Oferta de espacio construido

Specific m utations in the ret oncogene have been associated with defined morphological variants o f thyroid cancer. Our data dem onstrates for the first tim e R E T rearrangem ents in vivo outside o f the thyroid gland. Santoro's (Santoro et al., 1993) initial study o f 528 non-thyroid tumours which did not identify R E T activation used Southern blot analysis o f genomic DNA. Even though Southern blot analysis is a reliable m ethod for the detection o f chrom osom al rearrangements it has limited sensitivity and requires at least approxim ately 10% o f tum our cells within the sample for detection. The small num ber o f nuclei with split signals above the threshold level in our tum our cohort indicates that the RET/PTC rearrangem ent is a low-level event in prim ary peritoneal carcinom a and m ay have been m issed by Southern blot analysis. Furthem iore, some o f their results were not validated by RT-PCR, a highly sensitive technique which is capable o f detection o f a small proportion o f cells carrying a particular genetic event. M oreover, Santoro’s study included only a small sample o f ovarian (n = l2 ) and renal cell carcinom a (n=10), from which the authors acknowledge that no inference could be drawn on particular subsets o f tumours not included in their study.

A single case in this study (case 25) showed neither R E T protein expression nor dem onstrable RET/PTC \ mRNA by RT-PCR. However this case exhibited split FISH signals in more than 10.2% o f cells analysed. It is likely, that this case harbours a R E T rearrangem ent other than H 4IRET {R ET/PTC \) w hich is not expressed at the protein level, possibly as a result o f post- transcriptional regulation. Similarly, alternate chimeric ret oncogenes have been dem onstrated in papillary thyroid cancer and the prevalence o f ret/PTC rearrangem ents in thyroid cancer varies widely am ong studies, w ith rates between 3 and 85% being reported. (Asa, 2001; Kondo et al., 2006; Nikiforov, 2002; Tallini and Asa, 2001) Furthermore, let-7 m icroRN A (which is a noncoding ~22 nucleotide RNA that negatively regulates gene expression at the

posttranscriptional level by repressing translation) has recently been demonstrated to critically affect cell growth and differentiation in PTC harbouring RET/PTC rearrangements and highlights a potential mechanism for post-transcriptional regulation o f ret protein expression. (Croce, 2008; Ricarte-Filho 2009) Alternatively in this case, the sensitivity o f both the RT-PCR

and immunohistochemistry was not high enough to detect a few RET/PTC\ rearranged cells

which may also be a reflection o f tumour heterogeneity. (Unger et al., 2004)

The last subgroup in this study (8 cases in total) had neither demonstrable RET/PTC \ niRNA by

RT-PCR nor split FISH signals greater than the cut-off level. However they all demonstrated ret

protein expression which must logically be the wild-type protein. Ret protein expression in non neuroendocrine tumours has not has not been widely studied. How'ever, recent evidence has demonstrated wild tj'pe ret protein expression in renal, breast, pancreatic and lung tumours which supports the observations in this current study. (Flavin et al, in press; Boulay et al, 2008; Ceyhan et al, 2006; Thomas et al, 2007)

The occurrence o f RET/PTC in primary peritoneal and papillary renal cell carcinoma has

interesting implications. Somatic mutations in cancer have been called driver when they are positively selected and causally related to tumour development and passenger when not directly

implicated with tumour growth. (Davies et al., 2005) RET/PTC rearrangements in our study can

hardly be considered as driver mutations in tumours with minimal levels o f detectable fusion gene. These rearrangements may reflect R E T instability in epithelial cells and point to the existence o f secondary cell subclones, which are unlikely to be o f any pathological consequence in determining biological behaviour.

Chapter 3 RET/PTC rearrangement in primary peritoneal carcinoma

It is interesting to hypothesize on the association between RET/PTC rearrangements and primary peritoneal carcinoma. One hypothesis is that inflammation favours the occurrence o f the rearrangement as a secondary phenomenon. The propensity o f thyrocytes to undergo R E T recombination has been explained by the peculiar arrangement o f chromatin that juxtaposes R E T and its fusion partners in interphase nuclei. It is possible that free radical production, cytokine secretion, cellular proliferation and other events related to inflammation trigger the occurrence o f the rearrangement predisposed to it by an unstable chromatin confirmation. (Gandhi et al., 2006) O f note, a proinflammatory tumour microenvironment has been described in ovarian cancer which contributes to immune cell recruitment and differentiation ( ‘tumour-immune cell education m odel’). (Chen et al., 2008) Conversely, the occurrence o f RET/PTC may directly influence the inflammation within these tumours. O f note, RET/PTC protein expression produces a strong inflammatory response in experimental animal models (Powell et al., 2003) and activates numerous inflammatory mediators and molecules within thyroid epithelial cells. (Melillo et al., 2005; Puxeddu et al., 2005; Russell et al., 2004; Shinohara and Rothstein, 2004) RET/PTC transformed cells can modify their microenvironment to promote autonomous cell proliferation in neighbouring nonneoplastic thyrocytes. (Melillo et al., 2005) Similarly, ovarian cancer cells can ‘educate’ immune infiltrates to produce the type o f cytokines that will facilitate tumour growth and metastases as well as acquiring immune tolerance. (Chen et al., 2008)

