ACCELERATED DISCOVERY

Unknown Primary Head and Neck Squamous Cell Carcinoma: Molecular Identification of the Site of Origin

Joseph Califano, William H. Westra, Wayne Koch, Glenn Meininger, Andre Reed, Lin Yip, Jay O. Boyle, F. Lonardo, David Sidransky

Affiliations of authors: J. Califano, W. Koch, G.Meininger, A. Reed, L. Yip, D. Sidransky (Department of Otolaryngology-Head and Neck Surgery), W. H. Westra (Department of Otolaryngology-Head and Neck Surgery and Department of Pathology), The Johns Hopkins Hospital, Baltimore, MD; J. O. Boyle (Head and Neck Division, Department of Surgery), F. Lonardo (Department of Pathology), Memorial Sloan-Kettering Cancer Center, New York, NY.

Correspondence to: David Sidransky, M.D., Division of Head and Neck Cancer Research, Department of Otolaryngology-Head and Neck Surgery, The Johns Hopkins University, Rm. 818, Ross Research Bldg., 720 Rutland Ave., Baltimore, MD 21205.


    ABSTRACT
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
BACKGROUND: Unknown primary head and neck squamous cell carcinoma (HNSCC) presents as a cervical lymph node metastasis without identification of the primary tumor, despite thorough diagnostic work-up that includes physical examination, computed tomography, esophagoscopy, laryngoscopy, bronchoscopy, and multiple surveillance biopsies. We investigated whether the site of origin of the primary tumor could be localized in the upper aerodigestive tract mucosa by detection of genetic alterations identical to those found in metastatic lesions. METHODS: Microsatellite analysis was performed on metastatic tumors obtained from 18 patients with unknown primary HNSCC. Histologically benign surveillance biopsy specimens were also analyzed. Patients were followed up to 13 years with continuing surveillance for primary mucosal tumors. Most patients were treated with neck dissection followed by radiation therapy to the affected neck and ipsilateral Waldeyer's ring. RESULTS: In 10 (55%) of the 18 patients, at least one histopathologically benign mucosal biopsy specimen from defined anatomic sites (i.e., most likely sites for an occult primary tumor) demonstrated a pattern of genetic alterations identical to that present in cervical lymph node metastases. One patient harboring genetic alterations in the base of the tongue and two patients harboring genetic alterations in a tonsillar fossa subsequently developed HNSCC in the identical or adjacent mucosal region; all three of the primary head and neck mucosal tumors that eventually appeared between 1 and 13 years later in these patients had genetic changes identical to those in the benign mucosal biopsy specimens and in the metastatic lymph nodes. CONCLUSIONS: These data support the hypothesis that histopathologically benign mucosa of the upper aerodigestive tract may harbor foci of clonal, preneoplastic cells that are genetically related to metastatic HNSCC and that such mucosal sites are the sites of origin of unknown primary HNSCC. Microsatellite analysis may represent a clinically useful tool for determining such sites.



    INTRODUCTION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Head and neck squamous cell carcinoma (HNSCC) of unknown primary tumor site presents as a cervical lymph node metastasis without an obvious mucosal lesion. The majority of these tumors arise from the upper aerodigestive tract; some originate in bronchopulmonary, esophageal, and other sites. Studies of HNSCC (1-4) estimate that the incidence of cervical lymph node metastasis with no obvious primary site ranges from 3% to 9%, with squamous histology constituting 75% of these tumors.

Clinical evaluation of patients includes a complete medical history and physical examination, chest radiograph, and indirect or flexible fiberoptic endoscopy. Computed tomography may be used to evaluate the chest and neck. If these tests are unrevealing, laryngoscopy, bronchoscopy, esophagoscopy, nasopharyngoscopy, and biopsy of all suspicious lesions are performed to evaluate pulmonary and upper aerodigestive tract sites. If no lesions are noted, directed bilateral biopsies of the most likely occult primary tumor sites (tongue base, nasopharynx, tonsils, and piriform sinus) and tonsillectomy are usually performed. Fine-needle aspiration biopsy of the cervical mass may be performed for diagnosis (1,5).

