesting to observe that the increase was similar when the analysis was separately performed in the four different groups of PTC patients (from Pisa, Milan, Perugia, and Catania). Because the reported series are really very heterogeneous from a geographical point of view, it is conceivable that the increased prevalence of BRAFV600E mutation is a worldwide phenomenon, making it plausible that only a common, or very similar, reason could explain this trend. It is known that higher iodine intake has been found to be associated with a higher incidence of PTC (9), and it is notable that programs of iodine prophylaxis appear to be associated with an increase in the prevalence of PTC and a decrease in follicular histology (follicular thyroid cancer [FTC]) (10–12). This evidence and the Chinese report (3) may raise the question of a link between iodine intake, BRAFV600E mutation, and the development of increased incidence of PTC, but up to now, no experimental evidence that iodine can induce somatic gene alterations in follicular cells has been reported. Nevertheless, it is known that the iodine radical (I●), iodinium ion (I ) and the hypoiodous acid intermediate (IO[IOH]) are commonly formed in the follicular cell and represent reactive elements that together or as an alternative to H2O2 can determine cell oxidative stress, which is required to create conditions for thyroid cell proliferation (13–15). Further experimental studies on this putative relationship between

Abstract CN07-04: Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling.

Abstract Thyroid cancers are associated with mutually exclusive point mutations of BRAF (57%) and RAS (NRAS >HRAS; 12%), and fusion events activating BRAF, or the RTKs RET, NTRK1 or NTRK3. The molecular taxonomy of the disease is different in more advanced forms of thyroid cancer. Although BRAF mutations also predominate, poorly differentiated (PDTC) and anaplastic thyroid cancers (ATC) are relatively enriched for RAS mutations (23-44%). As predicted from the genetics of the disease, thyroid cancers, like melanomas, are ERK-driven tumors. However, they exhibit lineage-specific properties that offer distinct therapeutic opportunities and challenges. The 10-year survival of patients with metastatic thyroid cancers that retain radioiodine (RAI) avidity is ∼60%, whereas it is only 10% if the metastases are refractory to RAI therapy (1). There are several lines of evidence indicating that constitutive MAPK activity causes decreased radioiodine responsiveness. Conditional expression of oncogenic RET/PTC, RAS or BRAF in thyroid PCCL3 cells downregulates expression of the sodium iodide symporter (NIS) and other key genes required to organify and retain iodide (2, 3), which is restored by treatment with RAF or MEK inhibitors (2, 4). Mice with dox-inducible expression of BRAFV600E in thyroid cells develop invasive papillary thyroid cancers with profoundly impaired expression of genes required for iodine uptake and metabolism and of 124-iodine uptake in vivo, which are reversed when dox is discontinued, but only partially restored by RAF or MEK inhibitors (5). Based on these findings we performed a phase 2 trial of the MEK inhibitor selumetinib (AZD6244) in patients with RAI-refractory thyroid cancer. Selumetinib increased iodide incorporation at metastatic sites in 12/20 patients as determined by 124I PET scanning, and in ∼40% (8/20) the increase in uptake was sufficient to enable treatment with 131I, with remarkable clinical responses (6). Responses were greater in patients whose tumors harbored RAS as compared to BRAF mutations. An international multicenter phase 3 registration study of selumetinib vs placebo to increase remission rates of high risk thyroid cancer patients receiving post-surgical adjuvant radioiodine therapy is now in progress (the ASTRA trial). There is concern that responses to monotherapy with MEK or RAF inhibitors may not be optimal, particularly in BRAF-mutant disease. We find that BRAF-mutant human thyroid cancer cell lines have decreased responsiveness to RAF and MEK inhibitors because of relief of negative feedback resulting in derepression of HER3 transcription, followed by neuregulin-1- induced HER3/HER2 signaling and reactivation of the MEK-ERK and PI3K-AKT pathways, which is overcome by co-treatment with HER kinase inhibitors (7). This mechanism of adaptive resistance differs in significant ways from the responses seen in BRAF-mutant melanoma and colorectal cell lines. Our preclinical data predicts that optimal restoration of RAI responsiveness would be best achieved by compounds that inhibit MAPK output in a profound and sustained way. Indeed, recent experiments show that such strategies have excellent potential to further augment the specialized functions of thyroid cells, and lead to even more effective therapies. Citation Information: Mol Cancer Ther 2013;12(11 Suppl):CN07-04. Citation Format: James A. Fagin. Maximizing the effectiveness of radioiodine therapy for metastatic thyroid cancer with selective inhibitors of MAPK signaling. [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2013 Oct 19-23; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2013;12(11 Suppl):Abstract nr CN07-04.