Another hypothesis is that a putative link may exist betw'een X-ray radiation and RET/PTC\ activation analogous to the known association in PTC. Increased risk o f epithelial ovarian carcinoma has been described following diagnostic X-rays (Harlap et al., 2002) and high frequency o f R E T rearrangements following radiation have been observed in PTC. (Klugbauer et al., 1995; Rabes et al., 2000)

M ore specifically preferential RET/PTC\ induction has been associated with X-rays, (M izuno et al., 2000) allowed by spatial juxtaposition o f the R E T and H4 proto-oncogenes, (Nikiforova et al., 2000) w hereas RET/PTC3 rearrangem ents were more prevalently associated with PTCs arising in children o f Belaraus and the Ukraine post-Chernobyl. Interestingly, one o f the patients with a demonstrable RET/PTC rearrangem ent in this study (case 53) had pre-operative chem otherapy, however no association between RET/PTC induction and this form o f therapy has been previously described.

It is interesting to speculate that RET/PTC) activation maybe contributing in part to the adaptation o f a papillary growth pattern. Yap et al (Yap et al., 1997) demonstrated that tyrosine phosphorylation alters thyroid epithelial organisation by interfering with actin-associated adhesive junctions. Under the influence o f tyrosine phosphorylation, thyroid cells lose their capacity to form follicles, spread and migrate into confluent monolayers and hence the capacity to form follicles in-vivo. Fischer et al (Fischer et al., 1998) cultured thyroid cells infected with a 7-<?/roviral vector expressing activated RET/PTC \ and dem onstrated alterations in the nuclear envelope and chromatin structure, found in later work to be induced by RET/PTC during interphase. (Fischer et al., 2003) It is plausible therefore that activated RET/PTC\ may influence the growth pattern in papillary tumours. Interestingly, the tum ours with activated RET, whilst having papillary m orphology did not have the classical nuclear clearing ('Orphan Annie') o f PTC, However, RET/PTC rearrangements have been found in tum ours (i.e. a subset o f Hurthle cell tumours) (Belchetz et al., 2002) that may lack both papillary architecture and/or classic nuclear features. As an adjunct all R E T rearranged cases were negative for mutated BRAF, suggesting distinct alternative pathways in the epipathogenesis o f these tumours akin to PTC. (Soares et al., 2003)

Chapter 3 RET/PTC rearrangement in primar>' peritoneal carcinoma

This study had some technical limitations. Degraded RNA isolated from form alin-fixed paraffin embedded tissue was used. Highly sensitive RT-PCR carries a higher risk o f false-positive amplification compared with other techniques. To minimise the effects o f these limitations, 3 methodologies were employed to identify RET rearrangements. Specifically, interphase FISH and RT-PCR analysis were employed on laser-microdissected cells (reducing the propensity for contam ination by RE T expressing macrophages) after careful review o f the samples before m olecular analysis. The num ber o f tum our nuclei with split FISH signals above background was also limited; a stringent cut-off level o f 10.2% positive cells was employed to detect the chrom osomal rearrangem ent. This cut-off level parallels previous studies which used a cut-off level o f between 5-10% to separate cases from false-positives. (Barr et al., 2002; Unger et al., 2004) Im m unohistochem ical detection o f RET protein expression was used to further corroborate our findings. The chim eric RET/PTC gene encodes a protein product that contains the cytoplasm ic portion o f ret. (Fusco et al., 1987) Therefore, immunohistochemical detection o f the carboxy term inus o f the RET protein should serve as a reliable m arker to detect rearranged RET.

(Cheung et al., 2001; Sugg et al., 1998)

However, one cannot entirely exclude the possibility o f the RET antibody detecting only wild- type RET as it is feasible that a steric change o f the epitope may occur after RE T rearrangement. Indeed, according to Rebelo et al, (Rebelo et al., 2003) positive staining m ay correspond to the expression o f the wild-type RET, RET rearrangement or both. M oreover, problem s with interpreting the specificity o f weak focal immunostaining for ^ 7 " have been reported. (Cerilli et al., 2002; Guyetant et al., 2003) To overcom e this problem we only deemed cases with diffuse

RET im m unostaining as being positive as recommended by previous authors. (Cerilli et al., 2002)

Any possibility that the tumours that expressed rearranged R E T in our series, represented

metastases from the thyroid is diminished given the fact no protein expression o f TTF-1 or

thyroglobulin was detected in any o f these tumours.

Importantly, our results have broad implications for molecular diagnostics. When trying to

diagnose metastatic PTC, the existence o f minor cell subclones with R E T rearrangements in non­

thyroid papillary tumours highlights the importance o f using quantitative methods to detect

RET/PTC rearrangements. In conclusion, this study demonstrates that RET/PTC rearrangement is

present in a small number o f non-thyroid papillary tumours, specifically primary peritoneal and

papillary renal cell carcinoma, therefore RET/PTC detection should not be equated per se with

PTC. It indicates that in some primary peritoneal and papillary renal cell carcinoma, RET/PTC

C hapter 3 R E T /P T C rearran g em en t in prim ary peritoneal carcinom a

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