P53 tumor suppressor gene mutations in laryngeal cancer and in recurrent disease following radiation therapy

Oral Oncology 38 (2002) 296–300 www.elsevier.com/locate/oraloncology

P53 tumor suppressor gene mutations in laryngeal cancer and in recurrent disease following radiation therapy G.W.A. Tjebbesa,*, P.A. Kreijveldb, M.G.J. Tilanusb, G.J. Hordijka, P.J. Slootwegb a

Department of Otorhinolaryngology (G05.101), University Hospital Utrecht, PO box 85500, 3508 GA Utrecht, The Netherlands b Department of Pathology, University Hospital Utrecht, Utrecht, The Netherlands Received 18 March 2001; received in revised form 26 March 2001; accepted 1 June 2001

Abstract In this study we performed p53 sequencing based mutation analysis in laryngeal cancers and matched recurrent disease following irradiation. The question is if irradiation affects the DNA and introduces or deletes mutations so that p53 cannot be used as a clonal marker anymore. P53 mutations were identified in fresh-frozen laryngectomy specimens with either primary laryngeal cancers, treated by surgery and irradiation post-operative with local failure during follow-up, or with recurrent laryngeal cancers following primary irradiation. In 21 tumors the p53 status was analyzed by direct sequencing full-length mRNA through RT-PCR. DNA sequencing analysis of exons 2 through 11 was performed when RNA isolation could not be performed. The marker mutation identified in this way was detected by DNA sequencing of the corresponding exon in formalin-fixed deparaffinized tumor biopsy samples in respectively matched recurrent disease following surgery and irradiation or primary tumor before irradiation. DNA sequencing analysis of the corresponding exon of peripheral blood leukocytes excluded the presence of germline mutations or polymorphisms. In 16 out of 21 tumors (71%), a mutation was identified. Fifteen of these marker mutations were detected in the matched tumor biopsy sample (94%). The only case lacking the marker mutation probably was a second primary tumor. We conclude that we find no direct evidence for induction or loss of p53 mutations following irradiation. Consequently, p53 may be used as a diagnostic tool when histological examination fails, for example in discriminating between the presence of a second primary tumor in the same area versus recurrent disease. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Head and neck squamous cell carcinoma; p53; Radiation therapy; Sequencing based mutation analysis

1. Introduction Radiation therapy plays an increasingly important role in the primary treatment of head and neck squamous cell carcinoma (HNSCC). Failure to respond results in recurrent disease, which requires additional therapy. Distinction between recurrent disease and radiation induced non-malignant tissue changes may be difficult clinically as well as histologically. Nonrepresentative tumor biopsy samples can be obtained during endoscopic inspection, due to deep submucosal tumor growth and/or growth in small dispersed epithelial fields, resulting in a delay in detection. Therefore other diagnostic modalities may be of use to demonstrate the presence or absence of recurrent disease. * Corresponding author. Tel.: +31-30-2506645; fax: +31-302541922. E-mail address: [email protected] (G.W.A. Tjebbes).

In recent studies we investigated whether p53 tumor suppressor gene, using molecular biological detection techniques, may be useful as a clonal marker in fingerprinting a tumor. Using sequencing based mutation analysis we found p53 tumor suppressor gene mutated in over 90% of HNSCC [1]. Mutations exhibited a tremendous variation and proved to be stable during tumor progression [2,3]. To be useful as a clonal marker in the detection of recurrent disease following radiation therapy, it is essential that p53 mutations, besides variability and stability, should remain present and identical in recurrent disease following irradiation. However, literature reports both loss and induction of p53 mutations following ionising radiation [4–8]. Gallo et al. [4], using single strand conformational polymorphism analysis (SSCP), demonstrated a loss of p53 mutations in 31% of recurrent head and neck malignancies following radiation therapy. This may be explained by clonal expansion: tumor cells carrying a

