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Molecular-cytogenetic characterization of head and neck cancer: Identification of novelprognostic factors and gene targets for therapy [double dissertation 2]

Wreesmann, V.B.; Singh, B.

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Citation for published version (APA):Wreesmann, V. B., & Singh, B. (2004). Molecular-cytogenetic characterization of head and neck cancer:Identification of novel prognostic factors and gene targets for therapy [double dissertation 2].

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Download date: 04 Oct 2020

Chapterr 6

Molecular-Cytogeneticc Characterization of Chernobyl-associatedd Thyroid Neoplasia in

Children n

Volkertt B. Wreesmann, Ronald A. Ghossein, Attil a Omeroglu, Tania Bogdanova,, Maria E. Dudas, Axel Hoos, Alexander Stojadinovic, Jatinn P. Shah, Pulivarthi H. Rao, R. Michael Tuttle, Bhuvanesh Singh h

Acceptedd for Publication to Head and Neck in 2004

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Molecularr Profiles of Radiation-Induced Thyroid Cancer

Abstract t Background:: Although the 1986 Chernobyl nuclear power plant accident helped to solidify the causall relationship between radiation exposure and development of thyroid neoplasia, the molecularr pathogenesis underlying the increase in tumorigenesis remains ill defined. We assessedd the contribution of chromosomal instability and abrogation in the components of the pS3pS3 pathway to the pathogenesis of Chernobyl-associated thyroid neoplasms in patients from the Ukrainee and The Russian Federation presenting during the period between 1995 and 1998. Methods:: Chromosomal aberrations were investigated in 28 Chernobyl-associated papillary thy-roidd carcinoma samples using comparative genomic hybridization (CGH). The expression of p53>p53> MDM.2,p21, Bcl-2, Cyclin Dl and Ki-67 was assessed in 73 Chernobyl-associated thyroid tumorss arrayed on tissue microarrays (TMA) by immunohistochemistry. Results:: Chromosomal instability was detected in 2 of 28 PTC (7%). Chromosomal aberrations includedd losses of chromosomal regions 6ql2-15,13q21-31,16q22-24 and X21 -26. p53 expression wass not detected in any of the radiation-induced tumors. However, dysregulation of several memberss of thep55 pathway was identified, including MDM2, p21 and BCL2 in 62%, 48% and 29%% of cases, respectively. Conclusions:: Our data suggest that chromosomal instability does not play a significant role in thee pathogenesis of radiation induced thyroid neoplasms. Although p53 aberrations are rare, expressionn of other members of the pS3 pathway is commonly abrogated, suggesting that this pathwayy plays a role in radiation-induced thyroid cancer pathogenesis.

A nn association between radiation exposure and childhood thyroid neoplasia wass first suggested in the early 1950s, with the description of antecedent his-toryy of radiation exposure in 10 of 28 children presenting with thyroid can-

cerr to Memorial Hospital in New York City.[7] The most persuasive clinical evidence implicatingg a causal role for radiation in thyroid tumorigenesis comes from the 1986 Chernobyll nuclear power plant accident.[2] The fallout from the reactor exposed the surroundingg population to large quantities of radioiodines, which are incorporated into thee thyroid gland after inhalation and/or ingestion. As a result, a more than 100-fold increasedd incidence of childhood papillary thyroid carcinomas (PTC) has been observed inn the population from highly exposed regions including parts of Belarus, Ukraine and Thee Russian Federation, with the first cases appearing as early as 4 years after expo-sure.. [3] Although the Chernobyl accident has substantiated the causal relationship betweenn radiation exposure and the development of thyroid neoplasms (PTC and fol-licularr thyroid adenomas), the mechanisms underlying tumorigenesis remain ill defined. .

