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Journal of Clinical Oncology, Vol 20, Issue 4 (February), 2002: 1105-1114
© 2002 American Society for Clinical Oncology

Determination of the Molecular Relationship Between Multiple Tumors Within One Patient Is of Clinical Importance

By Joost R.M. van der Sijp, Jan P.A.M. van Meerbeeck, Alex P.W.M. Maat, Pieter E. Zondervan, Hein F.B.M. Sleddens, Albert N. van Geel, Alex M.M. Eggermont, Winand N.M. Dinjens

From the Department of Surgical Oncology, Dr Daniel den Hoed Clinic, and Departments of Pulmonology and Thoracic Surgery, University Hospital Dijkzigt; and Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center Rotterdam, the Netherlands.

Address reprint requests to Winand N.M. Dinjens, PhD, Department of Pathology, Josephine Nefkens Institute, Erasmus University Medical Center Rotterdam, P.O. Box 1738, 3000 DR Rotterdam, the Netherlands; email: dinjens{at}path.fgg.eur.nl


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PURPOSE: To determine the molecular relationship between multiple tumors within one patient and to evaluate the impact of this knowledge on clinical management.

PATIENTS AND METHODS: In 25 consecutive patients with multiple tumors, proven by histology and immunohistochemistry to be identical, molecular aberrations were determined. Each patient had at least one lesion in the lung or head and neck region. Loss of heterozygosity (LOH) and p53 aberration analyses were carried out, and similar aberration profiles suggest clonality and metastasis whereas different profiles suggest independent primary tumors.

RESULTS: The molecular determinations indicated that 12 patients had a probable second primary tumor and 10 patients had a metastasis of the first lesion. In three patients, both an independent primary tumor and a metastasis were present. The molecular findings determined the course of additional treatment in all 10 patients with metastases, in all three patients with both a second primary tumor and a metastasis, and in seven of 12 patients with a second primary tumor.

CONCLUSION: By comparing DNA alterations of multiple tumors within one patient, the relationship between the tumors can be assessed. This study shows that in 20 of 25 patients, knowledge of the nature of both lesions was essential in clinical decision making. Furthermore, after thorough analysis of the five cases where clinical decision making was not influenced, there was in retrospect no clear indication for LOH or p53 analysis. Because these molecular analyses can be performed on routine specimens, they can be applied in almost all patients.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
PATIENTS WITH TWO synchronous or metachronous tumors may have two independent primary tumors or metastatic disease. Twenty percent to 30% of patients with head and neck malignancies and 10% of patients with lung cancer will develop second primary tumors.1 Conversely, patients with lung cancer will have metastatic disease in up to 70% of cases, depending on their stage at diagnosis, and in most instances, multiple metastases are present.2 Patients with head and neck cancer frequently develop solitary lung metastases. Moreover, in these patients, there is a higher prevalence for a second primary cancer, of which approximately 30% are located in the lung.3-5

A clinical problem arises in patients with synchronous or metachronous tumors where both lesions are solitary, treatable with curative intent, and have the same basic histology (eg, both adenocarcinoma or both squamous cell carcinoma). On routine hematoxylin and eosin (H&E) staining and immunohistochemistry, it is often difficult to determine whether a second lesion is a metastasis or a second primary tumor.

Most tumors are characterized by the accumulation of genomic aberrations. Among others, these aberrations often comprise large amplifications and deletions of DNA as well as small intragenic mutations (eg, point mutations) in specific genes.6 Carcinomas frequently have p53 mutations and loss of heterozygosity (LOH) at several chromosomes. Squamous cell carcinomas of the head and neck and lung and colorectal and bladder carcinomas harbor p53 gene mutations in more than 25% of cases.7-10 In these tumors, frequent LOH has been found at chromosomal regions 17p (p53), 13q (Rb), 9p (p16), 8p (gene unknown), and 3p (VHL and FHIT),11 and in almost 100% of these tumor types, at least one of these regions is deleted (unpublished data from more than 500 carcinomas). Therefore, p53 mutations and LOH profiles are valuable markers to determine the relationship between two lesions in one single patient.12-20 If the aberration profiles of two tumors from one patient are the same, the two lesions are most likely of the same clonal origin, one being a metastasis from the other. Different DNA aberration profiles suggest multiple independent primary tumors.

