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Genetic alterations in squamous cell carcinomas of the hypopharynx with correlations to clinicopathological features

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Genetic alterations in squamous cell carcinomas of the hypopharynx with correlations to clinicopathological features
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  Genetic alterations in squamous cell carcinomas of the hypopharynxwith correlations to clinicopathological features Juan P. Rodrigo a, *, Maria V. Gonza ´lez a , Pedro S. Lazo b , Sofı ´a Ramos b , Eliecer Coto c ,Ignacio Alvarez a , Luis A. Garcı ´a a , Carlos Sua ´rez a a Department of Otolaryngology, Hospital Central de Asturias, University of Oviedo, Instituto Universitario de Oncologı´ a, Oviedo, Spain b DepartmentofBiochemistryandMolecularBiology,HospitalCentraldeAsturias,UniversityofOviedo,InstitutoUniversitariodeOncologı´ a,Oviedo,Spain c Department of Molecular Genetics, Hospital Central de Asturias, University of Oviedo, Instituto Universitario de Oncologı´ a, Oviedo, Spain Received 2 May 2001; accepted 7 June 2001 Abstract The objective of this study is to describe the molecular alterations in carcinomas in one specific location of the head and neck, thehypopharynx. Thirty-seven hypopharyngeal squamous cell carcinomas were studied. The DNA from tumour and healthy tissue wasevaluated for amplification of the 11q13 region and of the  MYC   and  ERB B1 oncogenes, for integration of the Human Papilloma-virus (HPV), and for loss of heterozygosity (LOH) at  p53  and  NAT  2 loci.The most common alteration was the amplification of the11q13 region (78% of the cases), followed by LOH at  p53  locus (70%).  MYC   amplification was found in 19% of the cases,  ERB B1amplification in 29%, LOH at  NAT  2 locus in 25%, and integration of the HPV in 29%. 11q13 amplification was related with nodalmetastases and higher tumour recurrence rates. These findings confirm that 11q13 amplification is one of the most frequent geneticalterations in hypopharyngeal squamous cell carcinomas, and that it may have prognostic significance in these tumours. # 2002Elsevier Science Ltd. All rights reserved. Keywords:  Hypopharynx; Squamous cell carcinoma; Oncogenes; Tumour suppressor genes; Human papillomavirus 1. Introduction Many genes have been found to be implicated in thedevelopment and progression of different types of can-cers. These genetic alterations have a different patternaccording to the type of tumour and its location (e.g.colorrectal, breast, lung, bladder carcinomas, etc.) thatcould explain their different biological and clinicalbehaviour. Several genes have also been associated withthe development and progression of squamous cell car-cinomas of the head and neck (SCCHN) [1]. In moststudies SCCHN are considered as one tumour type.However, although being a histopathological entity,these carcinomas may behave differently according tothe different locations within this area. This is reflectedby differences in growth pattern, clinical behaviourand prognosis. This variability in the behaviouraccording to the location suggests different intrinsictumour properties. It may be expected that the geneticalterations responsible for these properties will reflectthese differences. Nevertheless, local anatomical circum-stances may also play a role. On the other hand, thepossible prognostic implications of the genetic altera-tions can be influenced by the larger or smaller relativeweight of each location in the different studies. Then, toobtain prognostic conclusions, studies on isolated loca-tions are needed.The objective of this study is to evaluate the geneticalterations of squamous cell carcinomas in a specificlocalisation, as is the hypopharynx. We have selectedsome of the most frequent alterations described in pre-vious studies [2–6]. We studied the amplification of theoncogenes  MYC  ,  ERB B1 (which encodes the epidermalgrowth factor receptor), and those located at the 11q13region ( CCN  D1, which encodes the cyclin D1,  FGF  3and  FGF  4, that encode proteins related to the fibro-blastic growth factor, and  EMS  1, which encodes thecortactin, a cytoskeletal protein). We also studiedthe loss of heterozygosity (LOH) of the genes  p53 ,located at 17p13, and N-Acetiltransferase-2 ( NAT  2), 1368-8375/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.PII: S1368-8375(01)00071-9Oral Oncology 38 (2002) 357–363www.elsevier.com/locate/oraloncology* Corresponding author. Tel.: +34-985-108000, ext. 38140; fax:+34-985-108015. E-mail address:  jrodrigo@hcas.insalud.es (J.P. Rodrigo).  located at 8p21-23, two regions frequently deleted inSCCHN. Finally, we studied the integration of theHuman Papillomavirus (HPV). Furthermore, it isattempted to establish the possible prognostic value of such alterations in this specific location. 2. Materials and methods 2.1. Patients Paired tumour and blood samples from 37 consecutiveprimary squamous cell carcinomas of the hypopharynxwere obtained prospectively from patients undergoingsurgical resection of their tumour following institutionalreview board guidelines. All patients included in ourstudy were male, had a single primary tumour, none hadundergone treatment prior to surgery, and had micro-scopically clear surgical margins. None of the patientswere thought to have had distant metastases at the timeof surgery. All patients were smokers and 34 of themwere also habitual alcohol drinkers. Atotal of 24 patientsreceived postoperative radiotherapy. As a general rule,this was administered to the patients with histologicallyN2 or N3 neck lesions, and also in N0–N1 neck lesionswith locally advanced stage (T4). The stage of diseasewas determined according to the TNM system of theInternational Union Against Cancer (4th Ed.). The clin-icopathologic data from the patients are shown inTable 1. Table 1Clinico-pathological characteristics of the patients, results of the genetic alterations studied and disease courseCasenumberAge Stage Pathologicalgrade CCN  D1  FGF  3  FGF  4  EMS  1  ERB B1  MYC p53 NAT  2 HPV Diseasecourse1 43 T4N2 P     + + +    NI NI    DT, 10 m2 78 T4N2 P    + + + +    NI NI    DOC, 8m3 52 T2N0 P    +       NI NI    DT, 15m4 60 T2N0 W         NI NI + AWD, 73m5 68 T4N0 W        + NI NI    DT, 12m6 67 T3N1 M         + + + DOC, 24 m7 51 T3N2 W + + + +     NI NI    DT, 34 m8 61 T4N2 W         +    + DT, 22 m9 47 T3N3 W    + +    +       DT, 10 m10 54 T1N1 P    + +    +    NI NI    AWD, 69 m11 62 T4N1 W + + +    + + +    + DOC, 3 m12 53 T2N1 W    + + +     +     DOC, 48 m13 76 T4N0 M     +     +    NI    AWD, 65 m14 56 T3N0 M       +    + +    DT, 34 m15 48 T2N3 M    +         + DT, 38 m16 62 T3N0 M + + +      NI NI + DOC, 26 m17 59 T3N2 W          NI    DT, 10 m18 49 T4N0 M     + +    +    NI    DT, 16 m19 54 T2N3 M +        NI NI    DT, 7 m20 67 T3N0 W +        NI NI    AWD, 36 m21 58 T3N1 M +    +      +     DT, 9 m22 63 T4N0 M       NI NI +    NI DT, 18 m23 50 T4N2 M +     + NI NI +    NI DT, 5 m24 69 T3N1 M    + +    NI NI + NI NI AWD, 40 m25 58 T4N3 P + + + + NI NI NI + NI DT, 18 m26 42 T4N1 W + + +    NI NI NI    NI AWD, 40 m27 62 T2N2 M    + +    NI NI NI NI NI DT, 17 m28 49 T4N2 W + + +    NI NI NI + NI DT, 16 m29 57 T4N2 M + + +    NI NI + NI NI DT, 9 m30 45 T4N2 M + + +    NI NI +    NI AWD, 36 m31 42 T3N0 W       NI NI NI NI NI DT, 17 m32 62 T4N3 M + + +    NI NI NI NI NI DOC, 24 m33 53 T4N3 M    + +    NI NI + NI NI AWD, 36 m34 59 T3N1 P + + +    NI NI NI    NI DT, 27 m35 62 T4N3 P +     + NI NI    NI NI DT, 12 m36 71 T3N0 M    +     NI NI +    NI AWD, 38 m37 62 T4N3 W + + + + NI NI + NI NI DT, 13 mPathologic grade: W, well differentiated; M, moderately differentiated; P, poorly differentiated. The positive sign (+) indicates the existence of onco-gene amplification, loss of heterozygosity, or integration of the HPV. The negative sign (  ) indicates the absence of such alterations. NI indicates thatthere is no information available. Disease course: AWD, alive without evidence of disease; DT, dead by the tumour; DOC, dead by other causes.358  J.P. Rodrigo et al./Oral Oncology 38 (2002) 357–363  2.2. DNA extraction Tissue specimens were frozen immediately in liquidnitrogen in the surgical room and stored at   70   C untilDNA extraction. High-molecular weight DNA was iso-lated by standard phenol-chloroform extraction. Sec-tions of tumours used for the study were selected fromareas that had a high proportion of malignant tissue.For each patient we also obtained DNA from cells in 10ml of blood following a salting-out method. 