InIn vitro evidence suggests that radiation exerts its carcinogenic effect through the inductionn of genomic damage.[4-6] Accordingly, prior studies focused on identification off genetic aberrations in radiation-induced thyroid cancers in an attempt to define underlyingg pathogenetic mechanisms.[7-77] A landmark study by Zitzelsberger and col-leaguess identified a unique recurrent pattern of structural chromosomal alterations in radiation-inducedd thyroid cancers using conventional cytogenetic karyotyping.[72] In

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addition,, Richter and colleagues detected an array of genetic abnormalities in Chernobyl-associatedd PTC including loss of heterozygosity (LOH) events and instabili-tyy of microsatellite repeats (MSI).[13] The accumulation of DNA damage in radiation inducedd thyroid cancers appears to be independent of p53 dysfunction, as prior studies demonstratee a low rate of pS3 abrogation in these tumors.[l 4-16] However, no studies havee performed an unselected, genome-wide assessment of Chernobyl associated thyroid neoplasia.. In addition the p53 signaling pathway, which is involved in the response to radiationn induced DNA damage through the regulation of cell cycle control, apoptosis andd genomic stability [17], has not been assessed inclusively. We applied comparative genomicc hybridization (CGH) [18] to study the molecular-cytogenetic composition of Chernobyl-associatedd neoplasms. In addition, we assessed the integrity of thepSJ path-wayy including p53, MDM2, p27, Bcl2, cyclin Dl and ki-67 by immunohistochemical analysiss of Chernobyl-associated thyroid tumors arrayed on tissue microarrays (TMA)[79]] to begin to define the pathogenetic mechanisms of radiation induced thyroid neoplasms. .

Materialss and Methods: Casee collection and clinicopathological profile:

Thee study population included 85 cases of radiation-induced thyroid neoplasia; 27 follicularr adenomas and 58 papillary carcinomas. Patients with PTC included 42 females andd 16 males. Patients with follicular adenoma included 21 females and 9 males. All patientss included were children below the age of 15 that had been exposed to radioactive falloutt from the Chernobyl nuclear power plant accident in 1986. Patients with PTC rangedd in age between 9 and 15 years with a median of 12 years. Patients with follicular adenomaa ranged in age between 2 and 15 years with a median of 13 years. At the time of thee Chernobyl accident, 73 of our patients were living in the oblasts of Kiev, Chernigov, Zhytomyr,, Sumy, Nikolaev, and Volyn in the Ukraine and 12 patients were living in Bryansk,, Russia. All of these patients underwent thyroidectomy between 1995 and 1998. Patientss with PTC ranged in age between 1 and 16 years. The clinicopathological and morphologicall characteristics of our cases have been described previously.[20] Comparativee genomic hybridization:

Twenty-eightt cases of radiation-induced papillary carcinomas were analyzed by CGH.. This cohort included 12 cases from Russia and 16 cases from the Ukraine, based onn availability of cancer tissue. Genomic DNA was extracted from paraffin-embedded tumorr tissue as described previously.[2/] Tumor DNA was labeled by nick translation (Lif ee Technologies, Inc., Rockville, MD) with fluorescein- 12-dUTP. Reference DNA wass extracted from normal placenta and labeled with Texas red-5-dUTP (New England Nuclear-DuPont,, Boston, MA). We have previously validated the use of paraffin embed-dedd tissue derived DNA for CGH in our laboratory. [22] CGH was performed as described.[23,, 24] For analysis, 7-10 separate metaphases were captured and processed usingg the Quantitative Image Processing System (Quips Pathvysion System; Applied Imaging,, Santa Clara, CA). Red, green, and blue fluorescence intensities were analyzed

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forr all metaphase spreads, normalized to a standard length, and statistically combined to showw the red:green signal ratio and 95% confidence intervals for the entire chromosome. DNAA copy number changes were detected based on the variance of the red:green ratio profilee from the standard of 1. Ratio values of 1.2 and 2.0 were defined as thresholds for gainss and amplifications, respectively, and losses were defined as a ratio value of 0.8 or less. . Tissuee micro-array construction and immunohistochemical analysis:

