Original Contributions
c-myc Oncogene Copy Number in Squamous Carcinoma of the Head and Neck BRUCE H. HAUGHEY, MB, CHB, MS, FRACS, DANIEL D. VON HOFF, MD, BRADFORD E. WINDLE, PHD, GEOFFREY M. WAHL, PHD, AND PRESLEY M. MOCK, MD Purpose:Altered resident cellular genetic sequences (oncogenes) may result in malignant transformation, maintenance of tumor growth, and metastatic propensity. In this pilot study, we have elected to probe c-myc oncogene in evaluating specimens from human squamous cell carcinoma. Materialsand Methods:Samples were obtained from 24 patients with squamous cell carcinoma of the head and neck. The ratio of tumor DNA values to that of control DNA was used to estimate the c-myc copy number. Results:Data from material obtained from eight patients was analyzed to the point of c-myc copy number. Tumors varied from stage II through IV. Five originated in the oral cavity and three in the larynx. Analysis of primary tumors demonstrated that two of eight had increased c-myc copy numbers. Histologically positive neck specimens were encountered in five of the study patients. Three demonstrated elevated c-myc copy numbers, two
of which had had increased copy number at the primary site. study confirms that c-myc amplification can be present in squamous cell carcinoma of the head and neck. c-myc Amplification may also be present in neck metastasis. Oncogene amplification in neck metastasis may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification.
Conclusion: This
Copyright
0 1992 by W.B. Saunders
Company
The oncogene hypothesis of malignant neoplasia has led to a unified concept whereby a variety of insults, including chemicals, radiation, and viruses, could induce malignancy. The final common pathway is a subtle alteration of identifiable sequences within the normal human gene, which in their undisturbed state, regulate cellular reproduction and growth in an orderly fashion. More than 40 such sets of sequences that reside in the hu-
From The Department of Otolaryngology-Head and Neck Surgery, Washington University Medical Center, St Louis, MO; The Division of Medical Oncology, The University of Texas Health Sciences Center, San Antonio, TX; and the The Salk Institute, San Diego, CA. Presented at the 31st Annual Meeting of the American Society for Head and Neck Surgery, April 5-6, 1989. Supported in pat-t by NIH Grant No. IUOlCA48405-01. Address reprint requests to Bruce H. Haughey, MB, ChB, MS, FRACS, 4932 Forest Park, Suite 6a, St Louis, MO 63108. Copyright 0 1992 by W.B. Saunders Company 0196-0709/92/l 303-0006$5.00/O 168
American Journal of Otolaryngology,
man DNA molecule (proto-oncogenes) have been proven to exist. They have been sequenced through hybridization with known, complementary sequences in retroviruses. When these genes, comprising hundreds of amino acids, are altered by amplification (multiple copies), point mutation, translocational rearrangement, or loss of flanking suppressor/regulator sequences, they may become oncogenes. These are then capable, either alone or in combination, of inducing and/or maintaining malignant growth. Oncogene products, oncoproteins, take a variety of forms and functions, including growth factors, growth factor receptors, cellular kinases, and binding proteins, any of which may influence cell replication activity. The detection of oncogene sequences in head and neck cancer tissue was first reported by Spandidos et al,l who found amplification of K-ras, H-ras, and myc in 14 squamous carcinomas. Friedman et al2 reported malignant Vol 13, No 3 (May-June), 1992: pp 168-171
c-myc HEAD/NECK
169
CANCER
transformation of transfected mouse fibroblasts by DNA extracted from three malignant squamous tumors of the head and neck (base of tongue, larynx, and pharynx). Kasid et al3 detected association of raf-1 with a radiationresistant laryngeal squamous carcinoma cell line (SQ-208) and subsequent reports have detected c-myc and H-ras or c-myc and K-ras4 amplification in squamous carcinoma of the head and neck. The significance of both myc and ras alterations in the same tumor demonstrates the potential importance of multiple perturbations contributing to malignant behavior. Amplification of c-myc has been detected in a broad spectrum of human tumors. The precise significance of this finding as it may indicate transformation to and maintenance of malignancy in vivo is unknown; however, augmented or rearranged c-myc expression is associated with acute transformation in avian,5 murine,6 and feline7 models. Given this background, we screened fresh human squamous cell carcinoma DNA for c-myc amplification according to the following.