2.3. Analysis of oncogene amplification The amplification of the  MYC, ERB B1, and 11q13oncogenes ( CCN  D1,  FGF  3,  FGF  4 and  EMS  1) wasdetermined using a semiquantitative method [differentialpolymerase chain reaction (PCR)], as previously descri-bed [2–4]. Briefly, two different sets of primers, one forthe target gene (the oncogene) and the other for the con-trol gene (located on the same chromosome as the targetgene), were present simultaneously in the reaction vessel.The control genes were the tissue plasminogen activatorgene (TPA) for the  MYC   gene, the beta-actin gene for the ERB B1, and the tyrosine-hydroxylase gene (TH) forthe 11q13 region. Samples of DNA from normal tissues(tonsils) obtained from noncancer patients were used asnegative controls. As positive controls, a mixture of DNA from normal tissue and increasing amounts of apreviously PCR-amplified sequence of the target gene,mimicking different degrees of amplification, was usedas template in the PCR reaction.The PCR-products were electrophoresed, stained withethidium bromide, and the images of the ultraviolet-illuminated gels were captured using a digital cameraand quantified by computerised densitometric analysistechniques (Kodak Digital Science 10, Eastman Soft-ware, Billerica, MA). The target gene:control generatios were determined. The results of densitometrywere corroborated by visual inspection of the gels. PCRwas carried out at least twice in the positive cases. 2.4. LOH analysis In the case of the  p53  locus, PCR was used to amplifyone dinucleotide (microsatellite) and one VTR (variabletandem repeat) polymorphism, both intragenic to  p53 [5]. Approximately 50 ng of genomic DNA were PCR-amplified with specific primers and electrophoresed on6% polyacrilamide sequencing gel. Gels were dried andautorradiographed. To identify the LOH at the  NAT  2locus we analysed the most frequent DNA polymorph-isms responsible for the rapid acetylator (RA) or slowacetylator (SA) phenotypes. A C to T change atnucleotide 481 is the most common mutation associatedwith the SA phenotype. Normal and tumour DNAswere PCR-amplified. After 25 cycles, reactions weredigested with the restriction enzyme KpnI, and elec-trophoresed on 6% polyacrilamide sequencing gel.Gels were dried and autoradiographed. The 481 T/Calleles were visualised as fragments of 290 bp (481T)and 170+120 bp (481C). The same method was used toanalyse the second most common mutation associatedwith the SA phenotype, a G to A change at position590. Normal and tumour DNAs were PCR-amplifiedand each reaction was digested with TaqI. Alleles werevisualised as fragments of 290 bp (590A) and 230+60bp (590G).For those individuals who were heterozygotes, auto-radiographic patterns of normal tissue and pathologicaltissue pairs were analysed by densitometry, and LOHwas defined as a reduction in the signal of more than80% in one of the alleles in the tumour tissue. 2.5. Detection of HPV-DNA PCR-amplification of HPV-6b and HPV-16 E6 and  L 1genes was accomplished through PCR, with specific pri-mers for each gene [6]. The  L 1 gene encodes one of theproteins of the viral capside and is only found in the cellssynthesising viral particles, indicating active infection bythe HPV. As positive control, we used the viral DNA,cloned in the bacterial plasmid pBR322. The negativecontrol in each case consisted of reaction mixtureswithout DNA. The PCR products were electrophoresedon a 2% agarose gel and visualised with ethidium bro-mide staining. 