Application of Molecular Diagnostics to the Evaluation of the Surgical Approach to Thyroid Cancer

Recent important studies that include long-term follow-up have shown that BRAF and RAS mutations can have negative implications for disease recurrence and survival. BRAF positivity has been shown to be associated with decreased survival and is an independent predictor of poor prognosis. Reliable pre-operative identification of high-risk papillary thyroid cancer (PTC) patients may productively guide initial surgical management since reoperative neck surgery is associated with increased morbidity. However, it is probably too early to conclude that at present it is possible to tailor surgical therapy patient by patient only on the basis of their mutational status. Other important parameters, not including molecular testing, represented by some specific morphological aspects, still play an important role, probably still more significant than molecular diagnostics, such as neck ultrasonography. Pre-operative knowledge of BRAF-positive PTC could alter the initial surgical treatment for at least 20% of patients and can potentially prevent the increased morbidity associated with reoperative neck exploration. However, additional multi-institutional and randomized studies will be needed to further define the role of the pre-operative identification of BRAF positivity to guide not only the initial extent of total thyroidectomy (TT) but also the need for and extent of lymphadenectomy.     

Mechanism and inhibition of BRAF kinase

The role of BRAF in tumor initiation has been established, however, the precise mechanism of autoinhibition has only been illustrated recently by several structural studies. These structures uncovered the basis by which the regulatory domains engage in regulating the activity of BRAF kinase domain, which lead to a more complete picture of the regulation cycle of RAF kinases. Small molecule BRAF inhibitors developed specifically to target BRAF^V600E have proven effective at inhibiting the most dominant BRAF mutant in melanomas, but are less potent against other BRAF mutants in RAS-driven diseases due to paradoxical activation of the MAPK pathway. A variety of new generation inhibitors that do not show paradoxical activation have been developed. Alternatively, efforts have begun to develop inhibitors targeting the dimer interface of BRAF. A deeper understanding of BRAF regulation together with more diverse BRAF inhibitors will be beneficial for drug development in RAF or RASdriven cancers.     

The RAS GTPase RIT1 compromises mitotic fidelity through spindle assembly checkpoint suppression

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New insights into the melanophilin (MLPH) gene controlling coat color phenotypes in American mink

The mutation causing the Silverblue color type (pp) is one of the most used recessive mutations within American mink (Neovison vison) fur farming, since it is involved in some of the popular color types such as Violet and Saphire which originate from a combination of recessive mutations. In the present study, the genomic and mRNA sequences of the melanophilin (MLPH) gene were studied in Violet, Silverblue and wild-type (wt) mink animals. Although breeding schemes and previous literature indicates that the Violet (aammpp) phenotype is a triple recessive color type involving the same locus as the Silverblue (pp) color type, our findings indicate different genotypes at the MLPH locus. Upon comparison at genomic level, we identified two deletions of the entire intron 7 and of the 5′ end of intron 8 in the sequence of the Silverblue MLPH gene. When investigating the mRNA, the Silverblue animals completely lack exon 8, which encodes 65 residues, of which 47 define the Myosin Va (MYO5A) binding domain. This may cause the incorrect anchoring of the MLPH protein to MYO5A in Silverblue animals, resulting in an improper pigmentation as seen in diluted phenotypes. Additionally, in the MLPH mRNA of wt, Violet and Silverblue phenotypes, part of intron 8 is retained resulting in a truncated MLPH protein, which is 359 residues long in wt and Violet and 284 residues long in Silverblue. Subsequently, our findings point out that the missing actin-binding domain, in neither of the 3 analyzed phenotypes affects the transport of melanosomes or the consequent final pigmentation. Moreover, the loss of the major part of the MYO5A domain in the Silverblue MLPH protein seems to be the responsible for the dilute phenotype. Based on our genomic DNA data, genetic tests for selecting Silverblue and Violet carrier animals can be performed in American mink.     

Pathologic Features Associated With Molecular Subtypes of Well-Differentiated Thyroid Cancer

ObjectiveTo determine the association between pathologic features and molecular classes (BRAF-like, RAS-like, and non-BRAF-like non-RAS-like [NBNR]).MethodsRetrospective review of a merged database containing 676 patients, 84% (571/676) were assigned to a molecular class from publicly accessible sequenced data of thyroid neoplasms.ResultsThe merged cohort included 571 neoplasms: 353 (62%) BRAF-like, 172 (30%) RAS-like, and 46 (8.1%) NBNR. Lymph node metastasis (any N1 disease) was present in 166/337 (49%) of BRAF-like, 23/164 (14%) of RAS-like, and 0/46 (0%) of NBNR and are significantly different (P < .001). Gross extra-thyroidal extension was observed in 27 patients, including 24/331 (7%) of BRAF-like, 2/160 (1%) of RAS-like, and 1/46 (2%) of NBNR (P = .01). N1B lymph node metastases or T4 disease was present in 74/333 (22%) of BRAF-like, 10/160 (6%) of RAS-like, and 1/46 (2%) of NBNR (P < .0001). Distant metastasis was present in 4/151 (2.6%) of BRAF-like, 2/50 (4%) of RAS-like and 0/46 for NBNR (P = .627). Angioinvasion was present in 0/81 (0%) of BRAF-like, 3/53 (6%) of RAS-like, and 3/46 (7%) of NBNR (P = .08); and multifocality was present in 27/81 (33%) of BRAF-like, 9/53 (17%) of RAS-like, and 1/46 (2%) for NBNR (P = .0001).ConclusionPathological features of metastasis, gross extra-thyroidal extension, and multifocality were more prevalent in BRAF-like samples compared to RAS-like and NBNR. A trend towards increased frequency of angioinvasion in RAS-like and NBNR cancers compared to BRAF-like samples was observed. Further studies are needed to evaluate if preoperative knowledge of molecular mutations in thyroid tumors aids in decision-making regarding extent of surgery.     

Lyso-PAF,a biologically inactive phospholipid,contributes to RAF1 activation

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Oxylipin biosynthesis reinforces cellular senescence and allows detection of senolysis

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Molecular basis of angiotensin- and thrombin-induced endothelium-independent vasoconstriction and proliferation

Summary In this report we describe our own attempt to explain the similarity of some effects of unique endogenous bioregulators such as angiotensin and thrombin at the molecular level. We also briefly review the cellular effects and signalling pathways of these molecules. Recent studies indicate that thrombin displays two opposing vascular effects: endothelium-dependent relaxation and endothelium-independent vasoconstriction. However, it should be noted that this enzyme possesses both vasocontractile and growth-stimulating activities which are similar to those of angiotensin. To examine a molecular basis for the functional duality of thrombin and similarity with angiotensin-induced effects, we analyzed the thrombin sequence and 3D structure. We found that (i) segment 84–135 of thrombin includes two angiotensin-like sites, which contain the repeats of the Tyr-Ile-His-Pro sequence and are identical to the functionally important fragment of angiotensin; and (ii) the thrombin 68–150 domain resembles the C-terminal tail of the macrophage migration inhibitory factor (MIF): five of the eight residues in angiotensin (Asp-Arg-Val-Tyr-Ile) are identical to fragment 94–98 of this one. Thus, angiotensin shares a surprisingly high degree of structural homology with both these molecules. We anticipated that angiotensin-like sites of thrombin (fragments 86–95 and 117–127) might be responsible for its vascular and growth-promoting activities on vascular smooth muscle cells. Taken together, the data obtained from a comparative analysis of thrombin, angiotensin and macrophage migration inhibitory factor-induced effects enable us to reveal the regulatory responsible domain of the enzyme and may prove useful for drug design.