- Email: [email protected]
Serologic determinants of survival in patients with squamous cell carcinoma of the head and neck
Serologic Determinants of Survival in Patients with Squamous Cell Carcinoma of the Head and Neck Gary L. Clayman, DDS, MD, Howard E. Savage, PhD, Nancy Ainslie, RN, Frank J. Liu, MD, Stimson P. Schantz, MD,Houston,Texas
Specific circulating serum proteins may reflect unique properties governing the growth and progression of head and neck cancers, One hundred three previously untreated patients with squamous cell carcinoma of the head and neck were prospectively evaluated for serum IgA, lgG, and lgM and Clq-binding macromolecules, lmmunoglobulins were assessed by the immunoturbidimetric technique. Clq-binding maeromolecules (CIqBM) were measured utilizing the iodine-125 assay of Zubler et al (J Immunol 1976; 116: 232-5). Neither the level of serum immunoglobulins nor CIqBM values were correlated with the primary site, AJC (American Joint Committee on Cancer) stage of disease, or size of primary lesion. Likewise, comparison of serum lgA with ClqBM values demonstrated that these laboratory parameters were independent variables (r = 0.15 by Pearson linear regression). Univariate statistical analysis, utilizing the Cox proportional hazard model, showed serum lgA and ClqBM values to each contribute significantly to the ability to predict survival in patients with advanced squamous cell carcinoma of the head and neck (p = 0.01 and 0.003, respectively). Furthermore, multivariate analysis reveals that both ClqBM and serum lgA levels contribute significantly to the hazards model beyond staging in predicting survival (p <0.001 ). Predictive results were most apparent in patients with stage IV disease and related to the probability of both regional and distant metastatic recurrences. Conversely, serologic analysis provided no information in patients who were staged early. These results support pretreatment multiparametric serologic analysis of patients with squamous cell carcinoma of the head and neck.
From the Departments Of Head and Neck Surgery (GLC, HES, NA, SPS) and Laboratory Medicine (FJL), the University Of Texas M: D. Anderson Cancer Center, Houston, Texas. Requests for reprints Should be addressed to Stimson P. Schantz, MD, Department of Head and Neck Surgery, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. Presented at the 36th Annual Meeting of the Society of Head and Neck Surgeons, Washington, DC, May 19-22, 1990.
434
THE AMERICAN JOURNAL OF SURGERY
A
growing body of evidence supports the integration of biologic parameters of host-tumor interaction into the assessment and treatment of patients with head and neck cancer. Initial investigations of circulating macromolecules capable of binding the first component of complement in vitro (Clq-binding maeromolecules, ClqBM) found a stage-independent relationship between elevated levels of ClqBM and lack of a clinical response to induction chemotherapy [1,2]. These results were subsequently verified by Blazar et al [3] who quantitated polyethylene glycol precipitates from sera of patients with head and neck cancer and found that a high level of precipitate predicted a low probability of response to chemotherapy. The clinical relevance of ClqBM was reinforced by the observation that its elevation was predictive of a high probability of death with disease, independent of the use of induction chemotherapy (i.e., patients with elevated ClqBM levels who received multimodality therapy that did not include anticancer drugs did just as poorly) (Schantz SP, et al, unpublished data). A considerable number of attempts have been made to correlate immunoglobulin levels and clinical stage or activity of disease in cancer patients. Elevation of IgA has been demonstrated at initial workup in sera from patients with nasopharyngeal carcinoma [4], perhaps reflecting a local immune response, and in sera and saliva of patients with head and neck squamous cell carcinoma [5]. Significant serum IgA elevations have also been found in patients with advanced or recurrent disease among a variety of tumors [6,7]. The current investigation was designed to determine whether serologic levels of C lqBM and immunoglobulin classes (specifically, IgA) could be predictive of survival in patients with squamous cell carcinoma of the head and neck, independent of standard clinical staging techniques. These parameters were evaluated for predicability both together and alone, and in a variety of patient subsets. PATIENTS AND METHODS Patients: A total of 103 patients with previously untreated squamous cell carcinoma of the upper aerodigestive tract were evaluated prospectively for serologic levels of ClqBM, IgG, IgA, and IgM. Only patients who received definitive therapy at this institution were eligible. Median patient age was 56 years (range: 21 to 81 years); the male-to-female ratio was 3:1. Primary disease was in the oral cavity in 25 patients, the pharynx in 51, and the larynx in 27. Disease staging was by the 1983 American Joint Committee on Cancer (AJC) criteria [8], yielding 12 patients with Stage I disease, 13 with stage II lesions,
VOLUME 160 OCTOBER 1990
SURVIVAL PREDICTORS IN HEAD AND NECK CANCERS
34 with stage III, and 44 with stage IV. All serologic samples were drawn by venipuncture before the initiation of therapy. The disease-free interval following therapy was calculated in months from the date of initial treatment to the last-noted physician contact at which the patient had no evidence of disease. Median follow-up of all patients was 18 months (range: 2 to 44 months). Twenty-seven of 103 patients died of disease. Of the patients who died of disease, 24 were patients with stage IV tumors. Twelve of the 103 patients died of other causes with a median follow-up of 24.5 months within this group. All patients were entered into this study before January 1, 1988. Follow-up occurred at 3-month intervals during the first year, 6-month intervals in the second year, and then annually. Disease status was assessed at follow-up by members of the Department of Head and Neck Surgery without knowledge of the serum ClqBM or immunoglobulin values. All data were collected in a prospective fashion and transformed into coded computer data before retrieval for the current analysis. Measurement of CIqBM: Sera were collected from clotted blood and