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G.W.A. Tjebbes et al. / Oral Oncology 38 (2002) 296–300

p53 mutation may be more sensitive to radiation due to defective p53 protein unable to induce cell-cycle arrest for repair of radiation induced DNA damage, resulting in an accumulation of potentially lethal DNA damages. Assuming a clonal heterogeneity of tumor cell populations, p53 wild type cell clones may survive resulting in recurrent disease with loss of p53 mutation. This theory, however, is controversial. Several studies show resistance to radiation of tumors carrying a p53 mutation possibly through the inhibition of p53 mediated apoptosis [9–11]. Another theory was put by Kemp et al. [5] who also found loss of p53 mutation following irradiation. Since they found an increase of p53 null clones, loss of the p53 gene, they postulated that p53 itself may be a target for radiation induced mutagenesis, being deleted or altered in cells surviving irradiation. On the other hand, data suggesting p53 mutation induction by radiation are offered by De Benedetti et al. [6], who conducted p53 mutation analysis in lung cancers following radiation therapy for Hodgkin’s disease. They found an absence of smoking related G:C!T:A transversions [12] and a prominence of G:C!A:T transitions, characteristic of radiation and oxidative damage [13,14]. It should be noted that the second lung cancers occurred 9.8 years (mean) after radiation treatment. Comparable data are shown from p53 analysis of lung cancers from uranium miners, exposed to radon [7] and radiation-induced sarcomas [8]. In the present study, we investigated p53 status using a sequencing based mutation analysis strategy, previously described [1], in matched pre- and post irradiation tumor samples of head and neck cancer patients who failed radiation therapy. The question put is whether p53 mutations remain present and identical in recurrent disease following irradiation. Stability of p53 mutations in recurrent disease following irradiation offers possibilities for molecular biological detection techniques as a diagnostic tool in the detection of presence or absence of recurrent disease following treatment and is also helpful in deciding whether a second tumor occurring in the same area as an earlier one represents local recurrence or second primary.

2. Materials and methods 2.1. Patients and methods Retrospectively, 21 consecutive fresh-frozen laryngectomy specimens were studied from patients surgically treated for (1) recurrent laryngeal cancer after primary irradiation or (2) primary laryngeal cancer followed by radiation therapy post-operative with a local failure during oncologic follow-up. In all cases, formalin-fixed, paraffin-embedded tumor biopsy samples of, respectively, the matched primary tumor before radiation

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therapy, or matched local recurrent disease following treatment were available. Ten millilitres of ethylenediaminetetracetic acid (EDTA) blood had been collected during surgery, serving as a control for polymorphisms and hereditary mutations in the p53 gene. Clinical data concerning these patients are listed in Table 1. No prior research concerning this series of laryngeal cancers has been performed or reported previously. Mutation identification was performed using a previously described [1] p53 mutation analysis strategy, sequencing full-length p53 mRNA, a technique best performed on fresh-frozen tissue. Fresh-frozen tissue was only obtained from laryngectomy specimens. Since most laryngeal cancers in our institute primarily are treated by radiation therapy, p53 mutation identification was most frequently performed in recurrent disease following radiation therapy. In this series of 21 laryngeal cancers, p53 mutation identification was performed in recurrent disease following irradiation in 19 cases, and in the primary tumor, with local recurrent disease following combination therapy of surgery and irradiation, in two cases. 2.2. RNA/DNA isolation Prior to DNA or RNA isolation, hematoxylin and eosin-stained histological slides were judged for the presence of at least 25% tumor cells. RNA isolation was performed in fresh frozen tissue of the surgical resection specimens. RNA was isolated from 10 sections of 20 mm thickness using the Trizol LS (liquid solvents) reagent procedure according to the manufacturers protocol (Life Technologies, Paisley, UK). DNA was synthesized using 3 mg RNA, 1 mg Oligo-(dT)15 primer (Promega, Madison, WI, USA) and 200 units SuperscriptTM RNAse H-reverse transcriptase (Life Technologies). DNA was extracted from 15 sections of 10-mm thickness fresh frozen tissue of PT and LNM using the QIAamp procedure according to the manufacturers protocol (Qiagen GmbH, Hilden, Germany). In all instances, 5-mm tissue sections obtained at the beginning and at the end of the series of sections from which DNA and RNA was isolated were histologically examined for presence of representative tumor tissue. Formalin-fixed paraffin-embedded tissue sections of 40 mm thickness were deparaffinized with xylene, rehydrated in serial-graded water-ethanol solution (100 and 70% respectively), and rinsed in deionized water. Subsequently, DNA was extracted by incubation for 2 nights at 55 C in 1 ml extraction buffer (100 mM NaCl, 10 mM Tris-HCl pH 8.0, 0.25 mM EDTA pH 8.0, 0.5% SDS) to which 20 ml proteinase K (10 mg/ml) was added every 12 h. A phenol-chloroform purification was performed and DNA precipitation was established by ethanol with help of the carrier glycogen.

