Molecular Rearrangements and Morphology in Thyroid Cancer

Abstract

Tumor development within the human thyroid gland provides an attractive experimental model with which to consider the pathogenesis of carcinoma, the most common and clinically significant cancer. Thyroid tumors are readily accessible to morphological and molecular study because their primary treatment is surgical. Investigations of thyroid cancer have expanded our knowledge of carcinoma biology. Thyroid carcinoma is the only adult epithelial malignancy in which specific chromosomal rearrangements have been identified.1Fusco A Grieco M Santoro M Berlingieri MT Pilotti S Pierotti MA Della Porta G Vecchio G A new oncogene in human thyroid papillary carcinomas and their lymph-nodal metastases.Nature. 1987; 328: 170-172Crossref PubMed Scopus (351) Google Scholar, 2Grieco M Santoro M Berlingieri MT Melillo RM Donghi R Bongarzone I Pierotti MA Della Porta G Fusco A Vecchio G Ptc is a novel rearranged form of the ret proto-oncogene and is frequently detected in vivo in human thyroid papillary carcinomas.Cell. 1990; 60: 557-563Abstract Full Text PDF PubMed Scopus (833) Google Scholar, 3Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA Pax8-Ppargamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000; 289: 1357-1360Crossref PubMed Scopus (738) Google Scholar RET and PPARγ rearrangements in thyroid carcinoma create fusion proteins that are hypothesized to play fundamental roles in thyroid oncogenesis.3Kroll TG Sarraf P Pecciarini L Chen CJ Mueller E Spiegelman BM Fletcher JA Pax8-Ppargamma1 fusion oncogene in human thyroid carcinoma [corrected].Science. 2000; 289: 1357-1360Crossref PubMed Scopus (738) Google Scholar, 4Santoro M Chiappetta G Cerrato A Salvatore D Zhang L Manzo G Picone A Portella G Santelli G Vecchio G Fusco A Development of thyroid papillary carcinomas secondary to tissue-specific expression of the Ret/Ptc1 oncogene in transgenic mice.Oncogene. 1996; 12: 1821-1826PubMed Google Scholar The fusion protein pathways are prime targets for new strategies directed at improving thyroid cancer diagnosis and treatment.5Mauro MJ O'Dwyer M Heinrich MC Druker BJ Sti571: a paradigm of new agents for cancer therapeutics.J Clin Oncol. 2002; 20: 325-334Crossref PubMed Scopus (197) Google Scholar, 6Druker BJ Perspectives on the development of a molecularly targeted agent.Cancer Cell. 2002; 1: 31-36Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 7Piazza F Gurrieri C Pandolfi PP The theory of Apl.Oncogene. 2001; 20: 7216-7222Crossref PubMed Scopus (94) Google Scholar The RET proto-oncogene is interesting because it is mutated by different mechanisms in different (endocrine and neuroendocrine) thyroid carcinomas. 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Our current concepts of chromosomal rearrangements in cancer are based primarily on investigations of blood cell malignancies. Chromosomal rearrangements in leukemias are genetic hits with complex functional consequences. 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A study reported by Fusco and colleagues22Fusco A Chiappetta G Hui P Garcia-Rostan G Golden L Kinder BK Dillon DA Giuliano A Cirafici A Santoro M Rosai J Tallini G Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.Am J Pathol. 2002; 160: 2157-2167Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar in this issue of The American Journal of Pathology correlates RET rearrangements with specific morphological patterns in thyroid tumors. The motivations are laudable but the undertaking is difficult because thyroid and other epithelial tumors typically consist of neoplastic cells intermingled irregularly with normal (connective tissue and vascular) and reactive (stromal and immune) cells in a solid tu-mor mass. Robust methods to analyze specific cell subpopulations within fresh and fixed epithelial tumors are needed.23Maitra A Wistuba II Virmani AK Sakaguchi M Park I Stucky A Milchgrub S Gibbons D Minna JD Gazdar AF Enrichment of epithelial cells for molecular studies.Nat Med. 1999; 5: 459-463Crossref PubMed Scopus (58) Google Scholar, 24Sugiyama Y Sugiyama K Hirai Y Akiyama F Hasumi K Microdissection is essential for gene expression profiling of clinically resected cancer tissues.Am J Clin Pathol. 