frozen at - 7 0 ~ until tested. Human Clq (Quidel, San Diego, CA) was labeled with iodine 125 by the iodobead method (Pierce Chemical Co., Rockford, IL). Five hundred micrograms of Clq was mixed with 1 mCi of sodium iodine 125 in 1 mL final volume of phosphate-buffered saline on ice, pH 7.4. One iodobead was added to this mixture and incubated for 5 minutes. Labeled C lq was separated from free iodine 125 on a 10-dentimeter Sephadex G-25 column (PD-10 column; Pharmacia, Piscataway, N J). The C 1q-binding test was performed as previously described, using EDTAtreated sera according to the method of Zubler et al [9]. Results were expressed as #g/mL equivalents of heataggregated IgG with reference to a standard curve made with purified IgG, aggregated at 63~ for 20 minutes at a concentration of 3 mg/mL, and diluted serially in heatinactivated (56~ 30 minutes) normal donor serum. Sera from healthy, age-matched normal donors were used as negative controls and sera from head and neck cancer patients with previously determined positive values provided serial controls in each test run. Normal values for ClqBM are less than 50 t~g/mL. Measurement of plasma immunoglobulins: Plasma IgG, IgA, and IgM concentrations were measured by an immunoturbidimetric technique, developed on the computer-programmable ROCHE, COBAS BIO centrifugal analyzer (Roche Analytical Instruments, Nutley, N J), using IgG, IgA, and IgM calibrators and anti-IgG, antiIgA, and anti-IgM sera (Atlantic Antibodies, A. Charles River Co., Scarborough, ME) as we have previously reported [10]. The normal values established in this laboratory for IgG, IgA, and IgM are 650 to 1,750, 75 to 330, and 30 to 225 mg/dL, respectively. Since antigen excess would lead to soluble antigen-antibody reaction product and then to an underestimation of plasma immunoglobulin concentrations, serum protein electrophoresis by the Beckman Paragon Electrophoresis System (Beckman Instruments, Fullerton, CA) was performed on each sample
TABLE I
Predlctlvlty of Serum ClqBM, Serum IgA, and/or Disease Stage for Death with Disease (n = 1 0 3 ) * Analysis Univariate ClqBM Serum IgA Stage ClqBM-I-, IgA Multivariate ClqBM by stage IgA by stage ClqBM-F, IgA by stage
Log Likelihood
p Value
--108.98 --109.49 --98.29 -106.02
0.003 0.01 0 0.0003
--95.62 --95.11 --92.62
0.0001 0.0001 0.0001
* Cox proportional hazards analysis. Disease stage is stages I, II, and Ill, collectively, versus stage IV.
to identify any highly elevated immunoglobulin before quantitation by the immunoturbidimetric method. Any samples with high immunoglobulin levels were appropriately diluted. Statistical analyses: Comparisons between populations as related to C lqBM or the respective immunoglobulins were performed using either the Student's t test or the Mann-Whitney U-test for nonparametric data. All p values reported in the text represent results of t-test analysis. Those significant by this test were likewise significant by the Mann-Whitney test. The Cox proportional hazards model was applied for investigating the significance of single and paired serologic tests in predicting death with disease and site of recurrence using the BMDP regression statistical package (BMDP Statistical Software, Inc., Los Angeles, CA). Differences in the probabilities of death with disease between populations categorized by stage of disease were determined using proportional hazards regression and log-rank testing similarly through the BMDP statistical package. In all analyses, only death with disease was considered. Death from other causes was treated as a censored event and excluded the patient from subsequent analyses. Any p value less than 0.05 were considered statistically significant. RESULTS The relationship between serum ClqBM and IgA levels and the probability of death with disease was assessed. Univariate analysis results revealed a direct relationship between elevated levels of ClqBM and serum IgA obtained prior to treatment and the likelihood of death with disease, that is, patients with the highest ClqBM or serum IgA values were at greatest risk as determined by the Cox proportional hazards model (p = 0.003 and 0.01, respectively) (Table I). Results of ClqBM levels and prognosis are presented in Figure 1. In a multivariate Cox proportional analysis, both ClqBM and serum IgA values simultaneously considered maintained their significance within the Cox proportional hazards model, that is, the combination was highly predictive of death with disease (p -- 0.0003). Table I demonstrates the prognostic significance of standard AJC staging,
THE AMERICAN J O U R N A L OF SURGERY
VOLUME 160
OCTOBER 1990
435
CLAYMAN ET AL
1.0-
1.0,
0.8\_
0.8-
\
0.6-
0.6-
%
0.4-
0.4-
0.20.0
--
ClqBM ( 16 (n=34)
--
ClqBM ) 47 (n=34) I I 24 36 Months
8
I 12
3~176 T 9
Io
9
o
~
I00 t
o I o
~
9
I"
: ,~_ :ao=a 100
200
300
3
" ~9 400
4
: 500
600
700
800
900
IgA ( r n g / d l )
Figure 3. Survival dot plot in stage IV disease by ClqBM and IgA median values (27 #g/mL against standard and 292 mg/dL, respactively). Mortality rates are 25%, 50%, 82%, and 36%, respectively, for quadrants 1 to 4.
which combined patients with stage I, II, and III disease as compared with patients with stage IV disease. When considered in light of standard AJC staging, both ClqBM and serum IgA concentrations were still highly significant in their association with death from disease (p <0.001). The most prognostic information was provided when all parameters (both staging and serologic factors) were considered together (p <0.0001). The mean serum IgA and ClqBM values by lesion distribution within the primary sites of the 103 patients were analyzed. No significant differences could be identified in either parameter between patient populations stratified by site of primary disease, although patients with oral cavity lesions had the lowest mean levels of both ClqBM and IgA. To determine if ClqBM or immunoglobulin levels were related to primary tumor size or tumor burden, serum levels of IgA, IgG, and IgM and ClqBM were analyzed against primary tumor (T) stage as well as standard TNM stages by AJC standards. Levels of the respective serum proteins did not vary significantly by these stratifications (data not shown). Furthermore, Pearson linear-regression analysis of these data showed elevated serum IgA and ClqBM values to be
436
-~__~
- \:: ~ -: ~ i._ ~ .