AA tissue micro-array containing 73 radiation-induced tumors from the Ukraine was constructedd as described in detail previously. [25] Briefly, two experienced pathologists conductedd a critical histologic slide review to confirm the diagnosis and identify repre-sentativee areas containing over 70% tumor cells in 73 specimens. Corresponding tissue blockss were collected and from the defined areas core biopsies were arrayed in triplicate onn a recipient paraffin block using a precision instrument (Beecher Instruments, Silver Spring,, MD). Five-/xm sections of these tissue array blocks were cut and placed on chargedd poly-L-lysine-coated slides. Thee content of individual cores was confirmedd by review of hematoxylin andd eosin stained slides every ten sections.. These sections were sub-jectedd to immunohistochemical analysis.. Normal thyroid tissue from patientss with sporadic papillary car-cinomass operated on at Memorial Sloan-Ketteringg Cancer Center servedd as baseline controls.

Mousee antihuman monoclonal antibodiess to p53 (Ab-2, clone 1801, 1:500;; Calbiochem, Cambridge MA), MDM 22 (Clone 2A10, 1:500; kindly providedd by Dr. A. Levine, Rockefellerr University, New York, NY),, £27 (Ab-1, clone EA10, 1:100; Calbiochem),, cyclin Dl (Ab-3, clone DCS-6,1:100; Calbiochem), Bcl-2 (clone 124,1:72; DAKO,, Glostrup, Denmark), and Ki-67 (Mib-1, 1:50; Immunotech, Marseille, France) weree used for immunohistochemistry as described in detail previously.[26] Tissue loss iss a significant factor for tissue array-based analysis, with previously reported rates of tis-suee damage ranging from 3 to 33%. [25, 27-29] In our analysis, the rate of lost cases attrib-utablee to tissue damage was 3-17% for the individual markers. Immunoreactivity was classifiedd as continuous data (undetectable levels or 0% to homogeneous staining or 100%)) for all markers. For every marker, the entire tumor tissue of the three core sec-tionss was evaluated. A consensus score was obtained between three investigators reading

Figuree 1. Ideogram showing DNA copy number changes identified byy comparative genomic hybridization in 28 cases of Chernobyl-associatedd papillary thyroid carcinomas . Thin vertical lines on eitherr side of the ideogram indicate losses (left) and gains (right) of thee chromosomal region. High-level amplifications were not identi-fied. fied.

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slidess under a multi-headed microscope (VBW, RAG, AO). Cut-off values used in this studyy were based on previously established cut-off values for well-characterized anti-bodiess used in our laboratory. [26] The cut-off values for tumor cell staining were defined ass follows: 1) high Ki-67 proliferative index if >5% tumor nuclei stained; 2)p53 nuclear overexpressionn if >5% tumor nuclei stained; 3) MDM2 overexpression if >50% tumor nucleii stained; 4) cyclin Dl overexpression if >5% of tumor nuclei stained; 5) Bcl-2 overexpressionn if >50% of tumor cells demonstrated cytoplasmic staining; 6)p21 over-expressionn if > 10% of tumor nuclei stained. Tumors were then grouped into two cate-goriess defined as follows: normal expression (neoplasms below the defined cut-off value off immunoreactivity in normal, benign, and tumor cells) and abnormal expression (nor-mall and neoplastic tissues above the defined cut-off values of immunoreactivity). Statisticall analysis:

Statisticall analyses were performed using the JMP4 statistical software package (SAS Institutee Ine, Cary, NC). Qualitative and quantitative differences between malignant andd benign thyroid tumors within our series were assessed using the Fisher's exact test andd Mann-Whitney £7-test, respectively. Statistical significance was defined as a two-tailedd p-value less than or equal to 0.05. Where appropriate, thep-value for accepting sig-nificancee was adjusted for the effect of multiple comparisons by Bonferroni's method. Resultss and Discussion:

Inn this study, we assessed the contribution of chromosomal instability and abroga-

