MICROSATELLITE ANALYSIS OF SERUM DNA IN PATIENTS WITH HEADAND NECK CANCERHomaira NAWROZ-DANISH

1, Claus F. EISENBERGER4, George H. YOO

1, Li WU4, Wayne KOCH

4, Carri BLACK2, John F. ENSLEY

2,Wei-Zen WEI

3 and David SIDRANSKY4*

1Department of Otolaryngology-Head and Neck Surgery, Wayne State University School of Medicine, Detroit, MI, USA2Department of Medicine, Division of Hematology/Oncology, Wayne State University School of Medicine, Detroit, MI, USA3Department of Immunology, Wayne State University School of Medicine, Detroit, MI, USA4Department of Otolaryngology-Head and Neck Surgery, Division of Head and Neck Cancer Research, Johns Hopkins UniversitySchool of Medicine, Baltimore, MD, USA

We have shown previously that microsatellite alterationsin serum DNA was predictive of distant metastasis in a studywith 21 primary head and neck squamous cell carcinomapatients. To further investigate serum microsatellite alter-ations as a prognostic tool, we carried out microsatelliteanalysis of serum DNA with 10 markers on 152 patients withhead and neck cancer. Forty-five percent (68/152) of patientshad microsatellite alterations of serum DNA identical tocorresponding tumor DNA. In 16 patients that had distantmetastasis, 11 patients had a positive serum test (microsat-ellite alterations detectable in their serum DNA with one ormore markers). The difference in distant metastasis ratesbetween the negative and positive serum tests (6.0% [5/84]vs. 16.2% [11/68], RR � 2.7) was clinically significant andalmost reached statistical significance (p � 0.06). When theanalysis was restricted to patients with recurrent disease, apositive serum test correlated with those who developeddistant metastasis (p � 0.04). Other parameters, such asdevelopment of recurrence, stage of the cancer, disease-freesurvival and overall survival, were not associated with a pos-itive serum test. Detecting tumor DNA in serum by micro-satellite analysis may help identify patients at risk for distantmetastasis. Therefore, circulating tumor cells may contrib-ute to the presence of tumor DNA in the serum. In the futureif a serum test is positive, therapeutic approaches may byintensified, such as platinum-based chemoradiation, to re-duce distant failures.© 2004 Wiley-Liss, Inc.

Key words: serum DNA; HNSCC; microsatellite alterations

In 1999 an estimated 40,400 new patients developed head andneck cancer.1 Head and neck squamous cell carcinoma (HNSCC)is the eighth most common malignancy overall, and the sixth mostcommon cancer in men.1 No significant improvement of survival(40%) has been reported in the past 2 decades.2 The major causeof failure and death is local-regional recurrence. Platinum-basedchemotherapy has shown to improve survival and local-regionalcontrol when used in conjunction with radiation for nasopharyn-geal carcinoma,3 unresectable and advanced head and neck squa-mous carcinoma (HNSCC).4–6 Failure from distant metastasisoccurs much less frequently than local-regional failure.2 A recentreport in advanced HNSCC, however, demonstrated that distantmetastasis was twice that of local-regional recurrence when che-moradiation was used.7 Therefore, as current modalities of therapyimprove local control, distant metastasis may become a predomi-nant cause of failure. The future goal of treatment is to intensifytherapy in high-risk patients and reduce toxicity in patients whoare being over treated. In the future, molecular tests may becomeavailable to predict which patients may benefit from tailoredtherapies.

Cancer develops through the accumulation of genetic alter-ations.8 The genetic transformation of normal cells to neoplasticcells occurs through a series of progressive steps. Genetic progres-sion models have been studied in HNSCC9 that show early lossesof chromosomal arms 9p, 3p and 17p followed by other chromo-somal arms.9,10 Genetic alterations include loss, gain or transloca-tion of chromosomal segments, activation of proto-oncogenes or

inactivation of tumor suppressor genes. The detection of thesegenetic alterations has been found by using newer techniques, suchmicrosatellite analysis, gene sequencing and genotyping. Micro-satellite markers are repeated, non-transcribed DNA sequences,usually di-, tri- or tetra-nucleotide that are finely dispersedthroughout the genome that are detected by using PCR.11 Micro-satellite alterations are an integral part of neoplastic progressionand are valuable in determining loss of heterozygosity (LOH),genetic mapping and genomic instability. An increased molecularunderstanding of the disease, however, has yet to translate into ascreening or prognostic test for head and neck cancer.