*--* -.... - -
Io ClqBU, Io ,o ClqB~, ~ hi ClqBM, hi ClqBM, hi
IgA (n=JO) tgA ~n=12) IgA {n=11) IgA (n=l I)
316
418
0.20.0
I
9 Dead ( n = 2 1 ) o Living ( n = 2 3 ) - - - Median values
9
200 1
0
-
1t2
48
Figure 1. Survival, from initiation of treatment, by serum ClqBM levels in all 103 patients.
0]
~ ,
.... ClqBM = 16-47 (n=35)
0
1 1
-C'. x% " ' ' ' " .
T H E A M E R I C A N J O U R N A L OF S U R G E R Y
214 Months
Figure 2. Survival in 44 patients with stage IV disease by high or low levels of ClqBM and IgA.
independent variables associated with death due to disease in patients with stage IV cancers of the head and neck (r -- 0.15). The distribution of patients by AJC standards was 12 stage I, 13 stage II, 34 stage III, and 44 stage IV. Logrank testing showed no significant difference in survival, assessed by the Kaplan-Meier method, in multiple comparisons between patients with stage I, II, or III disease. In contrast, patients with stage IV disease had a significantly worse prognosis as compared with any combination of patients with earlier stage I, II, or III disease (p -0.004, p <0.001, p <0.001, respectively). The risk of death with disease was then considered by stages I, II, and III taken together (group 1) versus stage IV patients (group 2). Overall, analysis of this grouping for its capacity to predict probability of death with disease showed it to be a model highly predictive by the Cox proportional hazard model (p <0.001). The Cox proportional hazard model was then utilized to determine risk of death with disease in these two groupings. Within group 1, neither serum IgA or ClqBM levels were significant in predicting survival in these patients; however, in group 2 (patients with stage IV lesions), elevated serum IgA and C 1qBM values significantly contributed to the survival model (p = 0.008 and -- 0.02, respectively). High (elevated) levels were considered above the median for stage IV patients; conversely low levels were less than the median. Multivariate analysis of ClqBM and serum IgA values contributed significantly to the hazards model beyond that currently defined by AJC staging methods (p <0.001) (Figure 2). Analysis of site of recurrence revealed that IgA, IgG, and IgM levels were not predictive for primary, regional/ nodal, or distant recurrence in groups 1 or 2 (data not shown). However, elevated ClqBM concentrations were predictive of regional or distant metastasis within group 2 (p = 0.01 and 0.02, respectively). In addition, the combined analysis of ClqBM and serum IgA was predictive of regional/nodal recurrence within group 2 (p -- 0.006). Group 2 patients were further analyzed to assess the relationship between IgA levels and ClqBM levels to subsequent death with disease. No relationship between
V O L U M E 160
OCTOBER 1990
SURVIVAL PREDICTORS IN HEAD AND NECK CANCERS
primary site and serologic IgA or C l q B M levels was found (data not shown). A survival dot plot is illustrated in Figure 3 for group 2 (stage IV) patients. Median values of the group 2 patients for ClqBM was 27 g g / m L and 292 m g / d L for serum IgA. These values are depicted graphically. Those patients with combined elevations above the median (stage IV patients) showed significantly increased mortality (82%). Notably IgG and IgM did not significantly contribute to survival within either group 1 or 2. It is evident, however, that patients with elevated C l q B M concentrations who died with disease were not necessarily the same patients with elevated IgA values who died with disease. Figure 3 emphasizes that each parameter m a y identify distinct populations at risk and thus may reflect distinct determinants of disease progression. COMMENTS The purpose of this study was to investigate whether analysis of serologic factors could assist us in predicting survival beyond standardized staging techniques in untreated patients with squamous cell carcinoma of the head and neck. In addition, we questioned whether these serologic factors could represent a function of tumor burden or if they could be predictive of recurrence sites and/ or rates. Although prior evidence relating to the prognostic implications of circulating immune complex assays, including the C lqBM assay, in cancer has been conflicting [11-13], our data strongly support a predictive value in advanced (stage IV) squamous cell carcinoma of the head and neck. Our results did not illustrate a correlation between tumor burden and ClqBM or serum IgA concentrations. However, the significance of C lqBM values in advanced-stage squamous carcinoma may more reflect a function of cell death, with circulating subcellular debris contributing to elevated values of C l q B M when quantitated in vitro. In support of this, we have found a high correlation between elevated C l q B M levels and advanced necrotic or cartilaginous destructive squamous cell carcinoma of the larynx (unpublished data). The lack of correlation between C l q B M and survival in early stage-disease probably reflects an alternative disease process, unrelated to the head and neck cancer, rather than a function of cancer progression [14]. Therefore, the current limitation of the C l q B M assay is its apparent lack of specificity. Efforts directed toward defining tumor-related antigens within the complement-binding substances may help to resolve this nonspecificity. The mechanism of elevated levels of C l q B M and serum IgA predicting decreased survival remains hypothetical. Certainly, the immunosuppressive role of immune complexes has been examined with regard to T cells [15], B cells [16], N K cells [17], and macrophages [18] and continues to be investigated in laboratory investigations as it relates to lymphocyte-target interaction as well as the activation of lymphocyte populations in vitro. Although serum IgA has been shown to be elevated in other series of head and neck carcinomas [5], its role in