Molecular aberrations in tumors can be assessed on routinely processed tiny tissue samples. Moreover, even cytologic smears are generally sufficient for genomic analysis. LOH and p53 mutations in samples from both tumors can be compared, and, with a high probability, a metastases or second primary lesion can be identified. Thus, in the clinical setting, a cytologic sample, safe and easy to perform, is sufficient to establish the nature of lesions. To establish whether clinical decisions were influenced by knowing the nature of lesions, we evaluated a consecutive series of 25 patients in whom there was a suspicion of solitary metastasis or second primary tumor.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
We investigated an unselected consecutive series of 25 patients (20 male and five female) with multiple synchronous or metachronous carcinomas who were referred during a 12-month period. Patient characteristics are listed in Tables 1 and 2. Median age was 67.5 years (range, 36 to 70 years). Six patients had a synchronous and 19 had a metachronous lesion, with a median interval after the first malignancy of 36 months (range, 6 to 208 months). The primary lesion was in most instances a head and neck (n = 11) or lung tumor (n = 7). Other primary tumor sites were colon (n = 4), breast, bladder, and kidney (N = 1 each). The site of the second tumor was in 17 cases the lung, four of these in combination with lymph node involvement, and in four cases head and neck. Other sites were brain, liver, chest wall, and bladder. Eighteen patients had squamous cell carcinomas, and six patients had adenocarcinomas. One patient had two large-cell nondifferentiated carcinomas. All six patients with synchronous lesions had squamous cell carcinomas.


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  		Table 1.  Patient Characteristics and Results
 

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  		Table 2.  Patient Characteristics and Results
 

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  		Table 4.  P53 Exon 5-8 PCR Primer Information
 
For genomic analysis, pathology specimens of the primary tumor of each patient were either acquired by cytology, biopsy, or excision at our own institution or were, if the patient was referred to us at a later time, obtained from the pathology files from the referral hospital. For analysis of the second lesion, we obtained in our institute cytologic or biopsy specimens from every case. In all lesions, LOH and p53 mutations were determined, and the results were used to define a second primary tumor or a metastatic lesion.

DNA Isolation
From routine formalin-fixed and paraffin-embedded tissues, H&E-stained sections were used to select parts of the tissues composed of more than 70% tumor cells (see Figs 1 and 4). From the same paraffin block, areas of normal tissue were also selected. The selected tissue parts were gently punched out of the tissue blocks. After this manipulation, a second H&E section was made to verify the isolated tissue parts (see Fig 4, T1A and T1B). In cases in which manual isolation of parts containing more than 70% tumor cells was impossible, laser capture microdissection was used on H&E-stained sections (see Fig 4, T2A, T2B, and T2C). The small tissue fragments were digested, without deparaffinization, in 100 to 200 µL of 50 mmol/L Tris/HCl (pH 8.0), to which 20 µL of proteinase K (20 mg/mL) was added. After overnight incubation at 56°C, the lysates were boiled for 10 minutes and subsequently centrifuged.



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  		Fig 1. Routine histologic sections from patient no. 5. T1 is the colon adenocarcinoma; T2, lesion in the liver; T3, lung adenocarcinoma. Arrows indicate tumor tissue; arrowheads, normal tissue. Bars = 200 µm.

 


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  		Fig 4. Routine histologic sections from patient no. 13 before and after microdissection. T1A and T1B indicate head and neck tumor before (T1A) and after (T1B) manual microdissection; T2A and T2B, lung tumor before (T2A) and after (T2B) laser capture microdissection; T2C, dissected fragment. Bars = 200 µm.