For those patients with disease confined to neck nodes, treatment includes complete neck dissection, high-dose radiation, or both. If no primary lesion can be identified, either bilateral or ipsilateral Waldeyer's ring is irradiated (1,5). Approximately half of patients are cured by initial therapy for all stages combined. Most recurrences involve the neck; less commonly, the primary mucosal tumor may develop (5-7).

Current theories of tumor progression have focused on the emergence of clonal populations of cells that undergo successive genetic alterations, producing a malignant phenotype with a selective growth advantage (8). A molecular model for primary HNSCC has shown genetic progression from premalignant to malignant lesions (9). In that study, minimally abnormal or apparently benign mucosa adjacent to premalignant lesions often shared clonal genetic changes found in HNSCC. Subsequent genetic events in various subclones result in the outgrowth of histopathologically diverse regions in a local area and are associated with the development of multiple primary tumors (10). These studies offer a molecular biologic basis for "field cancerization," a concept used to explain the predilection for patients with HNSCC to develop both multiple primary upper aerodigestive tract tumors and multiple areas of upper aerodigestive tract premalignant lesions. We hypothesize that lateral, mucosal, clonal expansion could be followed by rapid metastatic progression, resulting in a clinically detectable cervical lymph node metastasis without the outgrowth of an obvious primary tumor. A genetic relationship between benign directed biopsies and a metastatic focus would imply that a particular directed biopsy is anatomically related to the primary tumor site and that the biopsy specimen represents an earlier clonal outgrowth of neoplastic cells. In this study, we performed microsatellite analysis on directed biopsy samples and metastatic lymph nodes from patients with cervical metastatic squamous cell carcinoma of unknown primary origin to determine whether the site of origin can be identified by molecular genetic means.


    SUBJECTS AND METHODS
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Patient Selection Criteria

Patients were selected on the basis of review of diagnoses of archival biopsy specimens from the Department of Pathology, The Johns Hopkins Hospital (Baltimore, MD), from the Department of Pathology, Greater Baltimore Medical Center, from the Department of Pathology, Sinai Hospital (Baltimore), from the Department of Pathology, Memorial Sloan-Kettering Cancer Center (New York, NY), and from The St. Francis Community Hospital (Greenville, SC) as well as on the basis of review of the data from The Head and Neck Tumor Registry at The Johns Hopkins Hospital and the Tumor Registry at the Greater Baltimore Medical Center.

To be included, patients must have had the following: 1) neck dissection or excisional biopsy of cervical lymph nodes with a histopathologic diagnosis of metastatic squamous cell carcinoma; 2) a complete medical history, physical examination, panendoscopy including diagnostic laryngoscopy, flexible fiberoptic esophagogastroscopy, flexible fiberoptic bronchoscopy, multiple mucosal biopsies of head and neck sites, as well as bilateral tonsillectomy when indicated (not all patients had biopsy specimens taken from all eight standard mucosal sites including right and left piriform sinus, base of tongue, nasopharynx, and tonsils); and 3) imaging studies including computed tomography of the neck, chest radiograph, and computed tomography of the chest and abdomen when indicated. These evaluations did not reveal a primary tumor site for patients in this study, all biopsy specimens from patients in this study were interpreted as benign if they showed no morphologic evidence of carcinoma or dysplasia upon initial diagnosis, and adequate archival tissue was available for analysis for all patients.

In total, 18 of more than 200 patients with unknown primary HNSCC treated during the period from 1983 to 1996 fulfilled the above criteria. In addition, all patients except for patient 9 had a history of substantial tobacco exposure with or without ethanol exposure.