MATERIALS AND METHODS Samples were obtained from 24 human squamous cell carcinomas, with eight tumors being analyzed to the point of c-myc copy number data. Informed consent for molecular processing of tumor material and venesection was obtained from patients after full explanation. Tissue sampled from each patient includes (1) l-cm cubes of primary tumor [extracted from an intermediate position), (2) 1 cm2 of adjacent, normal mucosal lining excised with the primary tumor, (3) positive or first echelon lymph nodes, and TABLE 1.
Patient,
Age
Site
Stage
70 63 56 69 70 43 80 60
oc oc oc oc LNX LNX oc LNX
T3Nl T3N2b T3N3b T3NO T3Nl T3N2 T3NO T2NO
Abbreviations:
Tumor,
and c-myc Copy Number
OC, oral cavity;
Surgical Margins _ _ _ _ _ + LNX, larynx;
(4) normal peripheral blood lymphocytes. Specimens (2) and (4) were assumed to be control tissue. Tumor specimens, lymph nodes, and control mucosa were placed in transport medium (500 mL McCoy’s medium; 50 mL newborn calf serum; 5 mL P/S; 10 mL Hepes buffer; and 5 mL Na pyruvate) and processed by mechanical means to form a single-cell suspension of concentration 5.5 X lo5 cells/ml in freezing medium. The suspensions were then frozen. Total DNA extraction was then later performed via sodium dodechyl sulphate (SDS) cell lysis, proteinase K digestion of protein, extraneous protein eradication, phenol extraction, followed by ethanol precipitation and centrifugation. Quantitation of c-myc copy number was performed for each specimen by applying DNA to nitrocellulose using the S + S slot blot apparatus and hybridizing the blot with a 32P-labeled probe specific for c-myc sequences. Total DNA applied was quantitated by washing off the c-myc probe and rehybridizing the blot with a ribosomal DNAspecific probe. The blots were exposed to film and the slots scanned by a densitometer. The ratio of tumor DNA values to that of control DNA (normal mucosa, peripheral blood lymphocyte, and human placenta DNA) represented the c-myc copy number. A copy number (ratio) of 1.0 signifies no c-myc oncogene amplification, but greater than 1.0 suggests some degree of amplification, although copy numbers of between 0.5 and 3.0 have doubtful significance because of the potential effects of aneuploidy.’ Eight patients’ specimens were analyzed.
RESULTS Details of patient, site, stage, and histology for the eight patients are listed in Table 1. Tumors ranged from stage II through IV, with five of oral cavity and three of laryngeal origin. Margins of resected specimens were histologically clear of tumor on all but one case (T,N, larynx), and neck node histology was
Data
Node Malignancy + + + + + M, moderately;
DifferentiationN M-W M M M W W M-W W W, well; M-W, moderately
1” copy Number
Neck Node Copy Number
5.5 2.1 1.5 1.0 1.6 0.5 0.6 1.2
3.3 5.1 1.7 1.6 2.3 2.3 1.7 No specimen
well.
HAUGHEY
positive for metastasis in at least one node in five of the eight neck dissection specimens. In two cases, nodal histology was at variance with clinical N staging, an expected finding.g Table 1 further outlines copy numbers obtained for primary tumor tissue and neck nodes. It is noted that two of eight primary tumors demonstrated increased c-myc copy numbers (5.5 and 2.1), both of which also demonstrated neck metastasis with an elevated copy number (3.3 and 5.1, respectively). Of the five histologically positive neck specimens, three demonstrated elevated c-myc copy numbers, two of which had increased copy number at their respective primary site and one of which did not. One histologically negative neck (44 of 44 nodes clear) yielded a marginally significant copy number of 2.3, again with no amplification of the primary site. All normal mucosa, lymphocyte, and placental control analyses yielded copy numbers varying from 0.5 to 1.5, with the average defined as 1.0. A summary of copy numbers found in c-myc amplified neck nodes is found in Table 2. DISCUSSION The need to detect prognostic markers at presentation, so that appropriate therapy may be planned, has been fulfilled in some human tumors using oncogene analysis (N-myc,l’ erb B-2/neu). I1 Therefore, analysis at the DNA level of known oncogene sequences may represent a way of identifying metastatic propensity of individual tumors and, ultimately, of detecting clinically silent micrometastases.” Metastasis is the major prognostic determinant in head and neck cancer. Therapeutic implications include antibodies directed against oncogene protein products expressed at the cell surface,13 as well as
ET AL