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Table 1 p53 mutations in laryngeal cancer and recurrent disease following irradiationa Tumor

Age

Sex

TNM

Mutation identification Exon

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15f 16f 17f 18f 19f 20g 21g

71 57 42 75 58 54 69 63 52 53 64 46 68 48 54 51 72 53 56 58 72

v m m m m m m v v m m m m m m m m m m m m

cT2N0M0 cT2N0M0 cT2N0M0 cT2N0M0 cT1N0M0 cT1N0M0 cT2N0M0 cT4N0M0 cT3N0M0 cT2N0M0 cT1N0M0 cT1N0M0 cT2N0M0 cT3N0M0 cT1N0M0 cT2N0M0 cT3N0M0 cT1N0M0 cT2N0M0 pT3N1M0 pT3N1M0

7 6 5 6 5 8 5 7 4 6 5 5 7 4

6 4

Base

G!T A!G A!G A!T G! A G!T A!T G!C C!T Del. 3 Del. 19 Del. 22 Del. 11 Del. 1 Wild type Wild type Wild type Wild type Wild type G!A Del. 1

Mutation detection Mutation codon/resultb

Mutation matched tumor

R249M Y205C H179R H193L C135Y R273L H179L Splice site Q104e Del. 1xV 216/217/218 175 Frameshift 140 Downstream stop 252 Frameshift 48 Frameshift

Wild typed + + + + + + + + + + + + +

Splice site 90 Frameshift

+ +

Intervalc

3.1 2.0 0.7 0.6 3.0 0.8 1.0 0.11 1.5 1.2 5.8 4.4 1.7 0.10 2.4 1.0 2.7 3.8 0.6 2.2 0.6

a TNM stage in primary laryngeal cancer according to pTNM classification [18]. R, arginine; M, methionine; Y, tyrosine; C, cysteine; H, histidine; V, valine; L, leucine; Q, glutamine; del, deletion. b F.E. R249M means mutation in codon 249 resulting in substitution of arg by met. c Time interval primary tumor — recurrent disease in years.months. d No mutation detection in corresponding exon in matched (primary) tumor; subsequently full-length deparaffinized DNA sequencing analysis performed of primary tumor. e Stop. f No mutation identification in recurrent laryngeal cancers after full-length mRNA sequencing; subsequently no sequencing analysis performed in matched (primary) tumor. g Mutation identification in primary laryngeal cancer.

DNA from PBL was extracted using a conventional salting-out procedure [15]. 2.3. Polymerase chain reaction (PCR) amplification Full-length p53 mRNA was amplified using three overlapping PCRs. In DNA, exons 1 through 11 were amplified in nine different PCRs. With the exception of exon 7 amplification primers, all 50 primers had been elongated with a-21M13 template (50 TGT AAA ACG ACG GCC AGT 30 ), all 30 primers had been elongated with an M13-reverse template (50 CAG GAA ACA GCT ATG ACC 30 ), allowing sequencing with uniform primers. For exon 7, the 50 primer had been elongated with an M13-reverse template and the 30 primer had been elongated with a-21M13 template. PCR solutions for RNA and DNA amplification were generally the same: 500 ng DNA (or 3 ml of cDNA solution), 1 U AmpliTaq DNA Polymerase (PerkinElmer, Norwalk, CT, USA), 10 pmol of the tailed 50 and 30 primer and 10 mg dNTP was added to a buffer containing 50 mM KCl, 10 mM Tris-HCl pH 8,3 and 1,5 mM MgCl2 (1 mM MgCl2 for exon 6). Distilled water