2002; 117: 109-116Crossref PubMed Scopus (68) Google Scholar A main reason that blood cell malignancies have been so amenable to new molecular genetic approaches is that >90% pure populations of well-defined, viable tumor cells can often be obtained.25Golub TR Slonim DK Tamayo P Huard C Gaasenbeek M Mesirov JP Coller H Loh ML Downing JR Caligiuri MA Bloomfield CD Lander ES Molecular classification of cancer: class discovery and class prediction by gene expression monitoring.Science. 1999; 286: 531-537Crossref PubMed Scopus (9157) Google Scholar, 26Ferrando A Neuberg D Staunton J Loh M Huard C Raimondi S Behm F Pui C Downing J Gilliland D Lander E Golub T Look A Gene expression signatures define novel oncogenic pathways in T cell acute lymphoblastic leukemia.Cancer Cell. 2002; 1: 75-87Abstract Full Text Full Text PDF PubMed Scopus (882) Google Scholar, 27Armstrong SA Staunton JE Silverman LB Pieters R den Boer ML Minden MD Sallan SE Lander ES Golub TR Korsmeyer SJ Mll translocations specify a distinct gene expression profile that distinguishes a unique leukemia.Nat Genet. 2002; 30: 41-47Crossref PubMed Scopus (1587) Google Scholar, 28Rosenwald A Alizadeh AA Widhopf G Simon R Davis RE Yu X Yang L Pickeral OK Rassenti LZ Powell J Botstein D Byrd JC Grever MR Cheson BD Chiorazzi N Wilson WH Kipps TJ Brown PO Staudt LM Relation of gene expression phenotype to immunoglobulin mutation genotype in B cell chronic lymphocytic leukemia.J Exp Med. 2001; 194: 1639-1647Crossref PubMed Scopus (927) Google Scholar Immunohistochemistry was performed by Fusco and colleagues22Fusco A Chiappetta G Hui P Garcia-Rostan G Golden L Kinder BK Dillon DA Giuliano A Cirafici A Santoro M Rosai J Tallini G Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.Am J Pathol. 2002; 160: 2157-2167Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar with an affinity-purified polyclonal antiserum to detect RET protein expression. Because RET is not synthesized in normal follicular epithelial cells, immunoreactivity is thought to correlate with overexpression of the rearranged RET gene. However, the possibilities of confounding wild-type RET protein within thyroid tumors29Bunone G Uggeri M Mondellini P Pierotti MA Bongarzone I Ret receptor expression in thyroid follicular epithelial cell-derived tumors.Cancer Res. 2000; 60: 2845-2849PubMed Google Scholar, 30Fluge O Haugen DR Akslen LA Marstad A Santoro M Fusco A Varhaug JE Lillehaug JR Expression and alternative splicing of C-Ret RNA in papillary thyroid carcinomas.Oncogene. 2001; 20: 885-892Crossref PubMed Scopus (36) Google Scholar, 31Kjellman P Learoyd DL Messina M Weber G Hoog A Wallin G Larsson C Robinson BG Zedenius J Expression of the Ret proto-oncogene in papillary thyroid carcinoma and its correlation with clinical outcome.Br J Surg. 2001; 88: 557-563Crossref PubMed Scopus (41) Google Scholar and/or cross-reactivity of the antiserum with receptors bearing homologous tyrosine kinase domains need to be kept in mind. The immunohistochemistry was coupled with reverse transcriptase-polymerase chain reaction and laser capture microdissection of paraffin-embedded tumor tissues to assess RET rearrangement status. Degraded mRNA in paraffin-embedded tissue reduces sensitivity and in this study control housekeeping transcripts were detected in only 7 of 14 cases, a highly select sample of the original 46 cases. Such high-cycle nested polymerase chain reaction and hybridization protocols are prone to contamination and