the theoretical possibility of replacing the defective oncogene with its normal counterpart. This could ultimately furnish a method for selective eradication of metastases. Various oncogenes, including H-ras,4 K-ras,4 int-2,14 and rap have been reported as associated with squamous carcinoma of the head and neck. To this embryonic data we add confirmation that c-myc copy number is increased in some primary squamous aerodigestive carcinomas and add new data on the presence of c-myc amplification in neck node metastases and first echelon neck nodes. The importance of oncogene analyses in neck specimens lies in the known poor prognosis endowed by neck metastasis,14 especially in nodes demonstrating extracapsular spread.15 Further investigation of larger numbers of patients with squamous carcinoma of the head and neck will ultimately show the significance of oncogene amplification and its potential for correlation with relapse and survival times. CONCLUSIONS Our study confirms previous reports5’8pg that c-myc amplification can be present in primary squamous carcinoma of the head and neck. Amplification of c-myc also can be present in neck metastases from primary squamous carcinomas of the head and neck. To our knowledge, this is newly reported information. In this pilot series, the higher proportionate representation of c-myc amplified tumor in the neck metastases may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification. However, the possibility exists that this finding represents an epiphenomenon rather than a causal relationship. Therefore, further investigation is mandatory. REFERENCES
TABLE 2.
Elevated Versus N-Stage N-stage Nl N2 Nl NO
c-myc Copy Numbers
c-myc Copy Number 3.3 5.1 2.3 2.3
1. Spandidos DA, Lamothe A, Field JK: Multiple transcriptional activation of cellular oncogenes in human head and neck solid tumors. Anticancer Res 5221-224, 1985 2. Friedman WH, Rosenblum B, Larenstein P, et al: Oncogenes: Their presence and significance in squamous cell cancer of the head and neck. Laryngoscope 95:313316,1985 3. Kasid U, Pfeifer A, Weichselbaum R, et al: The raf
c-myc HEAD/NECK
CANCER
oncogene is associated with a radiation-resistant human laryngeal cancer. Science 237:1039-1041, 4. Shuler C. Kurran P. French BT. et al: Non-correlative c-myc and rasoncogene in squamous cell carcinoma cells with tumorgenic potential: Teratogenesis. Carcinog Mutagen 1053-65, 1990 5. Payne GS, Bishop JM, Varmus HE: Multiple arrangements of viral DNA and an activitated host oncogene (c-myc) in B lymphomas. Nature 295209-214, 1982 6. Adams JM, Harris A, Pinkert C, et al: The c-myc oncogene driven by immune globulin enhancers induced lymphoid malignancy in transgenic mice. Nature 318: 533-538, 1985 7. Neil JC, Hughes D, McFarlane R, et al: Transduction and rearrangement of the myc gene by feline leukemia virus in naturally occurring T-cell lymphomas. Nature 308:814-820, 1984 8. Seeger RG, Brodeur GM, Sather H: Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313:1111-1116, 1985 9. Collins SL: Controversies
in the management of cancer of the neck, in Thawley S, Panje WR (eds): Compre-
171
hensive Management of Head and Neck Tumors, vol 2. Philadelphia, PA, Saunders, 1982, p 1410 10. lohnson BE. Ihde DL. Makuch RW. et al: Mvc family Oncogene amplification in tumor cell lines- established from small cell lung cancer patients and its relationship to clinical status and course. J Clin Invest 79:1629-1634, 1987 11. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer: Correlation of relapse and survival with amplification of the HER-Zineu Oncogene. Science 235:177-182, 1987 12. Marx JL: How cancer cells spread in the body. Science 244:147-148, 1989 13. Sullivan LM, Quigley JP: An anticatalytic monoclonal antibody to avian plasminogen activator: Its effect on behavior of RSV-transformed chick fibroblasts. Cell 45:905-915, 1986 14. Schuller DE, McGuirt WF, McCabe BF: The prognostic significance of metastatic cervical lymph nodes. Laryngoscope 90:557-570, 1980 15. Johnson JJ, Barnes EL, Myers EN, et al: The extracapsular spread of tumors in cervical node metastasis. Arch Otolaryngol 107:725, 1981
c-myc Oncogene Copy Number in Squamous Carcinoma of the Head and Neck BRUCE H. HAUGHEY, MB, CHB, MS, FRACS, DANIEL D. VON HOFF, MD, BRADFORD E. WINDLE, PHD, GEOFFREY M. WAHL, PHD, AND PRESLEY M. MOCK, MD Purpose:Altered resident cellular genetic sequences (oncogenes) may result in malignant transformation, maintenance of tumor growth, and metastatic propensity. In this pilot study, we have elected to probe c-myc oncogene in evaluating specimens from human squamous cell carcinoma. Materialsand Methods:Samples were obtained from 24 patients with squamous cell carcinoma of the head and neck. The ratio of tumor DNA values to that of control DNA was used to estimate the c-myc copy number. Results:Data from material obtained from eight patients was analyzed to the point of c-myc copy number. Tumors varied from stage II through IV. Five originated in the oral cavity and three in the larynx. Analysis of primary tumors demonstrated that two of eight had increased c-myc copy numbers. Histologically positive neck specimens were encountered in five of the study patients. Three demonstrated elevated c-myc copy numbers, two
of which had had increased copy number at the primary site. study confirms that c-myc amplification can be present in squamous cell carcinoma of the head and neck. c-myc Amplification may also be present in neck metastasis. Oncogene amplification in neck metastasis may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification.