Selection of Loci for Microsatellite Analysis

The chromosomal band 9p21 contains the p16 (MTS1) gene, a cyclin/cyclin-dependent kinase inhibitor involved in cell cycle regulation, and corresponds to an area of genetic loss common to many solid tumors (11,12). At this time, this locus constitutes the region with the most frequent loss of heterozygosity (LOH) in HNSCC, and the p16 protein is not expressed in more than 80% of HNSCCs (13,14). The chromosomal band 11q13 includes the bcl-1/int-2 locus, an amplicon carrying the proto-oncogene cyclin D1, one of the few proto-oncogenes implicated in HNSCC (15). Apparent LOH in this region actually represents amplification of cyclin D1, as confirmed by studies using fluorescence in situ hybridization (16). The tumor suppressor gene p53 is commonly mutated in HNSCC (17). The p53 gene is found on chromosomal arm 17p13, which also corresponds to an area of frequent LOH in HNSCC (13). Chromosomal arm 3p has been shown to contain at least three putative HNSCC suppressor loci (18,19). Chromosomal band 13q21 contains an area with frequent LOH near the retinoblastoma protein gene (Rb) locus that is now thought to include a second, novel tumor suppressor gene locus (20).

The following microsatellite markers were included in this study: D3S647, D3S1067, D3S1284, D3S1038, and D3S1007 (all on chromosomal arm 3p); D8S273, D8S549, and D8S261 (all on chromosomal arm 8); IFN-{alpha}, D9S736, D9S1747, and D9S171 (all on chromosomal arm 9p21); D11S873, INT-2, and PYGM (all on chromosomal arm 11q13); D13S170 and D13S133 (both on chromosomal arm 13q21); and TP53 and CHRNB (both on chromosomal arm 17p13).

Tissue and DNA Extraction

Tissues were obtained from archival, paraffin-embedded blocks from the institutions above or from fresh-frozen tissue, obtained with written informed consent from patients at The Johns Hopkins Hospital according to protocol approved by the Institutional Review Board, The Johns Hopkins Hospital. Representative sections from tissue used for DNA extraction were stained with hematoxylin-eosin, and a pathologist (W. H. Westra) confirmed the diagnosis for each lesion. Fresh-frozen tissue from the metastatic lymph node was meticulously dissected on a cryostat to ensure that the specimen contained at least 75% tumor cells. Approximately thirty-five 12-µm sections were then collected and placed in 1% sodium dodecyl sulfate/proteinase K (0.5 mg/mL) at 58 °C for 24 hours. Paraffin-embedded tissues obtained from directed mucosal biopsies were sectioned into twenty-five 14-µm sections. Each section was placed on a glass slide and individually microdissected with the use of a dissecting microscope to obtain more than 75% epithelial cells. The samples were placed in xylenes overnight to remove the paraffin, pelleted in 70% ethanol, dried, and incubated in sodium dodecyl sulfate/proteinase K at 58 °C for 72 hours. Digested tissue from both sources was then subjected to phenol-chloroform extraction and ethanol precipitation as previously described (21). Normal, control DNA was obtained by 1) venipuncture and isolation of lymphocyte DNA as previously described (21), 2) isolation of DNA from microdissected nonepithelial, normal tissues from the previously mentioned archival, paraffin-embedded biopsy specimens, or 3) if necessary, isolation of DNA from nonepithelial, paraffin-embedded tissue from archival paraffin-embedded blocks other than the biopsy specimen blocks in the manner described above.

Microsatellite Analysis

Microsatellite markers suitable for polymerase chain reaction (PCR) analysis were obtained from Research Genetics (Huntsville, AL). Before amplification, 50 ng of one primer from each pair was end labeled with [{gamma}-32P]adenosine triphosphate (20 mCi/mL; Amersham Life Science, Inc., Arlington Heights, IL) and bacteriophageT4 kinase (New England Biolabs, Inc., Beverly, MA) in a total volume of 50 µL. PCR reactions were carried out in a total volume of 12.5 µL containing 10 ng of genomic DNA, 0.2 ng of labeled primer, and 15 ng of each unlabeled primer. The PCR buffer included 16.6 mM ammonium sulfate, 67 mM Tris (pH 8.8), 6.7 mM magnesium chloride, 10 mM ß-mercaptoethanol, and 1% dimethyl sulfoxide to which were added 1.5 mM deoxynucleotide triphosphates and 1.0 U of Taq DNA polymerase (Boehringer Mannheim Biochemicals, Indianapolis, IN). PCR amplifications of each primer set were performed for 30-35 cycles consisting of denaturation at 95 °C for 30 seconds, annealing at 50 °C to 60 °C for 60 seconds, and extension at 70 °C for 60 seconds as described. One third of the PCR product was separated on 8% urea-formamide-polyacrylamide gels and exposed to x-ray film from 4 to 48 hours. Analysis of allelic imbalance was performed in a blinded fashion for each patient in terms of biopsy location and time of biopsy. For informative cases, allelic loss (or allelic imbalance in the case of the 11q13 locus) was scored if one allele was decreased by greater than 40% in tumor DNA when compared with the same allele in normal control DNA.