Tumor DNA was found recently to be enriched in the serum ofpatients with various types of cancer including head and neck,lung, liver, breast, colorectal and pancreas.12–16 The significance oftumor DNA in the plasma has recently been reviewed, and thepostulated mechanisms are: (i) circulating tumor cells; (ii) passiveleakage of tumor DNA; and (iii) active secretion of tumor DNA.The etiology may be a combination of these mechanisms. Ourpreliminary study with 21 patients with HNSCC showed thatserum DNA microsatellite alterations were identical to tumorDNA changes in 29% of patients. A positive serum test waspredictive of future distant metastasis.18 This suggests that micro-satellite alterations in serum DNA may represent a simple test forassessing tumor burden and patient prognosis. In a larger cohort ofpatients, we carried out microsatellite analysis on the sera ofpatients with HNSCC treated with curative intent to determine iftumor DNA in the serum was predictive of clinical outcome.

MATERIAL AND METHODS

Patients and clinical dataOur study includes patients with head and neck squamous cell

carcinoma who were treated at Johns Hopkins Hospital or WayneState University-Karmanos Cancer Institute between 1995–1998.The patients’ charts were retrospectively reviewed to obtain dataon variables such as age, gender, tumor site, TNM stage (AJCC-1997). Follow-up information was obtained from charts. Time to

Grant sponsor: American Cancer Society; Grant number: CRTG-99-246-01-CCE. Grant sponsor: National Cancer Institute; Grant number:P50-CA96784.

*Correspondence to: Division of Head and Neck Cancer Research, TheJohns Hopkins University SOM, 818 Ross Research Building, 720 RutlandAvenue, Baltimore, MD 21205-2196.Fax: �410-502-5153; �410-614-1411. E-mail: dsidrans@jhmi.edu

Received 15 August 2003; Revised 26 November 2003; Accepted 5January 2004

DOI 10.1002/ijc.20240Published online 12 April 2004 in Wiley InterScience (www.interscience.

wiley.com).

Int. J. Cancer: 111, 96–100 (2004)© 2004 Wiley-Liss, Inc.

Publication of the International Union Against Cancer

local-regional recurrence, distant metastasis and death along withcauses of death were also obtained.

Sample collection and DNA isolationTumors from HNSCC patients obtained fresh from surgical

resection and blood (preoperatively) from venipuncture were col-lected from patients at Wayne State University Medical Institu-tions and Johns Hopkins University Medical Institutions with priorconsent. Tumor tissue was frozen and microdissected describedpreviously.17 Serum was obtained and DNA was extracted fromlymphocytes, tumor tissue and serum as described previously.16,18

Serum DNA concentrations raised from 10–900 ng/ml.19

Microsatellite analysisOligonucleotide markers for microsatellite analysis were ob-

tained from Research Genetics (Huntsville, Alabama) and includedD9s161, D9s200 on 9p; D3s1238, D3s1284 on 3p; Chrnb1 on 17p;D14s50 on 14p (all dinucleotides). Furthermore, trinucleotide andtetranucleotide markers were selected because they are highlypolymorphic and are located at regions lost frequently in head andneck cancer.10,18 Primers selected for increased probability of

FIGURE 1 – Microsatellite analysis autoradio-graphs. Representative microsatellite analysis ofpatient normal lymphocyte DNA, correspondinghead and neck squamous cell carcinoma tumorDNA, and serum DNA (arranged from left toright) with markers indicated at the bottom. (a,b)Serum and tumor DNA exhibit a shift (presenceof new top allele) with Li7686 in 2 differentpatient samples. (c) LOH of top alleles in thetumor and serum DNA with marker D3s1284. (d)LOH of lower alleles in tumor and serum DNAwith marker CSFIR.

TABLE I – STAGING AT TIME OF INITIAL DIAGNOSIS1

Stage Total Serum (�) Serum (�) % Serum (�)

I 6 1 5 17II 19 9 10 47III 27 14 13 52IV 87 38 49 44REC 13 6 7 46T1 10 3 7 30T2 36 13 23 36T3 43 20 23 46T4 42 21 21 50N0 53 19 34 36N1 45 20 25 44N2 29 16 13 55N3 12 7 5 58M0 136 57 79 42M1 16 11 5 69Male 119 52 67Female 33 16 17All 152 68 84 451Patients with recurrent cancer were designated REC and 8 patients

had unknown primaries.