TableTable 1. Molecular expression profiles of cell-cycle regulators in thyroid neoplasms (n=85) andd normal thyroid tissue (n = 33)

Marker r

p53 p53 Negative e Positive e

mdm-2 2 Negative e Positive e

p21 p21 Negative e Positive e

Bcl-2 2 Negative e Positive e

Cyclinn Dl Negative e Positive e

Normal l

31 1 0 0

29 9 0 0

28 8 0 0

1 1 31 1

31 1 0 0

RI-FA A

23 3 0 0

10 0 16 6

23 3 2 2

5 5 21 1

25 5 0 0

RI-PTC C

50 0 0 0

18 8 30 0

25 5 23 3

15 5 36 6

51 1 0 0

p-p- value

NS S

NS S

0.008* *

NS S

NS S

Normal,, matched normal thyroid tissue; RI-A, radiation-induced follicular thyroid adenoma; RI-PTC, radiation-inducedd papillary thyroid carcinoma

Numberr of patients in subgroups less than total number in respective group reflects tissue loss during tissue microar-rayy processing

*p-valuee of comparison between benign tumors vs. malignant tumors (Fischer's exact test)

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tionn in the components of the pS3 pathwayy to the pathogenesis of _ Chernobyl-associatedd thyroid neo-plasmss in patients from the Ukraine , #>(Kiev,, Chernigov, Zhytomyr, Sumy, » / ^ V "* Nikolaev,, and Volyn) and The l"« -1 Russiann Federation (Bryansk) pre- ? *

AA ,-U A U » Kl67 p21

sentingg during the period between - -19955 and 1998. The etiological ,'JT impactt of the Chernobyl accident on s" tumorigenesiss in these children is supportedd by the virtual absence of PTCC and follicular adenomas in chil- * . , drenn from these regions that were CDI Bci2 bornn at least 2 years after the , - < , - « . - -\ Chernobyll accident.[30] Application ; * lflf* J _ V* % ' off CGH to the genetic analysis of 28 „ ' p" -t* %y ̂ m** jjV^ casess detected chromosomal abnor- „\* * -" * ^*! malitiess in 2 out of 28 cases (7%). «*/ ?t» **/. Bothh of these cases had 2 chromoso- „ Ë,n,„ ? ' i f :

p5ii 'MDM 2 » * r L 4 *

mall abnormalities each, which are d e t a i l e dd i n F i g u r e 1. U n e Case e x h l b - Figure 2. Immunohistochemical analysis in radiation induced thy-

itedd losses Of chromosomal regions r o i d tumors showing representative results.

6ql2-155 and 16q22-24 and another casee had losses of 13q21-31 and Xq21-26. Because the aberrations were non-recurrent, wee cannot rule out the possibility that they represent random genetic events. However, itt is of note that Zitzelsberger and colleagues detected recurrent deletions of 13q to be associatedd with radiation-induced thyroid carcinogenesis both in vitro as well as in vivo.[12,vivo.[12, 31] In addition, Bauer and colleagues previously reported deletions of chromo-somess 6q and 13q in a case of PTC with a history of external beam radiation. [32] Taking intoo account that these chromosomal abnormalities are rarely present in non-radiation associatedd PTC,[33, 34] it is possible that these regions may harbor tumor suppressor geness involved in radiation-induced carcinogenesis. However, it needs to be taken into accountt that accurate comparison of radiation-induced PTC to sporadic PTC would requiree a matched-pair analysis, a difficult undertaking given the rarity of sporadic PTC inn children. Nonetheless, although the carcinogenic effect of radiation is mediated throughh the induction of genomic instability, it appears from our data that Chernobyl-associatedd PTC from the Ukraine and Russia do not exhibit a higher frequency of DNA copyy number alterations relative to sporadic PTC from Europe and the USA. [22, 32-35]

Ourr observations sharply contrast with previous findings suggesting that radiation inducedd PTC commonly contain chromosomal alterations.[7, 12, 13] Zitzelsberger and