was added to a total volume of 50 ml. Amplification of DNA extracted from paraffin sections was performed in a slightly adapted solution containing 1 U AmpliTaq Gold DNA Polymerase (Perkin-Elmer, Norwalk, CT, USA) and 2 mM MgCl2. The PCR solution was heated at 94 C for 3 min followed by 35 cycles of 30 s at 94 C, 1 min at 60 C, 1 min at 72 C, and terminated at 72 C for 2 min, using a Gene Amp PCR System 9600 thermal cycler (Perkin Elmer, Norwalk, CT, USA). For exon 10, the annealing temperature was 56 C. 2.4. Fluorescent cycle sequencing The PCR products were sequenced in two directions by dideoxy-chain termination method using a Dye Primer Cycle Sequencing Ready Reaction Kit with AmpliTaq Polymerase FS according to the manufacturers protocol (Perkin-Elmer, Norwalk, CT, USA). The sequence product was run on a 6% denaturing polyacrylamide gel in an automated DNA sequencer (model 373A, Applied Biosystems, ABI, Foster City, CA, USA). The gel was subsequently analyzed using ABI Prism multi locus sequence analysis software [16]. The

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mutated codons were numbered from the first coding nucleotides.

full-length DNA sequencing was performed. The primary tumor appeared to exhibit only wild type p53.

3. Results

4. Discussion

3.1. Mutation identification

To investigate if p53 mutations remain present in laryngeal cancers and matched recurrent diseases following radiation therapy we identified p53 mutations in freshfrozen surgical specimens of either primary head and neck cancer or recurrent head and neck cancer after unsuccessful primary irradiation. Subsequently, we investigated whether these marker mutations were also present in matched biopsies of, respectively, recurrent disease following combination therapy of surgery followed by irradiation or primary tumor before irradiation. Using sequencing based mutation analysis, mutations were identified in 14 of 19 laryngeal cancers with recurrent disease following irradiation and in both laryngeal cancers primarily treated by surgery and postoperative irradiation. The 16 out of 21 p53 mutations thus identified proved to be identical in the primary tumor and matched recurrent disease following irradiation in 15 cases (94%). In the remaining case, a mutation was detected in recurrent disease following radiation therapy in exon 7, a G to T transition, a smoking related mutation, whereas in the primary tumor, no mutation was detected after sequencing deparaffinized full-length p53 DNA. This case concerned a glottic laryngeal carcinoma, with presumed recurrent disease 3 years after irradiation at the supraglottic false vocal fold. The specific mutation found in the new lesion, the slightly different location and the disease free interval of over 3 years, all support the presence of a second primary tumor instead of recurrent disease in this case. Our results do not provide any evidence for p53 mutation induction by irradiation in HNSCC. Therefore, p53 mutations reported in irradiation induced malignancies probably are not a direct effect of irradiation. Our results are at variance with those of Gallo et al [4], who reported loss of p53 mutations in recurrent laryngeal cancer in 31% of cases and an initial mutation percentage of only 48%, using SSCP as mutation detection method. Previously, in studying p53 mutation stability in metastasized disease in our institute, we performed p53 mutation analysis in primary tumors and matched lymph node metastases, using different molecular biological detection techniques. Using denaturing gradient gel electrophoresis (DGGE) and exon analysis, we detected p53 mutations in only 40% of primary tumors and a concordance of p53 mutations between primary tumor and matched lymph node metastases of only 47% [17]. In contrast, with our current technique mutations were identified in 91% of PT and a concordance of 100%, showing the importance of sensitive