97HEAD AND NECK CANCER SERUM DNA ANALYSIS

detecting shifts included D9s242, D17s695, L17686 and CFS1R.One primer from each set was end labeled with (�-32P) ATP(Amersham, Piscataway, NJ) using T4 polynucleotide kinase(New England Biolabs, Beverly, MA). PCR amplification wascarried out with 30–60 ng DNA as described previously.10 Prod-ucts were separated in 8% denaturing urea-polyacrylamide-form-amide gels followed by autoradiography. Loss of heterozygositywas scored if the ratio of one allele was significantly decreased(�50%) in tumor or serum DNA compared to normal DNA fromlymphocytes. A shift was called if a new allele was visible. Aconservative ratio for calling imbalances was used providinghigher specificity. A serum test was defined positive when eitherLOH or shift were detected in at least one microsatellite markerand was reproducible. Assessment of microsatellite band intensitywas achieved visually by 2 independent determinations followedby a final review by the senior author. The experiments were allcarried out in 1999.

Statistical analysisAll contingency tables were analyzed with either �2 tests or

Fisher exact tests, as appropriate. Univariate survival analyseswere done with log-rank tests and displayed as Kaplan-Meierplots. All tests reported are 2-tailed. Analysis was done with JMP4.0 (SAS Institute, Carey, NC) on a Macintosh computer.

RESULTS

Patients were Stage I (6), II (19), III (27) and IV (87) orrecurrent (13) at the time of initial diagnosis (Table I). Themajority (84%) of patients had advanced (Stage III, IV or recur-rent) cancer. Microsatellite analysis was carried out on normallymphocyte, tumor and serum DNA samples from 152 patients.Forty-five percent (68/152) of patients were found to have micro-satellite alterations (LOH or shifts) for one or more marker in theirserum. In each case the identical alterations were confirmed in thecorresponding tumor DNA (Fig. 1). Patients with early disease(Stage I and II) had a lower rate of positive tests when comparedto advanced disease (Stage III and IV) and recurrence (40 vs. 46%,RR � 1.17). One of six (17%) Stage I patients had a positiveserum test whereas 9 of 19 (47%) Stage II patients had a positivetest. Fourteen of twenty-seven (52%) and 38 of 87 (44%) patientswith Stage III and IV, respectively, had a positive serum test. Ratesof a positive serum test increased markedly between Stages I andII, but did not vary between Stages II, III and IV. Similar resultswere found when T1/T2 were compared to T3/T4 (35 vs. 48%,RR � 1.42). Furthermore, advanced nodal disease (N0 vs. N1/N2/N3) was predictive of a positive test (36 vs. 50%, RR � 1.41). Themajority of patients were male (119). The mean follow up time inthis group of patients was 27 months with a median of 25 months.No significant correlation was found between stage (I-IV, T1/T2vs. T3/T4) and a positive serum test. Patients with cervical metas-tasis (N1–N3) had a higher rate of microsatellite alterations in theirserum (50% vs. 36%); however, this association is not statisticallysignificant (p � 0.12). Six of thirteen (46%) patients with initialrecurrent disease had a positive serum test. These results did notchange when stratified for site of recurrence (local, regional ordistant). Furthermore, all of these results did not change whenadjusted for follow up time.

Five patients with a negative serum test had distant metastasis(5/84), and 11 patients with a positive serum test had distantmetastasis (11/68). The difference in distant metastasis rates be-tween the negative and positive serum (6.0% vs. 16.2%, RR � 2.7)were clinically significant, and almost reached statistical signifi-cance by using Fisher’s exact test (p � 0.06). Sixty-five patients inthis group initially presented or later developed local-regionalrecurrence. Forty-three percent of these patients (29/68) had apositive serum test, whereas 43% of patients with a negative serumtest also had or developed recurrences (36/84). Of the 29 patientswho initially tested positive and subsequently developed a recur-rence, 11 eventually developed distant metastasis. In this popula-

tion restricted only to those who developed recurrences, a positiveserum test correlated with the development of distant metastasis(p � 0.04).