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colleaguess recently detected multiple structural chromosomal alterations in a panel of 56 Chernobyll associated childhood thyroid tumors from the Gomel region of Belarus.[72] Inn addition, Richter and colleagues detected multiple LOH and MSI events in Belarusiann cases.[73] The difference with our data may exist for several different reasons. Firstly,, it is possible that the lower rate of chromosomal alterations detected in this studyy reflects differences in radiation doses received by patients living in the Ukraine andd Russian Federation (intermediate dose) and those received by patients living in Belaruss (high dose) at the time of the accident.[3, 36] Since induction of chromosomal changess is linearly associated with radiation dose,[7, 12] the higher dose received by Belarusiann children may account for the more complex karyotypes detected in Belarusiann PTC. Alternatively, it is possible that the degree of chromosomal instability characterizingg post-Chernobyl papillary carcinomas is influenced by the latency period betweenn the Chernobyl accident and the time of clinical presentation. For example, the populationn studied by Zitzelsberger and colleagues includes cases appearing in the years betweenn 1993 and 1996 whereas our study includes cases operated on in the years betweenn 1995 and 1998. Taking into account that chromosomal instability is strongly associatedd with biologic potential in cancers from various sources including sporadic thyroidd cancer,[22, 37] it is possible that early occurring, more aggressive post-Chernobyll cases are characterized by higher rates of chromosomal instability relative to thosee occurring after longer latency periods. This hypothesis is supported by findings fromm Lohrer and colleagues reporting an association between the length of the latency periodd and degree of microsatellite instability in PTC from Belarus.[38] Several other factorss may account for the difference in detection rate of chromosomal instability betweenn studies including differences in resolution between the various molecular-cyto-geneticc techniques that were used (LOH, CGH, conventional cytogenetic karyotyping, microsatellitee instability, FISH).[39, 40]

Inn addition to the presence of chromosomal instability, we assessed the integrity of thee p53 pathway in Chernobyl-associated neoplasia. The p53 gene has been shown to playy a pivotal role in cellular protection against malignant transformation through its abilitiess to control the cell cycle, induce apoptosis and safeguard genomic stability.[7 7] Inn case of genomic damage, the p53 protein may arrest the cell cycle to allow DNA repair orr induce apoptosis through a cascade of downstream factors.[17] Accordingly, the crit-icall role of p53 is evident from the fact that it is mutated in a large fraction of human malignancies.[47]] Previous studies have shown that p53 mutations are rare in Chernobyl-associatedd thyroid neoplasia. [14-16] However, these studies have not taken intoo account the role of other components of the pS3 pathway in Chernobyl-associated neoplasia.. We characterized the expression patterns of p53 and some of its most impor-tantt cellular effectors including Murine double minute 2 (MDM2),[42] p21,[43] cyclin Dl,[44]] Bcl-2,[45] and Ki-6746 in a morphologically well-characterized cohort includ-ingg 73 radiation-induced thyroid tumors (follicular adenomas and PTC) arrayed using tissuee microarray technology. Our findings are detailed in Table I and Figure II .

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Ourr findings corroborate prior studies showing an absence of p53 aberrations in Chernobyl-associatedd thyroid tumors.[74-76] However, our data suggest that dysregula-tionn of cell cycle control and apoptosis in Chernobyl associated thyroid cells may be achievedd through abrogation of effectors of p53 pathway, for example, we found that a largee fraction (62%) of cases featured over expression of MDM2, a p53 binding protein thatt shuttles p53 into degradative pathways.[42] Amplification and overexpression of MDM 22 has been shown as an important mechanism to dysregulate p53 in tumors with wildtypee p53.[47] Importantly, we did not find significant differences between malig-nantt tumors (63%) and benign adenomas (62%) indicating that MDM2 dysregulation mayy be an early event in the development of thyroid neoplasia.