2.6. Statistical analysis Statistical analysis was performed using Chi-squared,with Yates’ correction where appropriate, and Fisher’sexact tests.  P  values of   4 0.05 were considered to bestatistically significant. 3. Results The results of the genetic alterations studied in the 37cases and the evolution of the patients are shown inTable 1. For each genetic alteration some of the casescould not be evaluated due to insufficient material.Amplification of the 11q13 oncogenes ( CCN  D1,  FGF  3, FGF  4 and  EMS  1) was studied in all the cases, and atleast one of them was found to be amplified in 29 (78%)cases. Specifically, the  CCN  D1 gene was found ampli-fied in 16 cases (43%), the  FGF  3 gene in 21 cases (57%),the  FGF  4 gene in 22 cases (59%), and the  EMS  1 gene innine cases (24%). Co-amplification of these genes wasfrequently found, especially the  FGF  3 and  FGF  4 genes(that are closely located in the 11q13 amplicon). Theamplification of   ERB B1 and  MYC   oncogenes wasstudied in 21 cases;  ERB B1 was found amplified in six J.P. Rodrigo et al./Oral Oncology 38 (2002) 357–363  359  cases (29%) and  MYC   in four cases (19%). Of 37 cases,20 were informative (heterozygotes) for the  p53  gene,presenting LOH 14 of them (70%). Sixteen cases wereinformative for the  NAT2  gene and LOH was found infour of them (25%). Six of the 21 (29%) analysed casespresented integration of the HPV (4 cases with integra-tion of the HPV type 6b and two cases with the HPVtype 16). Representative examples of the molecularalterations studied are shown in Figs. 1–3.The relationship between the studied genetic altera-tions and the different clinical and pathological para-meters is summarised in Table 2 ( FGF  3 and  FGF  4amplifications are considered simultaneously becausethey were found to be co-amplified in most cases). Withthe exception of HPV integration, the incidence of thestudied genetic alterations increased with the increase inT-stage. The cases with 11q13 amplification showed agreater incidence of nodal metastases.  ERB B1 amplifi-cation was also associated to a greater incidence of nodal metastases, although the differences did not reachstatistical significance. The other genetic alterationsanalysed did not show any association with nodalmetastases.Of 37 patients, 28 died: 22 due to the tumour and sixdue to other causes. Nine patients were alive and with-out evidence of disease between 36 and 73 months(mean, 41,6 months). The patients that died by othercauses than the tumour were excluded of the recurrenceanalysis. Of the 31 remaining patients, 22 (71%) pre-sented recurrence (16 cases with locoregional recur-rences and six with distant metastases). The statisticalanalysis did not show any correlation between theisolated genetic alterations and tumour recurrence(Table 2), except in the case of   EM  S1 gene amplifica-tion, which was associated with a greater incidence of tumour recurrence, although the differences did notreach statistical significance ( P =0,14) probably due toan insufficient number of cases with amplification. 4. Discussion Since the clinical behaviour of the SCCHN dependsnotably on their location, it is questionable to includeall the locations within a single entity. In addition toanatomical factors, different genetic alterations couldaccount for the varying aggressiveness of tumours aris-ing at different sites. However, most of the studies ongenetic alterations in the head and neck carcinomas donot make a distinction between the different locations,being scarce the number of studies on isolated loca-tions, and even more those which analyse severalalterations simultaneously. Our results show that thefrequency of the genetic alterations analysed in hypo-pharyngeal carcinomas is similar, though somewhathigher, to the numbers found in previous studies thatincluded all the locations of the head and neck area. Fig. 1. Differential polymerase chain reaction (PCR) analysis of oncogene amplifications from