immunomodulation remains unclear. The elevated serum IgA levels may represent a chronic B-cell inflammatory response to mucosal ulceration. Additionally, although ClqBM can be composed of numerous substances, the most common immunoglobulins involved are IgG and IgM; however, our study revealed no direct correlation between C 1qBM and these circulating immunoglobulins. Still, serum IgA may represent some less quantifiable tumor burden such as soft tissue, vascular, or lymphatic invasion. Therefore, the immunoregulatory role of IgA in squamous cell carcinoma requires further investigation. To summarize, previous studies have shown that serologic evaluation of C l q B M help discriminate patients with a low probability of responding to induction chemotherapy [1,2]. Additionally, this study has shown that the combined serologic analysis of IgA and C 1qBM is predictive of survival in patients with advanced-staged squamous cell carcinoma of the head and neck. Pretreatment serologic evaluation of patients with advanced disease may potentially assist the caregiver in providing the most timely and appropriate treatment regimen. REFERENCES 1. Schantz SP, Savage HE, Brown BW, Reuben JM, Hong WK, Rossen RD. Associationof levelsof circulatingC 1q bindingmacromoleculeswith induction chemotherapyresponsein head and neck cancer patients. Cancer Res 1988; 48: 5868-73. 2. Schantz SP, Savage HE, Racz T, et al. Immunologicdeterminants of head and neck responseto inductionchemotherapy.J Clin Oncol 1989; 7: 857-64. 3. Blazar B, Fried M, Strorne M, et al. Circulating immune complexes and chemotherapy response in patients with head and neck cancer. Head Neck Surg 1989; 11: 431-6. 4. BaskiesAM, Chretien PB, Weiss JF, et al. Serum glycoproteins and immunoglobulins in nasopharyngeal carcinoma. Correlations with Epstein-Barr-virus associated antibodies and clinical tumor stage. Am J Surg 1979; 138: 478-88. 5. VinzenzK, Pavelka R, Schonthal E. Zekert F. Serum immunoglobulin levels in patients with head and neck cancer (IgE, IgA, IgM, IgG). Oncology 1986; 43: 316-22. 6. Pettingale KW, Merret TG, Tee DEH. Prognostic value of serum levels of immunoglobulins (IgG, IgA, IgM and IgE) in breast cancer: a preliminary study. Br J Cancer 1977; 36: 550. 7. Lee YTN. Quantitative change of serum protein and immunoglobulin in patients with solid tumors. J Surg Oncol 1977; 9: 17487. 8. American Joint Committee on Cancer. Manual for staging of cancer. 2nd edition. Philadelphia: JB Lippincott, 1983. 9. Zubler RH, Lange G, Lambert PH, Meischer PA. Detection 125I-Clq binding test. Effect of heating on the binding of Clq by immune complexesand application of the test to systemic lupus erythematosus. J Immunol 1976; 116: 232-5. 10. Schantz SP, Liu FJ, Taylor D., BeddingfieldN, Weber RS. The relationship of circulating IgA to cellular immunity in head and neck cancer patients. Laryngoscope 1988; 98: 671-8. 11. Bukh A, Moller-Larsen A, Aquado MT, Moller-Larsen F, Moiler NPH. Prognosticvalue of serial determinationsof circulating immune complex levels in malignant melanoma. Cancer Immunol Immunother 1988; 26: 280-4. 12. Rossen RD, Crane MM, Morgan AC, et al. Circulating immune complexesand tumor cell cytotoxinsas prognosticindicators in malignant melanoma: a prospectivestudy of 53 patients. Cancer Res 1983; 43: 422-8. 13. TheotilopoulusAN, Andrews BS, Urist MM, Morton DL,
THE AMERICAN JOURNAL OF SURGERY VOLUME160 OCTOBER1990 437
CLAYMANET AL
Dixon FJ. The nature of immune complexes in human cancer sera. J Immunol 1977; 119: 657-66. 14. Highton J, Panayi GS, Shepherd P, Wooley P. Effect of sera from patients with rheumatoid arthritis on normal lymphocytes: a possible immunoregulatory role for immune complexes. Ann Rheum Dis 1982; 41: 563-8. 15. Bich-Thuy LT, Banchereau J, Revillard JP. Suppression of polyclonal human B cell activation by IgG binding factors: interference with the maturation of Ig-containing ceils into Ig-secreting cells. Cell Immunol 1984; 87: 231-9. 16. Suzuki T, Miyama-Inaba M, Masuda T, Uchino H, Ishii K.
438
Monoclonal SBF produced by a hybridoma: in-vitro and in-vivo suppression of B tumour-cell proliferation. Immunology 1983; 50: 595-603. I7. Karsh J, Dorval G, Osterland CK. Natural cytotoxicity in rheumatoid arthritis and systemic lupus erythematosus. Clin Immunol Immunopathol 1981; 19: 437-46. 18. Michl J, Unkeless JC, Pieczonka MM, Silverstein SC. Modulation of Fc receptors on mononuclear phagocytes by immobilized antigen-antibody complexes. Quantitative analysis of the relationship between ligand number and Fc receptor response. J Exp Med 1983; 157: 1746-57.