An Alternative Approach in Endocrine Pathology Research: MALDI-IMS in Papillary Thyroid Carcinoma

To the Editor, Many different molecular techniques (polymerase chain reaction (PCR), DNA sequencing, fluorescence in situ hybridization (FISH)) have been introduced in thyroid pathology [1]. Fewer studies evaluated the role of proteomic analysis in the research of new useful targets [2, 3]. Matrixassisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a unique technology that explores the spatial distribution of biomolecules directly in situ, thus integrating molecular and morphological information. Therefore, we are investigating the potential role of MALDIIMS in detecting new diagnostic targets in papillary thyroid carcinoma (PTC). We have addressed the issue of molecular stratification of PTC in a small cohort of samples, evaluating both the genomic profile of the main genes of interest (BRAF, N-H-K RAS point mutations, and PAX8/PPARγ or RET/PTC rearrangements) and the proteomic profile shown byMALDIIMS analysis. We have collected ex vivo cytological specimens (see “Technical Note”), in order to analyse a sample perfectly mimicking the in vivo fine-needle aspiration (FNA) biopsy, while respecting the ethical requirements. Unsupervised proteomic analysis of the samples was followed by comparison with histopathology and genetic classification of the patients (Table 1). Data generated by MALDI-IMS were submitted to hierarchical cluster analysis (HCA), in order to evaluate the different proteomic expressions in the cases under study. HCA allowed to cluster proteomic spectra based on their similarity on a dendrogram: spectra showing comparable features were grouped under the same node of the dendrogram; then, it was possible to select nodes and assign them a specific colour (Fig. 1) [4]. Node A groups together three cytological specimens (two collected from patient 1 and one from patient 2), all histologically diagnosed as hyperplastic benign nodules. Node B groups together specimens collected from patients 3, 4 and 5, who were affected by PTC comparable for stage and histotype (Table 1). Node C, instead, groups a microcarcinoma (follicular variant (fv) of PTC), from patient 6, and a nodule histologically classified as uncertain malignant potential (UMP) tumour (patient 7). The last one, originated in a multinodular goitre environment and showed ambiguous morphological features, defined as borderline between a hyperplastic nodule and an “incipient” fv,PTC. The three distinct nodes (A, B, C) generated by HCA analysis confirmed that MALDI-IMS can potentially discriminate between benign and malignant thyroid lesions. Moreover, the homologies between cases 6 and 7 highlight that MALDI-IMS is able to identify a PTC even when the classic diagnostic morphological aspects are still unclear and ambiguous (mild nuclear clearing, rare grooves, no pseudoinclusions). This finding is in agreement Veronica Mainini and Fabio Pagni equally contributed to this paper.

Mutational Testing and its Utility in Thyroid Cancer Management: The Need for Something More