low specificity, although appropriate negative and positive controls were reported. A common source of contamination/false-positives is purified plasmids and/or cell lines harboring rearrangements that are used to test assay sensitivity or to perform functional studies. Low specificity is often suggested by artifactual bands and smearing seen with ethidium bromide staining. The androgen receptor clonality assay was also conducted on DNA obtained from manually dissected paraffin-embedded tissue, and it is somewhat surprising that all four follicular adenomas were polyclonal. Nearly all follicular adenomas in the literature are monoclonal.32Namba H Matsuo K Fagin JA Clonal composition of benign and malignant human thyroid tumors.J Clin Invest. 1990; 86: 120-125Crossref PubMed Scopus (123) Google Scholar, 33Hicks DG LiVolsi VA Neidich JA Puck JM Kant JA Clonal analysis of solitary follicular nodules in the thyroid.Am J Pathol. 1990; 137: 553-562PubMed Google Scholar, 34Moniz S Catarino AL Marques AR Cavaco B Sobrinho L Leite V clonal origin of non-medullary thyroid tumours assessed by non-random X-chromosome inactivation.Eur J Endocrinol. 2002; 146: 27-33Crossref PubMed Scopus (41) Google Scholar On the other hand, this may be a special mixed group of tumors. Two of the hyperplastic nodules were monoclonal and two were polyclonal, consistent with the clonality reported in the literature.35Kopp P Kimura ET Aeschimann S Oestreicher M Tobler A Fey MF Studer H Polyclonal and monoclonal thyroid nodules coexist within human multinodular goiters.J Clin Endocrinol Metab. 1994; 79: 134-139Crossref PubMed Scopus (121) Google Scholar, 36Apel RL Ezzat S Bapat BV Pan N LiVolsi VA Asa SL Clonality of thyroid nodules in sporadic goiter.Diagn Mol Pathol. 1995; 4: 113-121Crossref PubMed Scopus (93) Google Scholar, 37Chung DH Kang GH Kim WH Ro JY Clonal analysis of a solitary follicular nodule of the thyroid with the polymerase chain reaction method.Mod Pathol. 1999; 12: 265-271PubMed Google Scholar Based on these considerations, this study must be interpreted conservatively and additional experiments with fresh tissue (fragments or frozen sections) and complimentary methods such as fluorescence in situ hybridization will be needed to corroborate RET rearrangement at the DNA level. Putting aside potential technical caveats, the data of Fusco and colleagues22Fusco A Chiappetta G Hui P Garcia-Rostan G Golden L Kinder BK Dillon DA Giuliano A Cirafici A Santoro M Rosai J Tallini G Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.Am J Pathol. 2002; 160: 2157-2167Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar show that RET rearrangement can occur within “morphologically benign” thyroid nodules. Two main tumor groups were seen. In the first group, three tumors exhibited RET rearrangement in focal areas of well-developed papillary carcinoma-like morphological change (type A), consistent with the idea that these are small papillary carcinomas arising within benign thyroid nodules. This contention would not be surprising to most pathologists who often observe small papillary carcinoma-like foci dispersed widely within thyroid tissue in both surgery and autopsy material. The distribution of these foci within the nodules seems inconsistent with the theoretical possibility that they preceded growth of the remaining tumor. In fact, all three tumors had foci with RET/PTC1 rearrangement, which is the most prevalent rearranged RET form in the small classic and occult/microcarcinoma38Viglietto G Chiappetta G Martinez-Tello FJ Fukunaga FH Tallini G Rigopoulou D Visconti R Mastro A Santoro M Fusco A Ret/Ptc oncogene activation is an early event in thyroid carcinogenesis.Oncogene. 