THE AMERICAN JOURNAL OFSURGERY VOLUME 160 OCTOBER 1990
Specific circulating serum proteins may reflect unique properties governing the growth and progression of head and neck cancers, One hundred three previously untreated patients with squamous cell carcinoma of the head and neck were prospectively evaluated for serum IgA, lgG, and lgM and Clq-binding macromolecules, lmmunoglobulins were assessed by the immunoturbidimetric technique. Clq-binding maeromolecules (CIqBM) were measured utilizing the iodine-125 assay of Zubler et al (J Immunol 1976; 116: 232-5). Neither the level of serum immunoglobulins nor CIqBM values were correlated with the primary site, AJC (American Joint Committee on Cancer) stage of disease, or size of primary lesion. Likewise, comparison of serum lgA with ClqBM values demonstrated that these laboratory parameters were independent variables (r = 0.15 by Pearson linear regression). Univariate statistical analysis, utilizing the Cox proportional hazard model, showed serum lgA and ClqBM values to each contribute significantly to the ability to predict survival in patients with advanced squamous cell carcinoma of the head and neck (p = 0.01 and 0.003, respectively). Furthermore, multivariate analysis reveals that both ClqBM and serum lgA levels contribute significantly to the hazards model beyond staging in predicting survival (p <0.001 ). Predictive results were most apparent in patients with stage IV disease and related to the probability of both regional and distant metastatic recurrences. Conversely, serologic analysis provided no information in patients who were staged early. These results support pretreatment multiparametric serologic analysis of patients with squamous cell carcinoma of the head and neck.
From the Departments Of Head and Neck Surgery (GLC, HES, NA, SPS) and Laboratory Medicine (FJL), the University Of Texas M: D. Anderson Cancer Center, Houston, Texas. Requests for reprints Should be addressed to Stimson P. Schantz, MD, Department of Head and Neck Surgery, M. D. Anderson Cancer Center, 1515 Holcombe Boulevard, Houston, Texas 77030. Presented at the 36th Annual Meeting of the Society of Head and Neck Surgeons, Washington, DC, May 19-22, 1990.
434
THE AMERICAN JOURNAL OF SURGERY
A
growing body of evidence supports the integration of biologic parameters of host-tumor interaction into the assessment and treatment of patients with head and neck cancer. Initial investigations of circulating macromolecules capable of binding the first component of complement in vitro (Clq-binding maeromolecules, ClqBM) found a stage-independent relationship between elevated levels of ClqBM and lack of a clinical response to induction chemotherapy [1,2]. These results were subsequently verified by Blazar et al [3] who quantitated polyethylene glycol precipitates from sera of patients with head and neck cancer and found that a high level of precipitate predicted a low probability of response to chemotherapy. The clinical relevance of ClqBM was reinforced by the observation that its elevation was predictive of a high probability of death with disease, independent of the use of induction chemotherapy (i.e., patients with elevated ClqBM levels who received multimodality therapy that did not include anticancer drugs did just as poorly) (Schantz SP, et al, unpublished data). A considerable number of attempts have been made to correlate immunoglobulin levels and clinical stage or activity of disease in cancer patients. Elevation of IgA has been demonstrated at initial workup in sera from patients with nasopharyngeal carcinoma [4], perhaps reflecting a local immune response, and in sera and saliva of patients with head and neck squamous cell carcinoma [5]. Significant serum IgA elevations have also been found in patients with advanced or recurrent disease among a variety of tumors [6,7]. The current investigation was designed to determine whether serologic levels of C lqBM and immunoglobulin classes (specifically, IgA) could be predictive of survival in patients with squamous cell carcinoma of the head and neck, independent of standard clinical staging techniques. These parameters were evaluated for predicability both together and alone, and in a variety of patient subsets. PATIENTS AND METHODS Patients: A total of 103 patients with previously untreated squamous cell carcinoma of the upper aerodigestive tract were evaluated prospectively for serologic levels of ClqBM, IgG, IgA, and IgM. Only patients who received definitive therapy at this institution were eligible. Median patient age was 56 years (range: 21 to 81 years); the male-to-female ratio was 3:1. Primary disease was in the oral cavity in 25 patients, the pharynx in 51, and the larynx in 27. Disease staging was by the 1983 American Joint Committee on Cancer (AJC) criteria [8], yielding 12 patients with Stage I disease, 13 with stage II lesions,
VOLUME 160 OCTOBER 1990
SURVIVAL PREDICTORS IN HEAD AND NECK CANCERS
34 with stage III, and 44 with stage IV. All serologic samples were drawn by venipuncture before the initiation of therapy. The disease-free interval following therapy was calculated in months from the date of initial treatment to the last-noted physician contact at which the patient had no evidence of disease. Median follow-up of all patients was 18 months (range: 2 to 44 months). Twenty-seven of 103 patients died of disease. Of the patients who died of disease, 24 were patients with stage IV tumors. Twelve of the 103 patients died of other causes with a median follow-up of 24.5 months within this group. All patients were entered into this study before January 1, 1988. Follow-up occurred at 3-month intervals during the first year, 6-month intervals in the second year, and then annually. Disease status was assessed at follow-up by members of the Department of Head and Neck Surgery without knowledge of the serum ClqBM or immunoglobulin values. All data were collected in a prospective fashion and transformed into coded computer data before retrieval for the current analysis. Measurement of CIqBM: Sera were collected from clotted blood and frozen at - 7 0 ~ until tested. Human Clq (Quidel, San Diego, CA) was labeled with iodine 125 by the iodobead method (Pierce Chemical