nodules are found to be benign at histology [1]. For this reason, ancillary diagnostic approaches for such nodules are needed. Genetic markers hold great promise in improving the accuracy of FNAC in patients with thyroid nodules. The last decade has seen advances in the understanding of the molecular basis of thyroid cancer, leading to the identifica­ tion of genetic alterations with a pathogenetic role, and thus these are the best candidates for thyroid cancer biomarkers. The thymine to adenine substitution at nucleo­ tide position 1799 of BRAF (BRAFV600E), con­ stitutes 98–99% of all BRAF mutations found in thyroid cancer [2]. Other alterations include a single point mutation at position 601, inframe insertions or deletions surrounding codon 600, and the AKAP9–BRAF rearrangement [3]. Many studies have focused their attention on the sensitivity and specificity of BRAFV600E as a hallmark of thyroid cancer in different clini­ cal settings [3,4]. The cumulative analysis per­ formed on more than a thousand tumors, with a few exceptions that are probably due to tech­ nical issues, assigns a positive predictive value of 100% to BRAFV600E. Despite the high specificity, the sensitivity of BRAF mutations for cancer is limited. Within thyroid tumors, BRAFV600E is restricted to the PTC histotype. Its prevalence is subjected to the sensitivity of detection methods and regional/ethnical fac­ tors of patients with PTC. While in the Korean population the prevalence of BRAFV600E has been reported to be 83%, its average prevalence worldwide is approximately 50% [5]. Other genetic alterations identified in thyroid cancer are RET–PTC, TRK and PAX8–PPARg rearrangements and RAS point mutations. These mutations not only exhibit a lower sensitivity, but they are also not infrequently Although it remains a rare disease, the incidence of thyroid cancer has been increasing and has almost doubled during the past 20 years in the USA and Europe. The increased incidence is limited to follicular cell­derived cancers, and within this category, the papillary histotype. The introduction of ultrasonography to clinical practice and the large access to this non invasive diagnostic procedure is partly responsible for the increased incidence of thyroid cancer and for its early detection. The result is that, now­ adays, the majority of thyroid cancers are of small size at the time of diagnosis and often less than 1 cm in diameter. The high prevalence of benign nodular goiter and the high incidence of clinically silent subcentimetric thyroid can­ cers raise relevant diagnostic and prognostic issues. Fine­needle aspiration cyto logy (FNAC) is currently the primary means to distinguish between benign and malignant nodules. Although FNAC is the most accurate and cost­ effective method for evaluating thyroid nod­ ules, it is a highly operator­dependent method, its accuracy is assured only by an experienced pathologist and it yields inconclusive results; that is, indeterminate or suspicious for malig­ nancy results, in approximately 20% of cases [1]. These two histo logical categories include follicular adenomas and follicular carcin omas (FTCs), which cannot be distinguished, and papillary carcinomas (PTCs), mostly folli cular variant PTCs that exhibit some cytologic char­ acteristics of malignancy, but not enough to be definitely classified. The number of FNACs performed yearly in Europe is estimated to be in the hundreds of thousands, yielding tens of thousands of inconclusive results. The impos­ sibility to rule out cancer in these nodules imposes a diagnostic thyroidectomy for most of these patients, although the majority of these

Cellular and molecular basis for thyroid cancer imaging in nuclear medicine

Papillary and follicular thyroid carcinomas (well-differentiated forms) are the most common follicular cell-derived thyroid malignancies, while poorly differentiated thyroid carcinomas and anaplastic thyroid carcinomas (also poorly differentiated) are the less common ones. Papillary carcinomas are morphologically and genetically different from follicular carcinomas: the former are associated, in up to 70 % of cases, with BRAF or RAS point mutations or RET/PTC rearrangements; the latter carry the RAS point mutation or the PAX8/PPARgamma rearrangement. The poorly differentiated forms have abnormalities in the TP53 and the CTNNB1 genes. The best way to image thyroid cancer cells is to exploit the capability of normal follicular thyroid cells to concentrate iodine 131I through the sodium–iodine symporter. Iodine is necessary for the production of the thyroid hormones triiodothyronine (T3) and thyroxine (T4). Unfortunately, the cells of poorly differentiated carcinomas lose the capability to concentrate iodine; at the same time, their basal metabolism increases to satisfy the energy demands of highly proliferating cells. These cells require more glucose and a glucose analog, namely 18F-2-fluoro-2-deoxy-d-glucose (18FDG), is used in place of glucose to study their metabolism. The increased intake of glucose is mediated by a transmembrane transporter called glucose transporter-1 located on the cell membrane. The alternation of 131I and 18FDG uptake observed in thyroid tumors and their metastases is known as the “flip-flop” phenomenon. This review looks at the cellular and molecular mechanisms underlying thyroid cancer and thyroid cancer imaging.