    RESULTS
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
Mucosal biopsy specimens from 18 patients with HNSCC metastatic to a cervical lymph node with unknown primary site were tested. In 10 (55%) of the 18 patients (patients 1-6, 12, 14, 15, and 17), benign mucosal sites contained genetic alterations corresponding to those found in a cervical metastasis (Table 1).Go In each case, identical losses on multiple chromosomal arms or chromosomal breakpoints (boundaries of chromosomal loss and retention) were shared between the metastasis and a mucosal site. These mucosal sites included tonsil only (three cases), pyriform sinus only (one case), tonsil and pyriform sinus (two cases), base of tongue (three cases), false vocal cord (one case), and floor of mouth (one case). In the remaining eight cases, biopsy specimens were histologically normal and genetic alterations, if any, did not correspond to those present in a cervical metastasis.


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Table 1. Clinical outcome and biopsy data*

 
The biopsy specimens from eight of the 10 patients that shared genetic alterations with a cervical metastasis showed completely normal histology. The patterns of allelic polymorphism at different chromosomal marker regions exhibited by different biopsy specimens, referred to hereafter as "allelograms," are shown in Fig. 1.Go In Fig. 1Go, a, an allelogram from patient 1 demonstrates identical patterns of LOH for a biopsy of the left pyriform sinus and metastatic lymph node on chromosomal arms 9p21, 11q13, and 17p13. (A representative autoradiograph is shown in Fig. 2Go, a, and a corresponding photomicrograph showing normal histology appears in Fig. 2,Go b.) Fig. 1Go, b, shows identical patterns of LOH on chromosomal arm 11q13 and 17p13 for a left tonsil biopsy and metastatic node from patient 2.






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Fig. 1. Representative allelograms from cervical lymph node metastases and directed biopsy sites. Only chromosomal arms for which microsatellite markers demonstrate genetic alteration in a biopsy specimen or a metastasis are shown. a) Allelogram from patient 1, demonstrating identical genetic alterations on chromosomal arms 9p21, 11q13, and 17p13 in metastatic lymph node (T) and left pyriform sinus (L PYR) biopsy specimen. b) Allelogram from patient 2, demonstrating identical genetic alterations on chromosomal arms 11q13 and 17p13 in metastatic lymph node (T) and left tonsil (L TON) biopsy specimen. c) Allelogram from patient 6, demonstrating identical genetic alterations in metastatic lymph node (T) and right floor of mouth (R FOM) biopsy specimen in chromosomal arms 3p and 17p13 and additional genetic alterations in the metastatic implant at arms 9p21 and 11q13 showing genetic progression. d) Allelogram from patient 3, demonstrating concordant loss of heterozygosity (LOH) in two loci; T = metastatic lymph node, and L, R BOT = left, right base of tongue. e) Allelogram from patient 4, demonstrating identical patterns of LOH; T = metastatic lymph node, and L TON, PYR = left tonsil, pyriform sinus. f) Allelogram from patient 5, demonstrating additional LOH at 9p21 and 13q21; T = metastatic lymph node; R TON, PYR = right tonsil, pyriform sinus. g) Allelogram from patient 12, demonstrating identical patterns of LOH and 6-centimorgan (cM) breakpoint at 9p21; T = metastatic lymph node, and R TON = right tonsil. h) Allelogram from patient 14, demonstrating identical 6-cM breakpoint at 17p13 and identical pattern of LOH at 3p and 13q21 with divergent LOH on 9p21; T = metastatic lymph node, and R FVC = right false vocal cord. i) Allelogram from patient 15, demonstrating 9-cM breakpoint at 9p21; T = metastatic lymph node, and L TON = left tonsil. j) Allelogram from patient 17, demonstrating identical patterns of LOH; T = metastatic lymph node, and L BOT = left base of tongue. The following microsatellite markers are shown: D3S647, D3S1007, D3S1284, D3S1067, and D3S1038 (all on chromosomal arm 3p); D8S549 (on chromosomal arm 8); IFN-{alpha}, D9S1747 (laboratory designation: D9S8162), and D9S171 (all on chromosomal arm 9p21); D11S873, INT-2, and PYGM (all on chromosomal arm 11q13); D13S133 and D13S170 (both on chromosomal arm 13p21); and TP53 and CHRNB (both on chromosomal arm 17p13).