1995; 11: 1207-1210PubMed Google Scholar, 39Tallini G Santoro M Helie M Carlomagno F Salvatore G Chiappetta G Carcangiu ML Fusco A Ret/Ptc oncogene activation defines a subset of papillary thyroid carcinomas lacking evidence of progression to poorly differentiated or undifferentiated tumor phenotypes.Clin Cancer Res. 1998; 4: 287-294PubMed Google Scholar, 40Sugg SL Ezzat S Rosen IB Freeman JL Asa SL Distinct multiple Ret/Ptc gene rearrangements in multifocal papillary thyroid neoplasia.J Clin Endocrinol Metab. 1998; 83: 4116-4122Crossref PubMed Scopus (257) Google Scholar papillary carcinoma subtypes. Two of these three nodules were polyclonal, as would also be expected if they contained co-existing tumors. Thus, tumor morphology and molecular genetics seem concordant in this group. In the second group, two of six tumors with widespread but weak papillary carcinoma-like morphological change (type D) exhibited RET/PTC3 rearrangement, consistent with the idea that these are carcinomas with variable papillary nuclear morphology. In support of this possibility, one tumor was monoclonal, as would be expected for a papillary carcinoma. The other tumor could not be tested. Two other tumors with type D morphology were negative for RET rearrangement and the remaining two tumors were not tested. Thus, tumor morphology and molecular genetics seem discordant in this group. Interestingly, other recent studies affirm a discordance between RET rearrangement status and papillary thyroid carcinoma-like morphology in thyroid tumors. For example, up to 50% of Hurthle cell thyroid tumors harbor RET rearrangements,41Chiappetta G Toti P Cetta F Giuliano A Pentimalli F Amendola I Lazzi S Monaco M Mazzuchelli L Tosi P Santoro M Fusco A The Ret/Ptc oncogene is frequently activated in oncocytic thyroid tumors (Hurthle cell adenomas and carcinomas), but not in oncocytic hyperplastic lesions.J Clin Endocrinol Metab. 2002; 87: 364-369Crossref PubMed Scopus (77) Google Scholar, 42Cheung CC Ezzat S Ramyar L Freeman JL Asa SL Molecular basis of Hurthle cell papillary thyroid carcinoma.J Clin Endocrinol Metab. 2000; 85: 878-882Crossref PubMed Scopus (116) Google Scholar despite the widely held view that they are more closely related to follicular tumors than to papillary carcinoma. In addition, papillary carcinomas containing RET/PTC3 rearrangement exhibit a spectrum of histologies (classic, solid, and tall cell) that have different biological tendencies.43Nikiforov YE Rowland JM Bove KE Monforte-Munoz H Fagin JA Distinct pattern of Ret oncogene rearrangements in morphological variants of radiation-induced and sporadic thyroid papillary carcinomas in children.Cancer Res. 1997; 57: 1690-1694PubMed Google Scholar, 44Thomas GA Bunnell H Cook HA Williams ED Nerovnya A Cherstvoy ED Tronko ND Bogdanova TI Chiappetta G Viglietto G Pentimalli F Salvatore G Fusco A Santoro M Vecchio G High prevalence of Ret/Ptc rearrangements in Ukrainian and Belarussian post-Chernobyl thyroid papillary carcinomas: a strong correlation between Ret/Ptc3 and the solid-follicular variant.J Clin Endocrinol Metab. 1999; 84: 4232-4238Crossref PubMed Scopus (226) Google Scholar, 45Basolo F Giannini R Monaco C Melillo RM Carlomagno F Pancrazi M Salvatore G Chiappetta G Pacini F Elisei R Miccoli P Pinchera A Fusco A Santoro M Potent mitogenicity of the Ret/Ptc3 oncogene correlates with its prevalence in tall-cell variant of papillary thyroid carcinoma.Am J Pathol. 2002; 160: 247-254Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar Thyroid tumors with mixed morphological and clinical features of papillary and follicular carcinoma have also been described.46Baloch ZW LiVolsi VA Encapsulated follicular variant of papillary thyroid carcinoma with bone metastases.Mod Pathol. 2000; 13: 861-865Crossref PubMed Scopus (129) Google Scholar These findings suggest that there is an imprecision in our morphological categorization of thyroid tumors and that unknown cellular factors cooperate with RET rearrangement to determine papillary carcinoma morphology and biology. The overall findings of Fusco and colleagues22Fusco A Chiappetta G Hui P Garcia-Rostan G Golden L Kinder BK Dillon DA Giuliano A Cirafici A Santoro M Rosai J Tallini G Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.Am J Pathol. 