RET/PTC and PAX8/PPARγ chromosomal rearrangements in post‐Chernobyl thyroid cancer and their association with iodine‐131 radiation dose and other characteristics

Childhood exposure to iodine-131 from the 1986 nuclear accident in Chernobyl, Ukraine, led to a sharp increase in papillary thyroid carcinoma (PTC) incidence in regions surrounding the reactor. Data concerning the association between genetic mutations in PTCs and individual radiation doses are limited. Mutational analysis was performed on 62 PTCs diagnosed in a Ukrainian cohort of patients who were < 18 years old in 1986 and received 0.008 to 8.6 Gy of (131) I to the thyroid. Associations between mutation types and (131) I dose and other characteristics were explored. RET/PTC (ret proto-oncogene/papillary thyroid carcinoma) rearrangements were most common (35%), followed by BRAF (15%) and RAS (8%) point mutations. Two tumors carrying PAX8/PPARγ (paired box 8/peroxisome proliferator-activated receptor gamma) rearrangement were identified. A significant negative association with (131) I dose for BRAF and RAS point mutations and a significant concave association with (131) I dose, with an inflection point at 1.6 Gy and odds ratio of 2.1, based on a linear-quadratic model for RET/PTC and PAX8/PPARγ rearrangements were found. The trends with dose were significantly different between tumors with point mutations and rearrangements. Compared with point mutations, rearrangements were associated with residence in the relatively iodine-deficient Zhytomyr region, younger age at exposure or surgery, and male sex. These results provide the first demonstration of PAX8/PPARγ rearrangements in post-Chernobyl tumors and show different associations for point mutations and chromosomal rearrangements with (131) I dose and other factors. These data support the relationship between chromosomal rearrangements, but not point mutations, and (131) I exposure and point to a possible role of iodine deficiency in generation of RET/PTC rearrangements in these patients.

Evidence of a Low Prevalence ofRASMutations in a Large Medullary Thyroid Cancer Series

Approximately 60% of sporadic medullary thyroid carcinomas (sMTC) remain orphan of a recognized genetic cause. Recently, a high percentage of RAS point mutations have been described in RET-negative sMTC. The aim of this study was to assess the prevalence of RAS point mutations in a large series of MTC collected in four Italian centers. For this purpose, we studied codons 12, 13, and 61 of H-, K-, and N-RAS genes in 188 MTC samples, either hereditary or sporadic, by direct sequencing. Correlations between the RAS mutational status and the clinical-pathological features of MTC patients as well as a meta-analysis of all published data were performed. The prevalence of RAS mutations in the present series of MTC was 10.1%, and 17.6% when considering only RET-negative cases. RAS mutations were found in MTC tumoral tissue, but not in peripheral blood indicating their somatic origin. A novel mutation in codon 72 (M72I) was found, but with a low or null transforming potential. No association was found between the presence of RAS mutations and the clinical-pathological features of the patients. Although not statistically significant, a positive association between the presence of RAS mutations and a better outcome was observed. The meta-analysis of all published studies confirmed a prevalence of 8.8% for RAS mutations in MTC. The prevalence of RAS mutations in our MTC series was relatively low and consistent with the meta-analysis data. Only somatic RAS mutations were found and only in RET-negative sMTC. Likewise, MTCs that harbor a RAS mutation identify a subgroup of tumors with less aggressive behavior. To our knowledge, this is the largest series of MTCs studied for the presence of mutations in RAS genes and the first meta-analysis on this specific topic.

Abstract 2544: Associations between RET/PTC rearrangements, BRAF and RAS mutations and radiation dose, age at exposure, and latency in post-Chernobyl thyroid cancer

Abstract Introduction: Childhood exposure to I-131 from the Chernobyl accident was associated with a dramatic increase in incidence of papillary thyroid cancer (PTC). PTCs are characterized by several genetic alterations including RET/PTC rearrangements and point mutations of BRAF and RAS. Association between mutations and radiation doses has not been studied in post-Chernobyl tumors. Design: We analyzed 70 PTCs diagnosed in a Ukrainian-American cohort who were &amp;lt; 18 y.o. at the time of Chernobyl and received 0.008-8.6 Gy of I-131 to the thyroid. Thyroid doses were estimated based on radioactivity measurements taken shortly after the accident, environmental transport models, and interview data. BRAF and RAS mutations were identified by melting curve analysis and RET/PTC1 and RET/PTC3 by real-time RT-PCR. All mutations were confirmed by sequencing. Results: Identified mutations and patient characteristics are shown in Table 1. Logistic regression analysis demonstrated a significant decrease in I-131 dose for both BRAF and RAS mutations (p=0.005 and 0.026). There were highly statistically significant (p&amp;lt;0.001) differences between the trends with dose for BRAF and RAS vs RET/PTC. Comparing with BRAF and RAS mutations, the age at exposure was lower in cases with RET/PTC (p=0.001). RET/PTC3 was associated with a shorter latency period as compared to other mutations (p&amp;lt;0.02). Table 1. Genetic alterations and corresponding patient characteristics Conclusions: Our results demonstrate for the first time that prevalence of BRAF or RAS point mutations in post-Chernobyl tumors significantly decreases with increasing radiation dose, in contrast to the lack of decrease for RET/PTC. RET/PTC rearrangements are associated with younger age at exposure than point mutations of BRAF and RAS. These data provide support for the potential relationship between chromosomal rearrangements, but not point mutations, and radiation exposure. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2544. doi:1538-7445.AM2012-2544