 



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Fig. 2. Molecular and histologic analysis of biopsy specimens. a) Representative autoradiograph from patient 1. Polymerase chain reaction amplification of normal lymphocyte DNA (N), tumor DNA (T), and DNA from microdissected mucosal biopsy specimen of left pyriform sinus (B). b) Photomicrograph of biopsy specimen of left pyriform sinus biopsy of patient 1, showing normal histology (original magnification x100).

 
In two of the patients (Nos. 5 and 6) with a biopsy specimen containing genetic alterations corresponding to a cervical metastasis, the biopsy specimens showed evidence of epithelial dysplasia (Fig. 1Go, f and c, respectively). We found identical losses on chromosomal arm 3p and 17p13 in mucosa and lymph node metastases from patient 6, but additional regions of loss on chromosomal arms 9p21 and 11q13 in the metastatic lymph node occurred during the progression from dysplasia to a metastatic phenotype.

Four patients (Nos. 2, 3, 12, and 16) eventually developed clinically detectable mucosal disease in their upper aerodigestive tract. One patient (No. 3; Fig. 1Go, d) had a biopsy specimen of the base of tongue that corresponded genetically to the primary tumor arising later in the same location. Another patient (No. 2; Fig. 1Go, b) had a biopsy specimen of the normal mucosa from the left tonsil that genetically matched a primary tumor that appeared 2 years later on the right base of the tongue. A biopsy of the right base of the tongue was not performed; however, during the interval between the original oropharyngeal left-sided biopsy and the appearance of the primary oropharyngeal tumor on the right side, the patient received radiation therapy directed only to the left Waldeyer's ring. The right tonsil biopsy specimen from patient 12 (Fig. 1Go, g) matched a contiguous right floor-of-mouth cancer that arose 13 years later. A biopsy of the floor of mouth was not performed initially; however, both specimens share a common 6-centimorgan boundary between an area of chromosomal loss and retention, indicating a clonal relationship. Patient 16 developed a primary tumor of the base of tongue 26 months after treatment of a genetically related neck metastasis. He did not receive initial biopsies of the base of tongue and did not exhibit genetic alterations in biopsy specimens that corresponded to cervical metastasis.

LOH was noted in histologically normal biopsy specimens that did not match patterns of genetic alterations found in metastatic lymph nodes in four patients (patient 3—one loss in one biopsy specimen; patient 5—one loss in each of two biopsy specimens; patient 7—one loss in one biopsy specimen; and patient 12—one loss in one biopsy specimen). Similar phenomena have been described in biopsy specimens from the lungs of current and former smokers but not from the lungs of nonsmoking control subjects (22). A genetic relationship between these biopsy specimens and a metastatic focus is neither demonstrated nor excluded by our analysis.

Patient 1 had a right-sided level I lymph node metastasis but a genetically related biopsy specimen of the left pyriform sinus. One interpretation of this unexpected result is a failure to do a biopsy of a related clonal population higher in the upper aerodigestive tract draining the nodal chain that contains the metastasis. Alternatively, nodal drainage patterns in the head and neck can vary considerably. Finally, this biopsy site may represent widespread distribution of epithelial clonal patches with genetic alterations.