 
LOH and p53 Aberration Analysis
Because the DNA retrieved from routine processed tissue specimens is highly degraded, we used small amplicon (< 200 bp) polymerase chain reaction (PCR) to investigate DNA aberrations (Tables 3 and 4). For LOH analysis, highly polymorphic dinucleotide repeat markers were amplified (for more information, see the Genome Database at www.GDB.org). The markers used are located at chromosomal regions known to be frequently deleted in carcinomas (Table 3). Exons 5 to 8 of the p53 gene were investigated by PCR single-strand conformation polymorphism (SSCP) analysis. Each exon was amplified in two overlapping fragments. The primers used are listed in Table 4. For LOH as well as for SSCP analyses, tumor DNA was always compared with normal DNA from the same patient. PCR was performed with 1 to 3 µL of isolated DNA in a final reaction volume of 15 µL containing, in mmol/L, MgCl2 1.5, dATP 0.02, dGTP 0.2, dTTP 0.2, and dCTP 0.2, {alpha}-32PdATP 0.8 µCi (Amersham, Buckinghamshire, United Kingdom), 20 pmol of each primer, and 0.2 units of Taq polymerase (Promega, Madison, WI). PCR was performed for 35 cycles (denaturing at 95°C for 30 seconds, annealing at 55°C for 45 seconds, and extension at 72°C for 1 minute) in a Biometra thermocycler (Biometra, Göttingen, Germany). A final extension was carried out at 72°C for 10 minutes. PCR products were diluted with loading buffer (95% formamide, 10 mmol/L EDTA [pH 8.0], 0.025% bromophenol blue, and 0.025% xylene cyanol), denatured at 95°C for 4 minutes and snap-cooled on ice. For LOH analysis, PCR products were separated on a denaturing 6% polyacrylamide gel, and for SSCP analysis, the samples were run overnight at 7 W on a nondenaturing 6% polyacrylamide gel containing 10% glycerol in 1 x TBE running buffer. After electrophoresis, gels were fixed in 10% acetic acid, dried on blotting paper on a vacuum gel dryer, and exposed to x-ray film overnight at -70°C using intensifying screens. Films were evaluated by visual inspection.


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  		Table 3.  LOH Marker Information
 
Clinical Assessment
A multidisciplinary team discussed all patients with a second lesion, in whom LOH and p53 mutation analyses were performed. For each patient, treatment policies were agreed on in the case of a second primary or a solitary metastasis. In all cases these treatment protocols were different. Hence, it was possible to determine whether the knowledge of having to deal with a second primary or a metastasis in the individual patient made a difference in choice of treatment.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The results and conclusions are listed in Table 2. In all 25 patients, LOH was detected in all tumors with at least three of the markers used (Table 5). LOH in different tumors from one patient was regarded as identical when the same markers demonstrated loss of the same allele (see Fig 2, T1 and T2 with markers D8S133 and D17S786, and Fig 5, T1 and T2). LOH was regarded as different when in different tumors from one patient different markers showed LOH (see Fig 2, T1/T2 and T3 with markers D13S155 and D8S133) or when the same marker had loss of different alleles (see Fig 2, T1/T2 and T3 with marker D17S786).


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  		Table 5.  LOH Marker and P53 SSCP Results
 


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  		Fig 2. LOH results from patient no. 5. Three LOH patterns from the three tumors (T1, colon; T2, liver; T3, lung) compared with patient’s normal DNA (N). Arrows indicate alleles; arrowheads, deleted alleles. T1 and T2 have an identical deletion pattern, and T3 has a different LOH pattern.

 


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  		Fig 5. LOH results from patient no. 13. Two LOH patterns (marker D9S161 and TP53CA) from the two tumors (T1, head and neck; T2, lung) compared with patient’s normal DNA (N). Arrows point to alleles; arrowheads, deleted alleles. Both tumors demonstrate deletion of the same alleles.

 
In 20 of 25 patients, tumor-specific p53 SSCP aberrations were found in at least one of the tumors. We regarded the same aberrant SSCP pattern in different tumors from the same patient as a strong indication for the presence of the same mutation (see Fig 3, T1 and T2 with p53 exon 8, and Fig 6, T1 and T2 with P53 exon 8), as is generally accepted. In the 25 patients investigated, no discrepancies were found between the LOH and the p53 SSCP analyses results (Table 5).