Co., Rockford, IL). Five hundred micrograms of Clq was mixed with 1 mCi of sodium iodine 125 in 1 mL final volume of phosphate-buffered saline on ice, pH 7.4. One iodobead was added to this mixture and incubated for 5 minutes. Labeled C lq was separated from free iodine 125 on a 10-dentimeter Sephadex G-25 column (PD-10 column; Pharmacia, Piscataway, N J). The C 1q-binding test was performed as previously described, using EDTAtreated sera according to the method of Zubler et al [9]. Results were expressed as #g/mL equivalents of heataggregated IgG with reference to a standard curve made with purified IgG, aggregated at 63~ for 20 minutes at a concentration of 3 mg/mL, and diluted serially in heatinactivated (56~ 30 minutes) normal donor serum. Sera from healthy, age-matched normal donors were used as negative controls and sera from head and neck cancer patients with previously determined positive values provided serial controls in each test run. Normal values for ClqBM are less than 50 t~g/mL. Measurement of plasma immunoglobulins: Plasma IgG, IgA, and IgM concentrations were measured by an immunoturbidimetric technique, developed on the computer-programmable ROCHE, COBAS BIO centrifugal analyzer (Roche Analytical Instruments, Nutley, N J), using IgG, IgA, and IgM calibrators and anti-IgG, antiIgA, and anti-IgM sera (Atlantic Antibodies, A. Charles River Co., Scarborough, ME) as we have previously reported [10]. The normal values established in this laboratory for IgG, IgA, and IgM are 650 to 1,750, 75 to 330, and 30 to 225 mg/dL, respectively. Since antigen excess would lead to soluble antigen-antibody reaction product and then to an underestimation of plasma immunoglobulin concentrations, serum protein electrophoresis by the Beckman Paragon Electrophoresis System (Beckman Instruments, Fullerton, CA) was performed on each sample
TABLE I
Predlctlvlty of Serum ClqBM, Serum IgA, and/or Disease Stage for Death with Disease (n = 1 0 3 ) * Analysis Univariate ClqBM Serum IgA Stage ClqBM-I-, IgA Multivariate ClqBM by stage IgA by stage ClqBM-F, IgA by stage
Log Likelihood
p Value
--108.98 --109.49 --98.29 -106.02
0.003 0.01 0 0.0003
--95.62 --95.11 --92.62
0.0001 0.0001 0.0001
* Cox proportional hazards analysis. Disease stage is stages I, II, and Ill, collectively, versus stage IV.
to identify any highly elevated immunoglobulin before quantitation by the immunoturbidimetric method. Any samples with high immunoglobulin levels were appropriately diluted. Statistical analyses: Comparisons between populations as related to C lqBM or the respective immunoglobulins were performed using either the Student's t test or the Mann-Whitney U-test for nonparametric data. All p values reported in the text represent results of t-test analysis. Those significant by this test were likewise significant by the Mann-Whitney test. The Cox proportional hazards model was applied for investigating the significance of single and paired serologic tests in predicting death with disease and site of recurrence using the BMDP regression statistical package (BMDP Statistical Software, Inc., Los Angeles, CA). Differences in the probabilities of death with disease between populations categorized by stage of disease were determined using proportional hazards regression and log-rank testing similarly through the BMDP statistical package. In all analyses, only death with disease was considered. Death from other causes was treated as a censored event and excluded the patient from subsequent analyses. Any p value less than 0.05 were considered statistically significant. RESULTS The relationship between serum ClqBM and IgA levels and the probability of death with disease was assessed. Univariate analysis results revealed a direct relationship between elevated levels of ClqBM and serum IgA obtained prior to treatment and the likelihood of death with disease, that is, patients with the highest ClqBM or serum IgA values were at greatest risk as determined by the Cox proportional hazards model (p = 0.003 and 0.01, respectively) (Table I). Results of ClqBM levels and prognosis are presented in Figure 1. In a multivariate Cox proportional analysis, both ClqBM and serum IgA values simultaneously considered maintained their significance within the Cox proportional hazards model, that is, the combination was highly predictive of death with disease (p -- 0.0003). Table I demonstrates the prognostic significance of standard AJC staging,
THE AMERICAN J O U R N A L OF SURGERY
VOLUME 160
OCTOBER 1990
435
CLAYMAN ET AL
1.0-
1.0,
0.8\_
0.8-
\
0.6-
0.6-
%
0.4-
0.4-
0.20.0
--
ClqBM ( 16 (n=34)
--
ClqBM ) 47 (n=34) I I 24 36 Months
8
I 12
3~176 T 9
Io
9
o
~
I00 t
o I o
~
9
I"
: ,~_ :ao=a 100
200
300
3
" ~9 400
4
: 500
600
700
800
900
IgA ( r n g / d l )
Figure 3. Survival dot plot in stage IV disease by ClqBM and IgA median values (27 #g/mL against standard and 292 mg/dL, respactively). Mortality rates are 25%, 50%, 82%, and 36%, respectively, for quadrants 1 to 4.
which combined patients with stage I, II, and III disease as compared with patients with stage IV disease. When considered in light of standard AJC staging, both ClqBM and serum IgA concentrations were still highly significant in their association with death from disease (p <0.001). The most prognostic information was provided when all parameters (both staging and serologic factors) were considered together (p <0.0001). The mean serum IgA and ClqBM values by lesion distribution within the primary sites of the 103 patients were analyzed. No significant differences could be identified in either parameter between patient populations stratified by site of primary disease, although patients with oral cavity lesions had the lowest mean levels of both ClqBM and IgA. To determine if ClqBM or immunoglobulin levels were related to primary tumor size or tumor burden, serum levels of IgA, IgG, and IgM and ClqBM were analyzed against primary tumor (T) stage as well as standard TNM stages by AJC standards. Levels of the respective serum proteins did not vary significantly by these stratifications (data not shown). Furthermore, Pearson linear-regression analysis of these data showed elevated serum IgA and ClqBM values to be
436
-~__~
- \:: ~ -: ~ i._ ~ .