As mentioned before, mutation identification was performed in fresh-frozen laryngectomy specimens. Most laryngeal cancers primarily are treated by radiation therapy, followed by surgery in case of recurrent disease. Therefore, in this series of 21 laryngeal cancers, p53 mutation identification was performed in recurrent disease following radiation therapy in 19 cases (Table 1, Nos. 1–19), and in the primary tumor, with local recurrent disease following combination therapy of surgery and radiation therapy in two cases (Table 1, Nos. 20– 21). Mutations were identified using full-length p53 RNA sequencing analysis. Mutations were confirmed by sequencing DNA of the corresponding exon. When full-length p53 RNA mutation-analysis did not reveal a mutation, or when RNA isolation was not successful, full-length DNA-sequencing of all exons was performed. Using this technique, mutations were identified in 16 out of 21 cases (71%). It concerned a point mutation in 10 cases (Table 1, Nos. 1–9, 20), of which two were located at a splice site (Table 1, Nos. 8, 20). In six cases it concerned a deletion (Table 1, Nos. 10–14, 21). Three mutations were located outside the core region (exons 5–8) (Table 1, Nos. 9, 14, 21). In the other five cases, wild type p53 was identified (Table 1, Nos. 15–19). In all cases, DNA sequencing analysis of the corresponding exon of PBL was performed, serving as a control, to exclude germline mutations and polymorphisms. PBL showed wild type p53 in all cases (results not shown). 3.2. Mutation detection Mutations identified in the fresh-frozen surgical resection specimen of either the recurrent head and neck cancer after unsuccessful primary RT or of the primarily surgically treated head and neck cancer were detected in the matched tumor sample by sequencing the corresponding p53 exon in material from, respectively, the primary tumor before radiation treatment or material from the local recurrent disease following combination therapy of surgery and post-operative irradiation. Of the 16 mutations identified, 15 proved to be identical in the matched tumor sample (94%). The resulting single case (Table 1, No. 1) concerned a point mutation in exon 7, identified in recurrent disease following irradiation. In this specific case, where the matched tumor biopsy before irradiation proved to be lacking the marker mutation in the corresponding exon,

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molecular biological detection techniques [2]. Moreover, in recurrent malignant disease following irradiation, mutations are even more difficult to demonstrate: Due to extensive fibrosis, necrosis or presence of inflammatory cells carrying wild type p53 only a relatively low percentage of mutated p53 may be encountered. Therefore when using less-sensitive molecular biological techniques in material with a low number of cancer cells, mutations may evade detection instead of being lost to irradiation. In this study wild type p53 was identified in five of 19 (26%) recurrent laryngeal cancers following radiation therapy which is slightly higher than our previous study in which p53 mutation analysis in primary HNSCC revealed wild type p53 in less than 10% [1]. One could wonder whether this is due to loss of p53 mutations during irradiation. Tumors in this previous study were located mainly in the oral cavity and oropharynx. One cannot exclude that p53 mutation percentages may vary among different subsites in the upper aerodigestive tract. Oral and oropharyngeal sites are not only exposed to the carcinogenic effects of tobacco but also of alcohol and therefore may show different mutational spectra and varying percentages of mutated p53. P53 mutation analysis in our institute of a series of 11 laryngeal cancers, primarily treated by surgery, revealed p53 mutations in eight cases (73%; data not shown), comparable to this study and thus supporting the idea that laryngeal cancers show a lower percentage of p53 mutations. Therefore, we conclude that loss of p53 mutations following irradiation does not account for the lower percentage of p53 mutations in this series of laryngeal cancers as compared with our previous data. Our results show that p53 mutations are stable in radiation-resistant laryngeal cancers. Also, we did not find evidence for induction or loss of p53 mutations following radiation therapy. Therefore, possibilities are offered for molecular biological detection techniques to be used as a diagnostic tool, for example when histological investigation fails in detection of recurrent disease following irradiation, or in discriminating between the presence of a second primary tumor versus recurrent disease.