2002; 160: 2157-2167Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar suggest that we have much to learn regarding thyroid cancer pathogenesis and the degree to which thyroid tumor morphology correlates with underlying molecular genetic events. The imprecise nature of papillary carcinoma morphology makes pathological diagnosis of thyroid tumors difficult. The issue is complicated by varied approaches of pathologists to follicular-patterned thyroid tumors.47Renshaw AA Gould EW Why there is the tendency to “overdiagnose” the follicular variant of papillary thyroid carcinoma.Am J Clin Pathol. 2002; 117: 19-21Crossref PubMed Scopus (38) Google Scholar, 48Chan JK Strict criteria should be applied in the diagnosis of encapsulated follicular variant of papillary thyroid carcinoma.Am J Clin Pathol. 2002; 117: 16-18Crossref PubMed Scopus (136) Google Scholar, 49Baloch ZW Livolsi VA Follicular-patterned lesions of the thyroid: the bane of the pathologist.Am J Clin Pathol. 2002; 117: 143-150Crossref PubMed Scopus (217) Google Scholar Even so, it is likely such imprecision has little clinical impact because nearly all low-stage thyroid cancers have excellent prognosis. Whereas it might be informative from a biological perspective to better define the biology of the tumors in this study, from a clinical perspective they will likely behave at worst like well-differentiated thyroid cancer, a readily curable disease. In fact, 80 to 90% of thyroid nodules now removed by surgery are benign, and we therefore may actually be overtreating many patients with thyroid nodules to exclude the possibility of cancer. The most rational long-term clinical goal is to increase our ability to differentially diagnose benign from malignant/precursor thyroid nodules before surgery so that appropriate, more individualized treatments can be rendered. In summary, the results of Fusco and colleagues22Fusco A Chiappetta G Hui P Garcia-Rostan G Golden L Kinder BK Dillon DA Giuliano A Cirafici A Santoro M Rosai J Tallini G Assessment of RET/PTC oncogene activation and clonality in thyroid nodules with incomplete morphological evidence of papillary carcinoma: a search for the early precursors of papillary cancer.Am J Pathol. 2002; 160: 2157-2167Abstract Full Text Full Text PDF PubMed Scopus (118) Google Scholar suggest that some thyroid nodules with a predominance of benign morphological features have RET rearrangement. Techniques such as fluorescence in situ hybridization will be needed to document the existence, frequency, and geographic distribution of RET rearrangements in these relatively rare tumors. The study highlights an imprecision in our morphological classification of papillary carcinoma-like tumors, making it more likely that molecular endocrine tumor markers will help us subdivide thyroid and other endocrine tumors into more distinct biological subgroups. Regardless of whether morphological or molecular markers are considered, their clinical utility is dependent strictly on tumor biology and clinical context. It is therefore critical that well-organized clinical databases containing comprehensive patient information and clinical follow-up data be constructed to rigorously define clinicopathological correlates of putative biomarkers identified with the new molecular genetic techniques.50Becich MJ The role of the pathologist as tissue refiner and data miner: the impact of functional genomics on the modern pathology laboratory and the critical roles of pathology informatics and bioinformatics.Mol Diagn. 2000; 5: 287-299PubMed Google Scholar Thyroid cancer is no exception in this respect.