*--* -.... - -
Io ClqBU, Io ,o ClqB~, ~ hi ClqBM, hi ClqBM, hi
IgA (n=JO) tgA ~n=12) IgA {n=11) IgA (n=l I)
316
418
0.20.0
I
9 Dead ( n = 2 1 ) o Living ( n = 2 3 ) - - - Median values
9
200 1
0
-
1t2
48
Figure 1. Survival, from initiation of treatment, by serum ClqBM levels in all 103 patients.
0]
~ ,
.... ClqBM = 16-47 (n=35)
0
1 1
-C'. x% " ' ' ' " .
T H E A M E R I C A N J O U R N A L OF S U R G E R Y
214 Months
Figure 2. Survival in 44 patients with stage IV disease by high or low levels of ClqBM and IgA.
independent variables associated with death due to disease in patients with stage IV cancers of the head and neck (r -- 0.15). The distribution of patients by AJC standards was 12 stage I, 13 stage II, 34 stage III, and 44 stage IV. Logrank testing showed no significant difference in survival, assessed by the Kaplan-Meier method, in multiple comparisons between patients with stage I, II, or III disease. In contrast, patients with stage IV disease had a significantly worse prognosis as compared with any combination of patients with earlier stage I, II, or III disease (p -0.004, p <0.001, p <0.001, respectively). The risk of death with disease was then considered by stages I, II, and III taken together (group 1) versus stage IV patients (group 2). Overall, analysis of this grouping for its capacity to predict probability of death with disease showed it to be a model highly predictive by the Cox proportional hazard model (p <0.001). The Cox proportional hazard model was then utilized to determine risk of death with disease in these two groupings. Within group 1, neither serum IgA or ClqBM levels were significant in predicting survival in these patients; however, in group 2 (patients with stage IV lesions), elevated serum IgA and C 1qBM values significantly contributed to the survival model (p = 0.008 and -- 0.02, respectively). High (elevated) levels were considered above the median for stage IV patients; conversely low levels were less than the median. Multivariate analysis of ClqBM and serum IgA values contributed significantly to the hazards model beyond that currently defined by AJC staging methods (p <0.001) (Figure 2). Analysis of site of recurrence revealed that IgA, IgG, and IgM levels were not predictive for primary, regional/ nodal, or distant recurrence in groups 1 or 2 (data not shown). However, elevated ClqBM concentrations were predictive of regional or distant metastasis within group 2 (p = 0.01 and 0.02, respectively). In addition, the combined analysis of ClqBM and serum IgA was predictive of regional/nodal recurrence within group 2 (p -- 0.006). Group 2 patients were further analyzed to assess the relationship between IgA levels and ClqBM levels to subsequent death with disease. No relationship between
V O L U M E 160
OCTOBER 1990
SURVIVAL PREDICTORS IN HEAD AND NECK CANCERS
primary site and serologic IgA or C l q B M levels was found (data not shown). A survival dot plot is illustrated in Figure 3 for group 2 (stage IV) patients. Median values of the group 2 patients for ClqBM was 27 g g / m L and 292 m g / d L for serum IgA. These values are depicted graphically. Those patients with combined elevations above the median (stage IV patients) showed significantly increased mortality (82%). Notably IgG and IgM did not significantly contribute to survival within either group 1 or 2. It is evident, however, that patients with elevated C l q B M concentrations who died with disease were not necessarily the same patients with elevated IgA values who died with disease. Figure 3 emphasizes that each parameter m a y identify distinct populations at risk and thus may reflect distinct determinants of disease progression. COMMENTS The purpose of this study was to investigate whether analysis of serologic factors could assist us in predicting survival beyond standardized staging techniques in untreated patients with squamous cell carcinoma of the head and neck. In addition, we questioned whether these serologic factors could represent a function of tumor burden or if they could be predictive of recurrence sites and/ or rates. Although prior evidence relating to the prognostic implications of circulating immune complex assays, including the C lqBM assay, in cancer has been conflicting [11-13], our data strongly support a predictive value in advanced (stage IV) squamous cell carcinoma of the head and neck. Our results did not illustrate a correlation between tumor burden and ClqBM or serum IgA concentrations. However, the significance of C lqBM values in advanced-stage squamous carcinoma may more reflect a function of cell death, with circulating subcellular debris contributing to elevated values of C l q B M when quantitated in vitro. In support of this, we have found a high correlation between elevated C l q B M levels and advanced necrotic or cartilaginous destructive squamous cell carcinoma of the larynx (unpublished data). The lack of correlation between C l q B M and survival in early stage-disease probably reflects an alternative disease process, unrelated to the head and neck cancer, rather than a function of cancer progression [14]. Therefore, the current limitation of the C l q B M assay is its apparent lack of specificity. Efforts directed toward defining tumor-related antigens within the complement-binding substances may help to resolve this nonspecificity. The mechanism of elevated levels of C l q B M and serum IgA predicting decreased survival remains hypothetical. Certainly, the immunosuppressive role of immune complexes has been examined with regard to T cells [15], B cells [16], N K cells [17], and macrophages [18] and continues to be investigated in laboratory investigations as it relates to lymphocyte-target interaction as well as the activation of lymphocyte populations in vitro. Although serum IgA has been shown to be elevated in other series of head and neck carcinomas [5], its role in