Forty-eight people died in our study within 3 years after theirdiagnosis. The overall survival (Fig. 2) in patients with positiveand negative serum test did not significantly differ (p � 0.5).Disease-free survival (Fig. 2) was worse in patients with thepositive test as opposed to the negative groups (44% vs. 63%), butdid not reach statistical significance (p � 0.13). Sixteen patientshad distant metastasis and 13 patients died due to unrelated causes.Combining the endpoints of distant metastasis and death did notshow a significant difference between the test positive and testnegative group (p � 0.3).

DISCUSSION

Over the past 4 decades, DNA in the sera of normal individualsand in patients with advanced cancer and rheumatic diseases hasbeen confirmed.20 We demonstrated the presence of serum tumorDNA in patients with head and neck cancer by using microsatelliteanalysis and have shown that these alterations in serum DNA wereidentical to the tumor DNA alterations. Microsatellite alterationsof serum DNA and corresponding tumor DNA were detected innearly half of all patients. Tumor DNA has been found to beenriched in the serum of patients with cancers of the lung, liver,breast, colorectal and pancreas.12–16 Although the exact mecha-nism of concentrated tumor cell DNA in the serum is not clearlyestablished, the proposed mechanisms include: (i) circulating tu-mor cells; (ii) passive leakage of tumor DNA; and (iii) activesecretion of tumor DNA.12,20

FIGURE 2 – Overall survival curves. Patients with positive serum test(gray line) and negative serum test (black line) (p � 0.5).

98 NAWROZ-DANISH ET AL.

Because our group of patients with tumor DNA in their serumhad a higher rate of distant metastasis, circulating tumor cells mustin part contribute to serum tumor DNA. We have not, however,excluded the release of tumor DNA into the circulation by tumorcells. In our preliminary study, we identified that the presence ofmicrosatellite alterations in serum DNA was predictive of distantmetastasis (p � 0.015). Although a positive microsatellite serumtest was not found to be a statistically significant (p � 0.06)predictor of distant metastasis, a positive test was clinically sig-nificant (RR � 2.7). In most patients with distant metastasis(69%), the sera demonstrated microsatellite alterations in one ormore markers. Among the recurrent patient population, a positiveserum test predicted metastatic disease (p � 0.04).

Platinum-based chemotherapy has been shown to improve sur-vival and local-regional control when used in conjunction withradiation for nasopharyngeal carcinoma,3 unresectable and ad-vanced HNSCC.4–6 When local-regional recurrence is loweredwith chemoradiation in advanced HNSCC, distant metastasis ismore common than local-regional recurrences.7 Because a positive

serum test predicts distant metastasis and chemotherapy reducesrates of distant metastasis,2 a positive test may lead to the recom-mended use of adjuvant chemotherapy. A negative test reduced theneed for multimodality therapy and reduces toxicity. Beau-Falleret al.21 found that serum DNA of lung cancer patients may allowmonitoring of disease progression, possibly changing choices oftreatment. In the future, molecular test may become available topredict which patients may benefit from specific therapies.

Detecting serum DNA alterations did not correlate with stage andother clinical parameters consistent with a similar study.22 Overallsurvival rates were not significantly (p � 0.5) different in patientswho had either a positive or negative serum test. In contrast, diseasefree survival was worse in the group with the positive test as opposedto the negative groups; however, this did not reach statistical signif-icance (p � 0.13). The high rate of local regional recurrence (43%) inour group overshadowed the lower rate of distant metastasis (10.5%)in our study. Because a positive test was predictive of distant metas-tasis and rate of distant metastasis was not high, a test that predictsdistant metastasis and not local-regional recurrence may not predict

FIGURE 3 – Disease-free survival curves. Pa-tients with positive serum test (gray line) andthose with negative serum test (black line). Nostatistical significant difference is apparent (p �0.13).

99HEAD AND NECK CANCER SERUM DNA ANALYSIS

overall survival. A large number (48) of patients who did not developdistant metastasis died in our study.

We carried out microsatellite analysis using 10 highly sensitive andspecific markers on 152 patients with primary HNSCC treated withcurative intent. We found microsatellite alterations in the serum ofnearly half of patients with HNSCC and a correlation with the pres-ence of serum tumor DNA and distant metastasis. With the knowl-

edge that there is tumor DNA in the patient’s serum, other moleculargenetic analysis techniques may be of value in the future.

ACKNOWLEDGEMENT

G.H.Y. is supported by the American Cancer Society (CRTG-99-246-01-CCE).

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