various tumour samples in which geneamplification was found. The PCR products were separated by agar-ose gel electrophoresis and quantified by image analysis densitometry.Two representative cases of each oncogene are depicted. N indicatescontrols with normal tissue DNA; TH, the tyrosine hydroxylase gene;TPA, the tissue plasminogen activator gene;  b -actin, the  b -actingene; and bp, base pairs.Fig. 2. Agarose gel electrophoresis of the HPV-6b/ E  6 gene poly-merase chain reaction (PCR)-products in the four cases in whom itwas detected. C+ indicates the positive control.Fig. 3. Representative examples of cases with loss of heterozygosity(LOH) at the  p53 -VTR and  NAT  2-T/C markers. Each figure depictsone patient’s paired blood (B) and tumour (T) DNA examined at the  p53  and  NAT  2 loci. Substantial loss of intensity of one allele is seen inthe tumour specimen compared with the blood specimen, as indicatedby an arrow.360  J.P. Rodrigo et al./Oral Oncology 38 (2002) 357–363  Chromosome 11q13 amplification has been describedin 20–50% of the head and neck carcinomas [4,7–9],being the most frequent oncogene alteration in thesetumours. The higher amplification rate found in thisstudy (78% of the cases) is concordant with other worksthat have described a greater amplification rate of the11q13 region in hypopharyngeal carcinomas [8]. Thiscould explain, in part, the greater aggressiveness of these tumours, since 11q13 amplification has been rela-ted in several studies to the presence of nodal metas-tases, greater local aggressiveness and a higher incidenceof tumour recurrence [4,8,9]. In our study, as in pre-vious series [8], 11q13 oncogenes have not been uni-formly amplified in all cases. These differences inamplification of 11q13 oncogenes are in agreement withthe structure of the 11q13 amplicon, as described in aprevious work [4].The  MYC   oncogene has been found amplified in thehead and neck carcinomas in 4–15% of the cases [2,10].In our study we also found a slightly superior fre-quency of amplification in hypopharyngeal carcinomas(19%). The  ERB B1 oncogene, which encodes the epi-dermal growth factor receptor (EGFR), has beenfound amplified in 7–25% of the cases in SCCHN[3,10–12]. Again, our cases showed a slightly higheramplification rate (29%). Therefore, it appears thatoncogene amplification is more frequent in hypo-pharyngeal carcinomas. However caution must be taken Table 2Relationship between the different molecular alterations, the T and N stages, and the disease courseT Stage N Stage Recurrence a T1–T2 T3 T4 N0 N+ No Yes CCN  D1Amplified 1 (6%) 5 (31%) 10 (63%) 2 (13%) 14 (87%) 3 (23%) 10 (77%)Non amplified 6 (28%) 7 (33%) 8 (38%) 9 (43%) 12 (57%) 6 (33%) 12 (67%)P b 0.48 0.10 0.82 FGF  3/ FGF  4Amplified 5 (21%) 6 (25%) 13 (54%) 3 (13%) 20 (87%) 7 (35%) 13 (65%)Non amplified 7 (39%) 6 (33%) 5 (28%) 8 (57%) 6 (43%) 2 (18%) 9 (82%)P b 0.21 0.01 0.56 EMS  1Amplified 1 (11%) 1 (11%) 7 (78%) 1 (11%) 8 (89%) 0 7 (100%)Non amplified 6 (22%) 11 (39%) 11 (39%) 10 (36%) 18 (64%) 9 (37%) 15 (63%)P b 0.12 0.32 0.14 MYC  Amplified 0 0 4 (100%) 3 (75%) 1 (25%) 1 (33%) 2 (67%)Non amplified 6 (35%) 8 (47%) 3 (18%) 5 (29%) 12 (71%) 3 (23%) 10 (77%)P b 0.007 0.26 0.71 ERB B1Amplified 1 (17%) 2 (33%) 3 (50%) 1 (17%) 5 (83%) 1 (25%) 3 (75%)Non amplified 5 (33%) 6 (40%) 4 (27%) 7 (47%) 8 (53%) 3 (27%) 9 (73%)P b 0.55 0.43 0.5  p 53LOH 1 (7%) 5 (36%) 8 (57%) 3 (21%) 11 (79%) 4 (36%) 7 (64%)No LOH 1 (17%) 2 (33%) 3 (50%) 2 (33%) 4 (67%) 1 (17%) 5 (83%)P b 0.8 0.9 0.76 NAT  2LOH 0 2 (50%) 2 (50%) 1 (25%) 3 (75%) 0 3 (100%)No LOH 2 (17%) 4 (33%) 6 (50%) 2 (17%) 10 (83%) 3 (30%) 7 (70%)P b 0.64 0.71 0.76 HPV  Positive 2 (33%) 2 (33%) 2 (33%) 2 (33%) 4 (66%) 1 (33%) 2 (67%)Negative 4 (27%) 6 (40%) 5 (33%) 6 (40%) 9 (60%) 3 (30%) 10 (60%)P b 0.94 0.83 0.71 a Six patients that died from other causes were excluded of the recurrence analysis. b Chi-squared test. J.P. Rodrigo et al./Oral Oncology 38 (2002) 357–363  361
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