Molecular Analysis of Thyroid Tumors

Thyroid cancer is the most common endocrine malignancy, and its incidence is rising in the USA and other countries. Papillary and follicular thyroid carcinomas are the two most common types of thyroid cancer. Non-overlapping genetic alterations, including BRAF and RAS point mutations, and RET/PTC and PAX8/PPARγ rearrangements, are found in more than 70% of papillary and follicular thyroid carcinomas. These represent the most common genetic alterations in thyroid cancer, as well as molecular markers of diagnostic and prognostic significance. The use of these and other emerging molecular markers will likely improve the diagnosis of malignancy in thyroid nodules as well as facilitate more individualized operative and postoperative management. Herein, we provide a brief overview of the common genetic alterations in papillary and follicular thyroid carcinoma and discuss the diagnostic and prognostic significance thereof.

Molecular Diagnostics of Thyroid Tumors

Thyroid cancer is the most common type of endocrine malignancy and its incidence is steadily increasing. Papillary carcinoma and follicular carcinoma are the most common types of thyroid cancer and represent those tumor types for which use of molecular markers for diagnosis and prognostication is of high clinical significance. To review the most common molecular alterations in thyroid cancer and their diagnostic and prognostic utility. PubMed (US National Library of Medicine)-available review articles, peer-reviewed original articles, and experience of the author. The most common molecular alterations in thyroid cancer include BRAF and RAS point mutations and RET/PTC and PAX8/PPAR γ rearrangements. These nonoverlapping genetic alterations are found in more than 70% of papillary and follicular thyroid carcinomas. These molecular alterations can be detected in surgically resected samples and fine-needle aspiration samples from thyroid nodules and can be of significant diagnostic use. The diagnostic role of BRAF mutations has been studied most extensively, and recent studies also demonstrated a significant diagnostic utility of RAS, RET/PTC, and PAX8/PPAR γ mutations, particularly in thyroid fine-needle aspiration samples with indeterminate cytology. In addition to the diagnostic use, BRAF V600E mutation can also be used for tumor prognostication, as this mutation is associated with higher rate of tumor recurrence and tumor-related mortality. The use of these and other emerging molecular markers is expected to improve significantly the accuracy of cancer diagnosis in thyroid nodules and allow more individualized surgical and postsurgical management of patients with thyroid cancer.

Molecular Diagnostics of Thyroid Tumors

Abstract Context.—Thyroid cancer is the most common type of endocrine malignancy and its incidence is steadily increasing. Papillary carcinoma and follicular carcinoma are the most common types of thyroid cancer and represent those tumor types for which use of molecular markers for diagnosis and prognostication is of high clinical significance. Objective.—To review the most common molecular alterations in thyroid cancer and their diagnostic and prognostic utility. Data Sources.—PubMed (US National Library of Medicine)–available review articles, peer-reviewed original articles, and experience of the author. Conclusions.—The most common molecular alterations in thyroid cancer include BRAF and RAS point mutations and RET/PTC and PAX8/PPARγ rearrangements. These nonoverlapping genetic alterations are found in more than 70% of papillary and follicular thyroid carcinomas. These molecular alterations can be detected in surgically resected samples and fine-needle aspiration samples from thyroid nodules and can be...

Acute myeloid leukemia with t(9;11)(p21-22;q23): common properties of dysregulated ras pathway signaling and genomic progression characterize de novo and therapy-related cases.