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  		Fig 3. P53 exon 5 and 8 PCR-SSCP results from patient no. 5. SSCP patterns from the three tumors (T1, colon; T2, liver; T3, lung) compared with patient’s normal DNA (N). Arrows indicate aberrant migrating fragments. T3 has a unique tumor-specific p53 exon 5 aberration; T1 and T2 demonstrate an identical p53 exon 8 aberration.

 


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  		Fig 6. P53 exon 8 PCR-SSCP results from patient no. 13. SSCP patterns from the two tumors (T1, head and neck; T2, lung) compared with patient’s normal DNA (N). Arrow indicates aberrant migrating fragment. T1 and T2 have the same p53 exon 8 aberration.

 
In five patients (nos. 5, 15, 16, 17, and 25), there were three tumor locations, one of which in each case seemed to be a lymph node or liver metastasis. The LOH and p53 gene aberrations found in these metastases were in all cases identical to the DNA alterations in one or both of the other tumors from the same patient (Table 5).

The molecular aberration profiles were used as clonal markers to define whether the second lesion in one patient should be regarded as a metastasis or a second primary tumor. In 20 patients, this knowledge had impact on clinical decision making (Tables 1 and 2). Two patients in whom the determination of LOH and p53 mutations influenced clinical management are briefly described as examples.

Patient No. 5
Patient no. 5 (born in 1923) underwent a left hemicolectomy in 1993 for a Duke’s B colon carcinoma (Fig 1, T1). Three years later, he was operated on for a single liver metastasis from his colon carcinoma (Fig 1, T2), and he underwent a segmentectomy of segment 8. Forty-eight months later, a single nodule in the left lower lobe of the lung was found on chest x-ray. On histologic examination, this lesion was also an adenocarcinoma (Fig 1, T3). LOH and p53 aberration analyses on these three lesions revealed identical aberration patterns in the colon tumor and liver tumor and a clearly different profile in the lung adenocarcinoma (Fig 2 and 3). Therefore, the colon tumor and the liver lesion were regarded as a primary tumor and a metastasis, respectively, and the lung adenocarcinoma was regarded as a second independent primary lesion. Because of these findings, the patient underwent a lobectomy instead of a segmentectomy, which would have been the treatment of choice had the lesion been a metastasis from the colon cancer.

Patient No. 13
Patient no. 13 had a PT4N2bM0 sinus piriformis squamous cell carcinoma (Fig 4, T1) for which he underwent chemotherapy and radiotherapy in combination with surgery and lymph node dissection. Thirty-four months later, he was found to have a squamous cell carcinoma in the right upper lobe of the lung (Fig 4, T2), with multiple enlarged lymph nodes in the mediastinum. In the case of a T4 lung carcinoma, he was eligible for chemoradiotherapy, but if this lesion was a metastasis from his head and neck tumor, additional aggressive treatment was not appropriate. LOH and p53 mutation analyses showed identical aberration patterns (Figs 5 and 6), indicating that the lesion in the lung (and most probably in the mediastinum also) was a metastasis, and thus unnecessary chemoradiotherapy was avoided. All five patients for whom the p53 mutation and LOH determination did not influence clinical management are described below.

Patient No. 11
This 74 year-old patient, with a squamous cell carcinoma of the buccal mucosa, underwent CO2 laser coagulation of the lesion. Two years later on chest x-ray, a lesion was found in the left upper lobe of the lung. After mediastinoscopy, which was negative, he underwent a left-sided thoracotomy. A left upper lobectomy was performed for a squamous lesion that was found by molecular characteristics to be a second primary. A radical resection with curative intent had already been performed in this case, not warranting additional treatment. Follow-up by a pulmonologist and ear, nose, and throat surgeon was advised.