One of the patients, patient 13, developed an esophageal cancer after presenting with a low, neck metastasis in level IV, and extensive directed biopsies did not demonstrate any genetic alterations, indicating a likelihood that this patient's neck metastasis arose from the primary esophageal site. None of the remaining patients have developed clinically apparent mucosal disease of the upper aerodigestive tract after a mean follow-up interval of 37 months.


    DISCUSSION
 Top
 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 
These data provide evidence that mucosal sites of origin can be molecularly identified in patients with occult primary HNSCC presenting as a cervical lymph node metastasis. Molecular progression models, including those for HNSCC, indicate the following: 1) Tumor progression involves the development of a selective growth advantage for a clonal population of cells resulting from successive genetic alterations; 2) diverse genetic alterations follow a generalized temporal order; and 3) the accumulation of alterations ultimately determines the expression of a malignant phenotype (8,9). These models rely on the study of premalignant or benign precursor lesions to establish genetic progression.

Previous studies (9,22) and the data presented here demonstrate that critical genetic alterations that result in clonal outgrowth can occur in histopathologically normal epithelium, and these clonal populations can be genetically related to malignant populations that result in metastatic foci. It is probable that small, clinically occult, neoplastic foci were present in these patients but escaped biopsy because of their minute size or sampling error. However, clonal, genetically altered, but normal-appearing mucosa would have preceded these lesions. Mucosal biopsy specimens with genetic alterations in these patients represent an early clonal population of cells on the progression pathway to clinically detectable cancer and a corresponding cervical metastasis. In agreement with this theory, some metastatic lesions contain additional genetic alterations other than those found in common with the mucosal biopsy specimens, although discordant genetic alterations unrelated to progression cannot be excluded.

These data suggest a novel strategy for tumor localization that uses genetic comparison of mucosal sites with metastatic implants. More precise mapping in patients with occult HNSCC may be accomplished, guiding future surveillance. Indeed, three patients developed a primary tumor in the region from which a benign biopsy specimen was taken that contained genetic alterations common to a metastatic implant. These data support extensive and systematically directed biopsies as part of an evaluation of unknown primary HNSCC to increase the likelihood of obtaining a biopsy specimen that may yield a histopathologic or molecular diagnosis of the site of origin. This approach may also be used to map widespread areas of clonally related precursor cell populations to allow for treatment of large areas of altered epithelium.

This approach may also be useful in differentiating primary head and neck tumors from intra-abdominal and pulmonary sites with the use of directed biopsies of the lower aerodigestive tract in an analogous fashion. Directed biopsies may be able to detect clonal populations of epithelial cells that are genetically related to the metastasis, despite inability to detect a clinically obvious tumor. Therapy may then be directed on the basis of the origin of clonally related cell populations. In one patient (No. 2), a subsequent primary tumor arose in a nonirradiated bed of tissue that did not undergo biopsy. In this patient, recurrence did not appear at the site of the altered clone that was treated with radiation (left tonsil), but it did appear at a nearby, contralateral site (right base of tongue) just beyond the radiation field.

Extensive use of directed biopsies may allow mapping of mucosal sites that harbor genetically altered clones, allowing more precise direction of radiation portals in order to avoid morbidity or treat widespread subclinical disease. These data imply that clonal expansion and tumor progression may theoretically be detected before any histopathologic abnormality or clinically detectable lesion has appeared in patients at risk for HNSCC. Clonal genetic alterations can be identified in body fluids, including saliva (23,24). Detection of genetically altered, benign-appearing mucosa may allow for heightened surveillance and detection of clonal populations of cells at earlier, more curable stages, before they have acquired malignant behavior (25).


    REFERENCES
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 Abstract
 Introduction
 Subjects and Methods
 Results
 Discussion
 References
 

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Manuscript received February 3, 1999; revised March 1, 1999; accepted March 2, 1999.


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