Inn addition to a high rate of MDM2 dysregulation, we detected p21 abnormality in 48%% of Chernobyl associated PTC. p21 is a key downstream p53 target that is transcrip-tionallyy regulated by p53.[43] In case of genomic damage, pS3 mediates cell cycle arrest throughh p21. [43] However, several lines of evidence support the presence of a p53 inde-pendentt pathway for p21 activation with divergent biological and clinical activities. [4S] Interestingly,, we found that expression of p21 was rare in benign adenomas and very commonn in PTC CP=0.008), suggesting that p21 dysregulation may contribute to the malignantt phenotype of the radiation-induced thyroid tumors. No significant correla-tionn was present between MDM2 expression and p21 expression. Our findings suggest thatt p21 is dysregulated independent of pS3 or MDM2 in radiation-induced thyroid neoplasia. .

Cyclin-Dll is an important antagonist of pS3 functional activity.[49] Cyclin Dl reg-ulatess the Gl checkpoint of the cell cycle, which is the point of arrest by p53. Cyclin Dl amplificationn and subsequent overexpression is associated with cell cycle dysregulation inn a wide variety of tumors including sporadic thyroid cancers.[50] Our data show that cyclinn Dl dysregulation is rare in Chernobyl associated thyroid tumors, suggesting that celll cycle dysregulation occurs independently.

Inn addition to regulation of the cell cycle, p53 mediates regulation of apoptosis in partt through the BAX/Bcl-2 apoptotic pathway. [45, 51] In this balanced system, Bcl-2 inhibitss apoptosis and promotes tumorigenesis while BAX acts as a pro-apoptotic factor. Basedd on previous studies, a cut-off value of for Bcl-2 expression was defined as > 50% tumorr cells being positive for Bcl-2.[26] As described previously, normal thyroid tissue wass characterized by strong Bcl-2 expression, contrary to observations made in other tumorr types.[52] This paradoxical finding could hypothetically be explained by the simultaneouslyy strong expression of pro- and anti-apoptotic components of the apoptot-icc pathway in normal thyroid tissue. In tumors, the balance of these counteracting mol-eculess may be shifted in favor of inhibiting apoptosis. This hypothesis is supported by recentt in vivo studies in mice using gain and loss of function models of BAX and BC1-2,[57]] but needs to be confirmed in the context of the human thyroid. Indeed, in Chernobyll associated neoplastic thyroid tissues, we found downregulation of Bcl-2 in 19%% of adenomas and 29% of papillary carcinomas. The difference between adenomas

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andd carcinomas was not significant. BCL-2 under-expression did not correlate with expressionn of any other factors that we investigated.

Ki-677 is a nuclear factor associated with cellular proliferation.[46] We chose to inves-tigatee the expression of Ki-67 since it is affected by a variety of pathways involved in cell cyclee regulation. Moreover, Ki-67 expression has been shown to correlate with clinical behaviorr in a variety of human tumors including thyroid carcinomas.[52-54] Sporadic PTCC and follicular adenomas rarely manifest Ki-67 overexpression consistent with their loww biologic potential. Interestingly, our data indicate an absence of Ki-67 expression in radiation-inducedd thyroid cancers. The absence of Ki-67 in our cases suggests that radi-ation-inducedd tumors have an equally low-grade nature as their sporadic counterparts, suggestingg excellent overall survival in this patient population.

Inn conclusion, our data suggest that unbalanced chromosomal abnormalities are rare inn Chernobyl associated thyroid tumors from Ukraine and Russia. In addition, our find-ingss corroborate the paucity of p53 alterations in Chernobyl-associated thyroid neo-plasms.. Dysregulation of cell cycle control, apoptosis and genomic stability may be achievedd through abrogation of other effectors in the p53 pathway, including MDM2, p21p21 and Bcl2. Additional investigations of the molecular-genetic composition of Chernobyll associated cancers are required to further define the involved oncogenetic pathways. .

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Acknowledgments s Bhuvaneshh Singh is a recipient of the George H. A. Clowes, Jr., M.D. Memorial Researchh Career Development Award from the American College of Surgeons.

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