Patient No. 19
Patient no. 19 (80 years of age) was treated for a right-sided lung carcinoma pT1N0M0 with a bilobectomy. Eighteen months later, a lesion in the bladder was diagnosed that proved to be a second primary, grade III with muscle invasion. In principle, this patient would have undergone a bladder resection. However, she was found to be at high risk for such a procedure. Therefore, in retrospect, the molecular analyses were not necessary. The patient was treated with radiotherapy to the bladder.

Patient No. 22
Patient no. 22 (54 years of age) had synchronous lesions of the oropharynx and the lung. The oropharynx tumor was staged as cT4Nx; the lung tumor, which proved to be a second primary on LOH and p53 aberration analyses, was staged as IIIa. As a single entity, both these lesions would have been treated with chemotherapy and, on response, with additional radiotherapy. The knowledge that this patient suffered from two independent primary tumors and not from metastatic disease had no impact on the treatment. The treatment scheme for both situations is the same, ie, induction chemotherapy and consolidation radiotherapy after response.

Patient No. 23
This patient (69 years of age) underwent a right-sided pneumonectomy for the presence of a squamous cell carcinoma. Ten years later, she was diagnosed with a second primary tumor in the left lung, and metastases in lymph nodes were also found. The patient refused additional treatment and died shortly after diagnosis. The results of the molecular investigations were of no influence in this case.

Patient No. 24
Patient no. 24, age 58 years, had lesions in both lungs. One lesion was located centrally in the left lower lobe; this would have necessitated a pneumonectomy. The other lesion was present in the right upper lobe, requiring lobectomy. Both tumors were squamous cell carcinomas, and molecular analyses demonstrated that these tumors were two independent entities. The patients’ pulmonary function did allow not for pneumonectomy in combination with a right upper lobectomy. The knowledge that this patient suffered from two primary lung tumors did not alter our treatment strategy, because this was dictated by the poor pulmonary function.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In 25 consecutive patients with multiple tumors, we evaluated the clonal relationship between the tumors within each patient. All patients had at least one lesion in the head and neck region or lung. In every patient, the histology, cytology, and immunohistochemistry of both lesions was the same on routine staining, hence making it impossible to discriminate between two independent primary tumors or metastatic disease. Tumors harbor genomic aberrations, and these can be used as tumor-specific clonal markers.6 We performed LOH and p53 aberration determinations on multiple tumors within one patient to obtain information on the relationship between the tumors. Both LOH and p53 aberration assays are based on the possibility to amplify specific genomic DNA fragments by PCR. Because the DNA retrieved from routinely processed tissue and cytology samples is highly degraded, we amplified short DNA fragments (< 200 bp) by PCR. Parts of tissue specimens were obtained with minimal invasive methods as ultrasound- or computed tomography (CT)-guided histologic biopsy or cytology. Ultrasound- or CT-guided biopsies, either for histology or cytology, are safe procedures and can be performed with low complication rates, even with intrapulmonary lesions.21-23 For LOH analyses, DNA has to be isolated from parts of the tumors with few normal cells. Such tissue was obtained by manual or laser capture microdissection. With these procedures, the routinely obtained and processed tissue specimens from all 25 patients were sufficient to perform molecular analyses.