[2]

[3]

[4]

[5]

[6]

[7] [8]

[9]

[10]

[11] [12] [13] [14]

[15]

[16]

[17]

References [18] [1] Kropveld A, Rozemuller EH, Leppers FGJ, Scheidel KC, de Weger RA, Koole R, et al. Sequencing analysis of RNA and

DNA of exons 1 through 11 shows p53 gene alterations to be present in almost 100% of head and neck squamous cell cancers. Lab Invest 1999;79(3):347–53. Tjebbes GWA, Leppers vd Straat FGJ, Tilanus MGJ, Hordijk GJ, Slootweg PJ. P53 tumor suppressor gene as a clonal marker in head and neck squamous cell carcinoma: p53 mutations in primary tumor and matched lymph node metastases. Eur J Cancer B Oral Oncol 1999;35:384–9. Van Oijen MG, Leppers vd Straat FGJ, Tilanus MGJ, Slootweg PJ. The origins of multiple squamous cell carcinomas in the aerodigestive tract. Cancer 2000;88(4):884–93. Gallo O, Chiarelli I, Bianchi S, Calzolari A, Simonetti L, Porfirio B. Loss of p53 gene mutation after irradiation is associated with increased aggressiveness in recurring head and neck cancer. Clin Cancer Res 1996;2:1577–82. Kemp CJ, Wheldon T, Balmain A. P53-deficient mice are extremely susceptible to radiation-induced tumorigenesis. Nature Gen 1994;8:66–9. De Benedetti VMG, Travis LB, Welsh JA, Van Leeuwen FE, Stovall M, Clarke EA, et al. P53 mutations in lung cancer following radiation therapy for Hodgkin’s disease. Cancer epid 1996;5:93–8. Taylor JA, Watson MA, Devereux TR. P53 mutation hotspot in radon-associated lung cancer. Lancet 1994;343:86–7. Brachman DG, Hallahan DE, Beckett MA, Yandell DW, Weichselbaum RR. P53 gene mutations and abnormal retinoblastoma protein in radiation-induced human sarcomas. Cancer Res 1991;51:6393–6. Lowe SW, Ruley HE, Jacks T, Housman DE. P53 dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993;74:957–67. Lowe SW, Bodis S, McClatchey A, Remington L, Ruley HE, Fisher DF, et al. p53 status and the efficacy of cancer therapy in vivo. Science 1994;266:807–10. Lee JM, Bernstein A. P53 mutations increase resistance to ionizing radiation. Proc Natl Acad Science USA 1993;90:5742–6. Hollstein M, Sidransky D, Vogelstein B, Harris CC. P53 mutations in human cancer. Science 1991;253:49–53. Little JB. Cellular, molecular and carcinogenic effects of radiation. Hematol Oncol Clin N Am 1993;7:337–52. Gajewski E, Rao G, Nackerdien Z, Dizdaroglu M. Modification of DNA bases in mammalian chromatin by radiation-generated free radicals. Biochemistry 1990;29:7876–82. Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res 1988;16:1215. Rozemuller EH, Eliaou JF, Baxter-Lowe LA, Charron D, Kronick M, Tilanus MG. An evaluation of a multicenter study on HLA-DPB1 typing using solid-phase Taq-cycle sequencing chemistry. Tissue Antigens 1995;46:96–103. Kropveld A, Van Mansveld AD, Nabben N, Hordijk GJ, Slootweg PJ. Discordance of p53 status In matched primary tumours and metastases in head and neck squamous cell carcinoma patients. Eur J Cancer B Oral Oncol 1996;32B:388–93. Spiessl B. UICC, International Union against Cancer. TNMatlas: illustrated guide to the TNM/pTNM-classification of malignant tumors. 3rd ed. New York: Springer, 1990.