We compared pathogenetic features of 32 de novo and 29 therapy-related (t) t(9;11)(p21-22;q23)/MLLT3-MLL acute myeloid leukemia (AML) cases to identify progression factors and to assess whether distinction between these manifestations is warranted. MLLT3-MLL rearrangement was commonly the sole karyotypic abnormality at diagnosis, with many secondary chromosomal changes emerging at relapse in both subgroups. Ras point mutations were common in both groups (overall, 18/50 [36%]) and associated with monocytic phenotype and aneuploid progression. Expression patterns of 675 microRNAs profiled in 7 cases were also similar, with let-7 species linked to Ras down-modulation expressed at low levels. Outcome for both groups was poor (relapsed or refractory in 49/61 [80%] cases); however, patients with t-AML were generally older and female, with worse outcome (P = .03), likely secondary to t-AML mostly arising in patients with breast cancer following topoisomerase inhibitor-containing chemotherapy. Ras activation seems to complement the MLLT3-MLL oncogene in transformation with features of de novo and t-AML with MLLT3-MLL being similar.

Detection of Minimal Malignant Clone in Lymphocytes and Bone Marrow Cells by Cytogenetic and Molecular Genetic Studies in Healthy Atomic Bomb Survivors

In order to assess the minimal malignant clone (MMC) for the development of leukemia, we studied the bone marrow and lymphocytes of total 44 healthy atomic - bomb survivors by cytogenetic technique. In the cytogenetic study, we compared the frequencies of chromosome aberration and quality of chromosome aberrations detected in the bone marrow and peripheral lymphocytes from 13 and 39 samples from 44 healthy atomic bomb survivors, respectively, relevant to the nonrandom aberrations prevalent in the human leukemic and malignant lymphoma cells. The frequencies of chromosome aberrations in bone marrow cells were slightly lower than those in lymphocytes and a few percent of clone were found in both bone marrow and lymphocytes. Although the completely same type chromosome aberrations as those found in leukemia and lymphomas were not observed, out of 3,846 cells with chromosome aberrations found in 52 samples for 44 survivors, 35 percent were related to the sites of oncogenes involving in the leukemogenesis and lymphomagenesis such as 8q24, 9q34 and 22q11. In addition, molecular genetic studies using PCR techniques for RAS point mutations in 3 survivors, specific chromosome translocations such as t(14;18), and t(7;14) in the bone marrow and peripheral lymphocytes of 3 healthy survivors revealed that any MMC was detected. On the other hand, by in vivo selection assay could detect transforming genes of N-and K-RAS in bone marrow samples from all 4 survivors. Three of the 4 survivors had development cancer or leukemia 7-10 years after present examination. Even though, we couldn't detect any MMC by chromosome analysis, the finding that 20 fragile sites were correlated with higher number of breakpoint of 52 atomic bomb survivors might also significantly an important role MMC in the initiation of radiation-related leukemogenesis and lymphomagenesis process, as seen presently, there is still an increase in the development

Detection of minimal malignant clone in lymphocytes and bone marrow cells by cytogenetic and molecular genetic studies in healthy atomic bomb survivors

In order to assess the minimal malignant clone (MMC) for the development of leukemia, we studied the bone marrow and lymphocytes of total 44 healthy atomic – bomb survivors by cytogenetic technique. In the cytogenetic study, we compared the frequencies of chromosome aberration and quality of chromosome aberrations detected in the bone marrow and peripheral lymphocytes from 13 and 39 samples from 44 healthy atomic bomb survivors, respectively, relevant to the nonrandom aberrations prevalent in the human leukemic and malignant lymphoma cells. The frequencies of chromosome aberrations in bone marrow cells were slightly lower than those in lymphocytes and a few percent of clone were found in both bone marrow and lymphocytes. Although the completely same type chromosome aberrations as those found in leukemia and lymphomas were not observed, out of 3,846 cells with chromosome aberrations found in 52 samples for 44 survivors, 35 percent were related to the sites of oncogenes involving in the leukemogenesis and lymphomagenesis such as 8q24, 9q34 and 22q11. In addition, molecular genetic studies using PCR techniques for RAS point mutations in 3 survivors, specific chromosome translocations such as t(14;18), and t(7;14) in the bone marrow and peripheral lymphocytes of 3 healthy survivors revealed that any MMC was detected. On the other hand, by in vivo selection assay could detect transforming genes of N-and K-RAS in bone marrow samples from all 4 survivors. Three of the 4 survivors had development cancer or leukemia 7-10 years after present examination. Even though, we couldn’t detect any MMC by chromosome analysis, the finding that 20 fragile sites were correlated with higher number of breakpoint of 52 atomic bomb survivors might also significantly an important role MMC in the initiation of radiation-related leukemogenesis and lymphomagenesis process, as seen presently, there is still an increase in the development of leukemia among survivors than the normal population. Keywords: Atomic bomb survivors, chromosome aberrations, bone marrow, lymphocytes, minimal clone, PCR, RAS gene