In all 25 patients, LOH of one of the markers used was found in at least one of the tumors. Tumor-specific p53 aberrations were detected in 20 of 25 patients. These results are in accordance with reported data for lung and head and neck carcinomas.7,8,11 Different or the same LOH or p53 aberration patterns in multiple tumors within one patient point to the presence of multiple primary tumors or metastatic disease, respectively. However, the results should be interpreted with caution. Within one tumor, heterogeneity with respect to the presence of molecular aberrations can be present.24-28 This can lead to the detection of different aberrations in clonally related tumors. Furthermore, in metachronous clonally related tumors, different DNA aberrations can be found, because genomic instability in tumors proceeds in time.29 This can result in detection of specific DNA aberrations present in the most recent lesion and not present in the first lesion. These differences could be interpreted as indicative of different tumors when, in fact, one is dealing with metastatic disease. Therefore, we regarded DNA aberrations present in the first lesion and absent in the second tumor of patients with metachronous cancers as a strong indication for independent tumors. Conversely, the presence of specific DNA aberrations in the most recent tumor and not in the first lesion of patients with metachronous cancers is regarded as a weak indication for independent tumors. To obtain information about whether these theoretical possibilities are present in our results, we scored every genomic aberration as a single indicator for relationship. To our surprise, no discrepancies were found between LOH patterns of different markers and p53 aberrations in multiple tumors within one patient. We did not observe, for example, LOH with one marker pointing to independent tumors and with another marker pointing to clonal-related tumors within one patient or LOH indicating one entity and p53 aberration suggestive for metastatic disease in the same patient. Obviously, the deletion of the investigated loci and the p53 gene aberrations are early pathogenic events in these tumors.7,27,30,31 and are not epiphenomena of the tumorigenic process. As a result, then, these molecular aberrations are present in the major fraction of the tumor cell population, as has also been suggested by other investigators.7,8 The versatility of molecular determinations to assess the relationship between multiple tumors is also indicated by the results of the five investigated cases (patient nos. 5, 15, 16, 17, and 25) with strong suspicion of having metastases. In all these cases, the apparent metastases were molecularly related to one of the patient’s other tumors.

Lau et al12 described the clonal origin of multiple lung cancers by k-ras and p53 aberrations determined by SSCP analysis, although only two cases were investigated. We obtained molecular evidence that in 15 of 25 patients, multiple independent tumors were present, and that 11 of 25 patients had metastatic disease. There was no correlation between synchronous or metachronous tumors and the presence of multiple primary lesions or metastases. In all patients with metastatic disease, as determined by molecular analysis, the clinical decision making was clearly influenced by this knowledge. In the 15 patients with a second primary tumor, clinical decision making was influenced in 10 patients. By assessing the patients in whom clinical decisions were not influenced, it became clear that in retrospect the molecular analyses were not indicated. However, the study design was to determine LOH and p53 aberration analyses in a consecutive series of 25 patients to assess the reliability of this method.

Whether survival is influenced remains to be seen, our patient group being too heterogeneous and too small for such an analysis. However, because we were able to differentiate between second primary tumors and metastases, the type of surgery or other treatment could be determined by this information, thus avoiding undertreatment or overtreatment.

In conclusion, in our opinion, every patient with a second tumor, whether synchronous or metachronous, should undergo determination of LOH and p53 aberrations if this knowledge bears potentially therapeutic implications.


   ACKNOWLEDGMENTS
 
We thank Frank van der Panne and Mustaffa Abbou for preparing the figures and tables and Patricia Ewing-Graham for critically reading the manuscript.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
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3. Leon X, Quer M, Diez S, et al: Second neoplasm in patients with head and neck cancer. Head Neck 21: 204-210, 1999[CrossRef][Medline]

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5. Kaneko S, Yamaguchi N: Epidemiological analysis of site relationships of synchronous and metachronous multiple primary cancers in the National Cancer Center, Japan, 1962-1996. Jpn J Clin Oncol 29: 96-105, 1999[Abstract/Free Full Text]

6. Lengauer C, Kinzler KW, Vogelstein B: Genetic instabilities in human cancer. Nature 396: 643-649, 1998[CrossRef][Medline]

7. Kropveld A, Rozemuller EH, Leppers FG, et al: Sequencing analysis of RNA and DNA of exons1 through 11 shows p53 gene alterations to be present in almost 100% of head and neck squamous cell cancers. Lab Invest 3: 347-353, 1999

8. Fujita T, Kiyama M, Tomizawa Y, et al: Comprehensive analysis of p53 gene mutation characteristics in lung carcinoma with special reference to histological subtypes. Int J Oncol 5: 927-934, 1999

9. Soong R, Powell B, Elsaleh H, et al: Prognostic significance of TP53 gene mutation in 995 cases of colorectal carcinoma: Influence of tumour site, stage, adjuvant chemotherapy and type of mutation. Eur J Cancer 16: 2053-2060, 2000

10. Friedrich MG, Riethdorf S, Erbersdobler A, et al: Relevance of p53 gene alterations for tumor recurrence patients with superficial transitional cell carcinoma of the bladder. Eur Urol 2: 159-166, 2001