Conclusion: This
Copyright
0 1992 by W.B. Saunders
Company
The oncogene hypothesis of malignant neoplasia has led to a unified concept whereby a variety of insults, including chemicals, radiation, and viruses, could induce malignancy. The final common pathway is a subtle alteration of identifiable sequences within the normal human gene, which in their undisturbed state, regulate cellular reproduction and growth in an orderly fashion. More than 40 such sets of sequences that reside in the hu-
From The Department of Otolaryngology-Head and Neck Surgery, Washington University Medical Center, St Louis, MO; The Division of Medical Oncology, The University of Texas Health Sciences Center, San Antonio, TX; and the The Salk Institute, San Diego, CA. Presented at the 31st Annual Meeting of the American Society for Head and Neck Surgery, April 5-6, 1989. Supported in pat-t by NIH Grant No. IUOlCA48405-01. Address reprint requests to Bruce H. Haughey, MB, ChB, MS, FRACS, 4932 Forest Park, Suite 6a, St Louis, MO 63108. Copyright 0 1992 by W.B. Saunders Company 0196-0709/92/l 303-0006$5.00/O 168
American Journal of Otolaryngology,
man DNA molecule (proto-oncogenes) have been proven to exist. They have been sequenced through hybridization with known, complementary sequences in retroviruses. When these genes, comprising hundreds of amino acids, are altered by amplification (multiple copies), point mutation, translocational rearrangement, or loss of flanking suppressor/regulator sequences, they may become oncogenes. These are then capable, either alone or in combination, of inducing and/or maintaining malignant growth. Oncogene products, oncoproteins, take a variety of forms and functions, including growth factors, growth factor receptors, cellular kinases, and binding proteins, any of which may influence cell replication activity. The detection of oncogene sequences in head and neck cancer tissue was first reported by Spandidos et al,l who found amplification of K-ras, H-ras, and myc in 14 squamous carcinomas. Friedman et al2 reported malignant Vol 13, No 3 (May-June), 1992: pp 168-171
c-myc HEAD/NECK
169
CANCER
transformation of transfected mouse fibroblasts by DNA extracted from three malignant squamous tumors of the head and neck (base of tongue, larynx, and pharynx). Kasid et al3 detected association of raf-1 with a radiationresistant laryngeal squamous carcinoma cell line (SQ-208) and subsequent reports have detected c-myc and H-ras or c-myc and K-ras4 amplification in squamous carcinoma of the head and neck. The significance of both myc and ras alterations in the same tumor demonstrates the potential importance of multiple perturbations contributing to malignant behavior. Amplification of c-myc has been detected in a broad spectrum of human tumors. The precise significance of this finding as it may indicate transformation to and maintenance of malignancy in vivo is unknown; however, augmented or rearranged c-myc expression is associated with acute transformation in avian,5 murine,6 and feline7 models. Given this background, we screened fresh human squamous cell carcinoma DNA for c-myc amplification according to the following.