Low frequency of RAS and absence of FLT3-ITD gene mutations in patients with Myelodysplastic Syndromes in India: AIIMS experience

e22231 Background: Chromosomal abnormalities and molecular detection has potential importance for diagnosis and prognosis of MDS, although the mechanisms underlying the development of MDS and their progressive evolution to AML are still largely unknown. Since, no studies have been reported from India on the prevalence of N-RAS, K- RAS point mutation in codon 12 and FLT3-ITD mutations in patients with MDS, we undertook this study. Methods: DNA and RNA were extracted from bone marrow /peripheral blood. Using RT-PCR the patients were screened for length mutations in FLT3 gene. PCR-RFLP and nested PCR-RFLP were used for the detection of point mutation in codon 12 of N-RAS and K-RAS. Results: A total of 53 patients (median age 39 yrs, range 9–78yrs; M: F 2:1; median TLC-3.9×109/l, range 0.8–116 ×109/l Median platelet count- 87 ×109/l, range 1–349 ×109/l, Median hemoglobin -6.8 g/dl, range 2.7- 16.1 g/dl, were studied. One out of 53 patients (2%) was found positive for N-RAS and four patients were positive for K-RAS (8%) mutation. FLT3-ITD mutation was studied in 47 patients; all the patients were found negative. The mean observation of all the patients was 30 months and the median overall survival was 28 months. Nine patients died during follow up. The presence of N-RAS codon 12 mutation was associated with the poor survival. FLT3-ITD mutation was not observed in any of our cases, which is in contrast to 3% reported from the West. Conclusions: Thus, it appears that the RAS and FLT3 mutations are uncommon in MDS patients in India. No significant financial relationships to disclose.

Genetics of therapy-related myelodysplasia and acute myeloid leukemia

Myelodysplasia (MDS) and acute myeloid leukemia (AML) are heterogeneous, closely associated diseases arising de novo or following chemotherapy with alkylating agents, topoisomerase II inhibitors, or after radiotherapy. Whereas de novo MDS and AML are almost always subclassified according to cytogenetic characteristics, therapy-related MDS (t-MDS) and therapy-related AML (t-AML) are often considered as separate entities and are not subdivided. Alternative genetic pathways were previously proposed in t-MDS and t-AML based on cytogenetic characteristics. An increasing number of gene mutations are now observed to cluster differently in these pathways with an identical pattern in de novo and in t-MDS and t-AML. An association is observed between activating mutations of genes in the tyrosine kinase RAS-BRAF signal-transduction pathway (Class I mutations) and inactivating mutations of genes encoding hematopoietic transcription factors (Class II mutations). Point mutations of AML1 and RAS seem to cooperate and predispose to progression from t-MDS to t-AML. Recently, critical genetic effects underlying 5q-/-5 and 7q-/-7 have been proposed. Their association and cooperation with point mutations of p53 and AML1, respectively, extend the scenario of cooperating genetic abnormalities in MDS and AML. As de novo and t-MDS and t-AML are biologically identical diseases, they ought to be subclassified and treated similarly.