immunomodulation remains unclear. The elevated serum IgA levels may represent a chronic B-cell inflammatory response to mucosal ulceration. Additionally, although ClqBM can be composed of numerous substances, the most common immunoglobulins involved are IgG and IgM; however, our study revealed no direct correlation between C 1qBM and these circulating immunoglobulins. Still, serum IgA may represent some less quantifiable tumor burden such as soft tissue, vascular, or lymphatic invasion. Therefore, the immunoregulatory role of IgA in squamous cell carcinoma requires further investigation. To summarize, previous studies have shown that serologic evaluation of C l q B M help discriminate patients with a low probability of responding to induction chemotherapy [1,2]. Additionally, this study has shown that the combined serologic analysis of IgA and C 1qBM is predictive of survival in patients with advanced-staged squamous cell carcinoma of the head and neck. Pretreatment serologic evaluation of patients with advanced disease may potentially assist the caregiver in providing the most timely and appropriate treatment regimen. REFERENCES 1. Schantz SP, Savage HE, Brown BW, Reuben JM, Hong WK, Rossen RD. Associationof levelsof circulatingC 1q bindingmacromoleculeswith induction chemotherapyresponsein head and neck cancer patients. Cancer Res 1988; 48: 5868-73. 2. Schantz SP, Savage HE, Racz T, et al. Immunologicdeterminants of head and neck responseto inductionchemotherapy.J Clin Oncol 1989; 7: 857-64. 3. Blazar B, Fried M, Strorne M, et al. Circulating immune complexes and chemotherapy response in patients with head and neck cancer. Head Neck Surg 1989; 11: 431-6. 4. BaskiesAM, Chretien PB, Weiss JF, et al. Serum glycoproteins and immunoglobulins in nasopharyngeal carcinoma. Correlations with Epstein-Barr-virus associated antibodies and clinical tumor stage. Am J Surg 1979; 138: 478-88. 5. VinzenzK, Pavelka R, Schonthal E. Zekert F. Serum immunoglobulin levels in patients with head and neck cancer (IgE, IgA, IgM, IgG). Oncology 1986; 43: 316-22. 6. Pettingale KW, Merret TG, Tee DEH. Prognostic value of serum levels of immunoglobulins (IgG, IgA, IgM and IgE) in breast cancer: a preliminary study. Br J Cancer 1977; 36: 550. 7. Lee YTN. Quantitative change of serum protein and immunoglobulin in patients with solid tumors. J Surg Oncol 1977; 9: 17487. 8. American Joint Committee on Cancer. Manual for staging of cancer. 2nd edition. Philadelphia: JB Lippincott, 1983. 9. Zubler RH, Lange G, Lambert PH, Meischer PA. Detection 125I-Clq binding test. Effect of heating on the binding of Clq by immune complexesand application of the test to systemic lupus erythematosus. J Immunol 1976; 116: 232-5. 10. Schantz SP, Liu FJ, Taylor D., BeddingfieldN, Weber RS. The relationship of circulating IgA to cellular immunity in head and neck cancer patients. Laryngoscope 1988; 98: 671-8. 11. Bukh A, Moller-Larsen A, Aquado MT, Moller-Larsen F, Moiler NPH. Prognosticvalue of serial determinationsof circulating immune complex levels in malignant melanoma. Cancer Immunol Immunother 1988; 26: 280-4. 12. Rossen RD, Crane MM, Morgan AC, et al. Circulating immune complexesand tumor cell cytotoxinsas prognosticindicators in malignant melanoma: a prospectivestudy of 53 patients. Cancer Res 1983; 43: 422-8. 13. TheotilopoulusAN, Andrews BS, Urist MM, Morton DL,
THE AMERICAN JOURNAL OF SURGERY VOLUME160 OCTOBER1990 437
CLAYMANET AL
Dixon FJ. The nature of immune complexes in human cancer sera. J Immunol 1977; 119: 657-66. 14. Highton J, Panayi GS, Shepherd P, Wooley P. Effect of sera from patients with rheumatoid arthritis on normal lymphocytes: a possible immunoregulatory role for immune complexes. Ann Rheum Dis 1982; 41: 563-8. 15. Bich-Thuy LT, Banchereau J, Revillard JP. Suppression of polyclonal human B cell activation by IgG binding factors: interference with the maturation of Ig-containing ceils into Ig-secreting cells. Cell Immunol 1984; 87: 231-9. 16. Suzuki T, Miyama-Inaba M, Masuda T, Uchino H, Ishii K.
438
Monoclonal SBF produced by a hybridoma: in-vitro and in-vivo suppression of B tumour-cell proliferation. Immunology 1983; 50: 595-603. I7. Karsh J, Dorval G, Osterland CK. Natural cytotoxicity in rheumatoid arthritis and systemic lupus erythematosus. Clin Immunol Immunopathol 1981; 19: 437-46. 18. Michl J, Unkeless JC, Pieczonka MM, Silverstein SC. Modulation of Fc receptors on mononuclear phagocytes by immobilized antigen-antibody complexes. Quantitative analysis of the relationship between ligand number and Fc receptor response. J Exp Med 1983; 157: 1746-57.
THE AMERICAN JOURNAL OFSURGERY VOLUME 160 OCTOBER 1990