MATERIALS AND METHODS Samples were obtained from 24 human squamous cell carcinomas, with eight tumors being analyzed to the point of c-myc copy number data. Informed consent for molecular processing of tumor material and venesection was obtained from patients after full explanation. Tissue sampled from each patient includes (1) l-cm cubes of primary tumor [extracted from an intermediate position), (2) 1 cm2 of adjacent, normal mucosal lining excised with the primary tumor, (3) positive or first echelon lymph nodes, and TABLE 1.
Patient,
Age
Site
Stage
70 63 56 69 70 43 80 60
oc oc oc oc LNX LNX oc LNX
T3Nl T3N2b T3N3b T3NO T3Nl T3N2 T3NO T2NO
Abbreviations:
Tumor,
and c-myc Copy Number
OC, oral cavity;
Surgical Margins _ _ _ _ _ + LNX, larynx;
(4) normal peripheral blood lymphocytes. Specimens (2) and (4) were assumed to be control tissue. Tumor specimens, lymph nodes, and control mucosa were placed in transport medium (500 mL McCoy’s medium; 50 mL newborn calf serum; 5 mL P/S; 10 mL Hepes buffer; and 5 mL Na pyruvate) and processed by mechanical means to form a single-cell suspension of concentration 5.5 X lo5 cells/ml in freezing medium. The suspensions were then frozen. Total DNA extraction was then later performed via sodium dodechyl sulphate (SDS) cell lysis, proteinase K digestion of protein, extraneous protein eradication, phenol extraction, followed by ethanol precipitation and centrifugation. Quantitation of c-myc copy number was performed for each specimen by applying DNA to nitrocellulose using the S + S slot blot apparatus and hybridizing the blot with a 32P-labeled probe specific for c-myc sequences. Total DNA applied was quantitated by washing off the c-myc probe and rehybridizing the blot with a ribosomal DNAspecific probe. The blots were exposed to film and the slots scanned by a densitometer. The ratio of tumor DNA values to that of control DNA (normal mucosa, peripheral blood lymphocyte, and human placenta DNA) represented the c-myc copy number. A copy number (ratio) of 1.0 signifies no c-myc oncogene amplification, but greater than 1.0 suggests some degree of amplification, although copy numbers of between 0.5 and 3.0 have doubtful significance because of the potential effects of aneuploidy.’ Eight patients’ specimens were analyzed.
RESULTS Details of patient, site, stage, and histology for the eight patients are listed in Table 1. Tumors ranged from stage II through IV, with five of oral cavity and three of laryngeal origin. Margins of resected specimens were histologically clear of tumor on all but one case (T,N, larynx), and neck node histology was
Data
Node Malignancy + + + + + M, moderately;
DifferentiationN M-W M M M W W M-W W W, well; M-W, moderately
1” copy Number
Neck Node Copy Number
5.5 2.1 1.5 1.0 1.6 0.5 0.6 1.2
3.3 5.1 1.7 1.6 2.3 2.3 1.7 No specimen
well.
HAUGHEY
positive for metastasis in at least one node in five of the eight neck dissection specimens. In two cases, nodal histology was at variance with clinical N staging, an expected finding.g Table 1 further outlines copy numbers obtained for primary tumor tissue and neck nodes. It is noted that two of eight primary tumors demonstrated increased c-myc copy numbers (5.5 and 2.1), both of which also demonstrated neck metastasis with an elevated copy number (3.3 and 5.1, respectively). Of the five histologically positive neck specimens, three demonstrated elevated c-myc copy numbers, two of which had increased copy number at their respective primary site and one of which did not. One histologically negative neck (44 of 44 nodes clear) yielded a marginally significant copy number of 2.3, again with no amplification of the primary site. All normal mucosa, lymphocyte, and placental control analyses yielded copy numbers varying from 0.5 to 1.5, with the average defined as 1.0. A summary of copy numbers found in c-myc amplified neck nodes is found in Table 2. DISCUSSION The need to detect prognostic markers at presentation, so that appropriate therapy may be planned, has been fulfilled in some human tumors using oncogene analysis (N-myc,l’ erb B-2/neu). I1 Therefore, analysis at the DNA level of known oncogene sequences may represent a way of identifying metastatic propensity of individual tumors and, ultimately, of detecting clinically silent micrometastases.” Metastasis is the major prognostic determinant in head and neck cancer. Therapeutic implications include antibodies directed against oncogene protein products expressed at the cell surface,13 as well as