11. Mertens F, Johansson B, Höglund M, et al: Chromosomal imbalance maps of malignant solid tumors: A cytogenetic survey of 3185 neoplasms. Cancer Res 57: 2765-2780, 1997[Abstract/Free Full Text]

12. Lau DH, Yang B, Hu R, et al: Clonal origin of multiple lung cancers: K-ras and p53 mutations determined by nonradioisotopic single-strand conformation polymorphism analysis. Diagn Mol Pathol 6: 179-184, 1997[CrossRef][Medline]

13. Ribeiro U, Safatle-Ribeiro AV, Posner MC, et al: Comparative p53 mutational analysis of multiple primary cancers of the upper aerodigestive tract. Surgery 120: 45-53, 1996[CrossRef][Medline]

14. Leong PP, Rezai B, Koch WM, et al: Distinguishing second primary tumors from lung metastases in patients with head and neck squamous cell carcinoma. J Natl Cancer Inst 90: 972-977, 1998[Abstract/Free Full Text]

15. Califano J, Leong PL, Koch WM, et al: Second esophageal tumors in patients with head and neck squamous cell carcinoma: Assessment of clonal relationships. Clin Cancer Res 5: 1862-1867, 1999[Abstract/Free Full Text]

16. Bot FJ, Sleddens HFBM, Dinjens WNM: Molecular assessment of clonality leads to identification of a new germ line TP53 mutation associated with malignant cystosarcoma phyllodess and soft tissue sarcoma. Diagn Mol Pathol 7: 295-301, 1998[Medline]

17. Matsuzoe D, Hideshima T, Ohshima K, et al: Discrimination of double primary lung cancer from intrapulmonary metastasis by p53 gene mutation. Br J Cancer 79: 1549-1552, 1999[CrossRef][Medline]

18. Oijen van MG, Leppers VD, Straat FG, et al: The origins of multiple squamous cell carcinomas in the aerodigestive tract. Cancer 88: 884-893, 2000[CrossRef][Medline]

19. Shimizu S, Yatabe Y, Koshikawa T, et al: High frequency of clonally related tumors in cases of multiple synchronous lung cancers as revealed by molecular diagnosis. Clin Cancer Res 6: 3994-3999, 2000[Abstract/Free Full Text]

20. Sozzi G, Miozzo M, Pastorino U, et al: Genetic evidence for an independent origin of multiple preneoplastic and neoplastic lung lesions. Cancer Res 55: 135-140, 1995[Abstract/Free Full Text]

21. Santambrogio L, Nosotti M, Bellaviti N, et al: CT-guided fine-needle aspiration cytology of solitary pulmonary nodules: A prospective, randomized study of immediate cytologic evaluation. Chest 112: 423-425, 1997[Abstract/Free Full Text]

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23. Akman C, Haider S, Ayarcan Y, et al: Percutaneous fine needle aspiration biopsy of the lung cancer and correlation with histologic results in 34 patients. Lung Cancer 29: 216, 2000 (suppl 1, abstr)

24. Mirchandani D, Zheng J, Miller GJ, et al: Heterogeneity in intratumor distribution of p53 mutations in human prostate cancer. Am J Pathol 147: 92-101, 1995[Abstract]

25. Ponten F, Berg C, Ahmadain A, et al: Molecular pathology in basal cell cancer with p53 as a genetic marker. Oncogene 15: 1059-1067, 1997[CrossRef][Medline]

26. Zhang J-S, Caplin S, Bosman FT, et al: Genetic diversity at the p53 locus between primary human colorectal adenocarcinomas and their lymph-node metastases. Int J Cancer 70: 674-678, 1997[CrossRef][Medline]

27. Waridel F, Estreicher A, Bron L, et al: Field cancerisation and polyclonal p53 mutation in the upper aero-digestive tract. Oncogene 14: 163-169, 1997[CrossRef][Medline]

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Submitted May 10, 2001; accepted October 4, 2001.


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