Nuclear Exclusion of Forkhead Box O and Elk1 and Activation of Nuclear Factor-κB Are Required for C2C12-RasV12C40 Myoblast Differentiation

Activating ras point mutations are frequently found in skeletal muscle tumors such as rhabdomyosarcomas. In this study we investigated the impact of two different H-ras mutants in skeletal muscle differentiation: RasV12, a constitutively active form, and RasV12C40, a mutant deficient in Raf1 activation. Stably transfected C2C12-RasV12 myoblasts actively proliferated as indicated by the sustained expression of proliferating cell nuclear antigen and retinoblastoma at the hyperphosphorylated state and failed to express differentiation markers. This differentiation-defective phenotype was a consequence of the chronic p44/p42MAPK phosphorylation and the inability of the cells to activate AKT. Moreover, we observed that p44/p42MAPK activation in C2C12-RasV12 myoblasts phosphorylated the ETS-like transcription factor (ELK) 1, which translocates to the nuclei and seemed to be involved in maintaining myoblast proliferation. C2C12-RasV12C40 myoblasts cultured in low serum repressed phosphorylation of p44/p42MAPK and ELK1, resulting in cell cycle arrest and myogenic differentiation. Under this condition, activation of AKT, p70S6K, and p38MAPK was produced, leading to formation of myotubes in 3 d, 1 d earlier than in control C2C12-AU5 cells. Moreover, the expression of muscle-specific proteins, mainly the terminal differentiation markers caveolin-3 and myosin heavy chain, also occurred 1 d earlier than in control cells. Furthermore, AKT activation produced phosphorylation of Forkhead box O that led to nuclear exclusion and inactivation, allowing myogenesis. In addition, we found an induction of nuclear factor-kappaB activity in the nucleus in C2C12-RasV12C40 myotubes attributed to p38MAPK activation. Accordingly, muscle differentiation is associated with a pattern of transcription factors that involves nuclear exclusion ELK1 and Forkhead box O and the increase in nuclear factor-kappaB DNA binding.

Network Analysis of Oncogenic Ras Activation in Cancer

To investigate the unregulated Ras activation associated with cancer, we developed and validated a mathematical model of Ras signaling. The model-based predictions and associated experiments help explain why only one of two classes of activating Ras point mutations with in vitro transformation potential is commonly found in cancers. Model-based analysis of these mutants uncovered a systems-level process that contributes to total Ras activation in cells. This predicted behavior was supported by experimental observations. We also used the model to identify a strategy in which a drug could cause stronger inhibition on the cancerous Ras network than on the wild-type network. This system-level analysis of the oncogenic Ras network provides new insights and potential therapeutic strategies.

Highly sensitive ECL-PCR method for detection of K- ras point mutation

A highly sensitive electrochemiluminescence-polymerase chain reaction (ECL-PCR) method for K- ras point mutation detection is developed. Briefly, K- ras oncogene was amplified by a Ru(bpy) 3 2+ (TBR)-labeled forward and a biotin-labeled reverse primer, and followed by digestion with MvaI restriction enzyme, which only cut the wild-type amplicon containing its cutting site. The digested product was then adsorbed to the streptavidin-coated microbead through the biotin label and detected by ECL assay. The experiment results showed that the different genotypes can be clearly discriminated by ECL-PCR method. It is useful in point mutation detection, due to its sensitivity, safety, and simplicity.

Molecular determination of benign and malignant thyroid tumors

Recent molecular studies have revolutionized our understanding of the pathogenesis of thyroid tumors and particular advances have been made in three areas. First, toxic thyroid nodules, which originate from constitutive activation of thyroid-stimulating hormone receptor/Gs α signaling and represent the dominant cause of thyrotoxicosis in regions with iodine deficiency. Second, papillary thyroid cancer, the most frequent thyroid malignancy, which is characterized by a common fingerprint of constitutive mitogen-activated protein kinase activation. Importantly, this is caused by distinct genetic alterations in radiation-induced (RET/PTC, NTRK and AKAP9/BRAF rearrangements) and sporadic tumors (BRAF and RAS point mutation) and, recently, there exciting in vitro have emerged explaining the structural basis for this. These findings suggest a scenario in which the fate of a thyroid tumor is determined by the specific genetic defect at the beginning. Third, application of microarray analysis in nodular pathologies in which the oncogenic pathway is less clear, notably follicular neoplasia, has led to the identification of a number of promising genetic markers (TFF-3, Gal-3, PLAB, CCND2 and PCKD2) for the diagnostic distinction of follicular adenoma and carcinoma. In addition to the diagnostic perspective, the identification of molecular fingerprints of thyroid tumors opens novel avenues for an improved therapeutic approach; for example, selective antagonism of cell signaling in treatment-refractory thyroid cancer.