ET AL
the theoretical possibility of replacing the defective oncogene with its normal counterpart. This could ultimately furnish a method for selective eradication of metastases. Various oncogenes, including H-ras,4 K-ras,4 int-2,14 and rap have been reported as associated with squamous carcinoma of the head and neck. To this embryonic data we add confirmation that c-myc copy number is increased in some primary squamous aerodigestive carcinomas and add new data on the presence of c-myc amplification in neck node metastases and first echelon neck nodes. The importance of oncogene analyses in neck specimens lies in the known poor prognosis endowed by neck metastasis,14 especially in nodes demonstrating extracapsular spread.15 Further investigation of larger numbers of patients with squamous carcinoma of the head and neck will ultimately show the significance of oncogene amplification and its potential for correlation with relapse and survival times. CONCLUSIONS Our study confirms previous reports5’8pg that c-myc amplification can be present in primary squamous carcinoma of the head and neck. Amplification of c-myc also can be present in neck metastases from primary squamous carcinomas of the head and neck. To our knowledge, this is newly reported information. In this pilot series, the higher proportionate representation of c-myc amplified tumor in the neck metastases may indicate an increased metastatic propensity for individual tumor cells demonstrating c-myc amplification. However, the possibility exists that this finding represents an epiphenomenon rather than a causal relationship. Therefore, further investigation is mandatory. REFERENCES
TABLE 2.
Elevated Versus N-Stage N-stage Nl N2 Nl NO
c-myc Copy Numbers
c-myc Copy Number 3.3 5.1 2.3 2.3
1. Spandidos DA, Lamothe A, Field JK: Multiple transcriptional activation of cellular oncogenes in human head and neck solid tumors. Anticancer Res 5221-224, 1985 2. Friedman WH, Rosenblum B, Larenstein P, et al: Oncogenes: Their presence and significance in squamous cell cancer of the head and neck. Laryngoscope 95:313316,1985 3. Kasid U, Pfeifer A, Weichselbaum R, et al: The raf
c-myc HEAD/NECK
CANCER
oncogene is associated with a radiation-resistant human laryngeal cancer. Science 237:1039-1041, 4. Shuler C. Kurran P. French BT. et al: Non-correlative c-myc and rasoncogene in squamous cell carcinoma cells with tumorgenic potential: Teratogenesis. Carcinog Mutagen 1053-65, 1990 5. Payne GS, Bishop JM, Varmus HE: Multiple arrangements of viral DNA and an activitated host oncogene (c-myc) in B lymphomas. Nature 295209-214, 1982 6. Adams JM, Harris A, Pinkert C, et al: The c-myc oncogene driven by immune globulin enhancers induced lymphoid malignancy in transgenic mice. Nature 318: 533-538, 1985 7. Neil JC, Hughes D, McFarlane R, et al: Transduction and rearrangement of the myc gene by feline leukemia virus in naturally occurring T-cell lymphomas. Nature 308:814-820, 1984 8. Seeger RG, Brodeur GM, Sather H: Association of multiple copies of the N-myc oncogene with rapid progression of neuroblastomas. N Engl J Med 313:1111-1116, 1985 9. Collins SL: Controversies
in the management of cancer of the neck, in Thawley S, Panje WR (eds): Compre-
171
hensive Management of Head and Neck Tumors, vol 2. Philadelphia, PA, Saunders, 1982, p 1410 10. lohnson BE. Ihde DL. Makuch RW. et al: Mvc family Oncogene amplification in tumor cell lines- established from small cell lung cancer patients and its relationship to clinical status and course. J Clin Invest 79:1629-1634, 1987 11. Slamon DJ, Clark GM, Wong SG, et al: Human breast cancer: Correlation of relapse and survival with amplification of the HER-Zineu Oncogene. Science 235:177-182, 1987 12. Marx JL: How cancer cells spread in the body. Science 244:147-148, 1989 13. Sullivan LM, Quigley JP: An anticatalytic monoclonal antibody to avian plasminogen activator: Its effect on behavior of RSV-transformed chick fibroblasts. Cell 45:905-915, 1986 14. Schuller DE, McGuirt WF, McCabe BF: The prognostic significance of metastatic cervical lymph nodes. Laryngoscope 90:557-570, 1980 15. Johnson JJ, Barnes EL, Myers EN, et al: The extracapsular spread of tumors in cervical node metastasis. Arch Otolaryngol 107:725, 1981
