IgE levels in sera of cancer patients Wendell

D. Winters,

Los Angeles,

Ph.D., and Douglas

C. Heiner,

M.D.,

Ph.D.

Cnlif.

Immunoglobnlin E (IgE) was measured in the sera of 18 healthy ad& volantee? donors, 67 a&&s with various types of solid neoplasms, and 17 adults with clinical allergy by means of a double-antibodys radioimmnnoassay. There was no significant difference in the geometric mean serum IgE level between all cancer subjects and the healthy control subjects except that cancer and noncancer patients who had definite clinical allergies shozced an increased mean IgE level. Similarly, there ulna no significant difference in the mean IgE level of any of the six cancer sub,groups studied when compared to the control mean. Thus, thare ws no evidence rcfiectcd in serum levels that IgE plays a role in the i,mmunopa%kology of the cancer populntion

tested.

Previous studies have established tha.t increased circulating levels of IgE are present in patients who suffer from allergic disorders, helminth infestations, Wiskott-Aldrich syndrome, and a few other disordersl-“, I5 Low levels of IgE are found in the sera of patients with severe immunologic deficiency diseases3and occasionally in their healthy counterparts. Bacterial and viral infections appear to have little effect on serum IgE levels.” IgE is a new class of immunoglobulin which is responsible for homocptotropic or reaginic antibody activity in serum. The possibility of a relationship between the incidence of cancer and allergy has been the subject of several studies; some authors suggest that cancer is less common in the allergic than in the nonallergic individual.G-8 Circulating levels of IgE in healthy donors vs levels in patients with advanced cancer studied 1~) solid-phase radioimmunoassay and by radial immunodiffusion” suggested that IgE levels may be low in certain untreated cancer patients. Unusually high levels were recorded by these authors for some patients, presumably due to a serum inhibitor that may have interfered with the proper performance of the assay. A recent paper by Waldmann and associates17indicated that serum IgE levels were normal in the majority of cancer types they studied, but were elevated in Hodgkin’s disease and reduced in chronic lymphatic leukemia, IgB myeloma, and IgD myeloma. In order to learn whether discernible correlations exist regarding serum From the Division of Oncology, Department of Surgery (Dr. Winters) and Department of Pediatrics (Dr. Heiner), UCLA School of Medicine and Harbor General Hospital. Supported by Grants Nos. HD05053-03 and CA15282 and Special Research-Resources Grant RR-3 from the National Institutes of Health. Received for publication Nov. 5, 1974. Accepted for publication March 20, 1975. Reprint requests to: Dr. Wendell D. Winters, Division of Oncology, Department of Surgery, UCLA School of Medicine, Los Angeles, Calif. 90024. Vol.

57, No.

a, pp.

181-186

182

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CLIN. IMMUNOL. FEBRUARY 1976

levels of IgE in a group of cancer patients available for stud,v, 67 patients with skeletal and soft tissue tumors, ie, sarcomas, melanomas, and carcinomas, were tested by a double-antibody radioimmunoassay known to bc scnsitirc and reproducible, and superior in our hands to other methods of quantitating srrum TgE. The other methods which have been studied in this laboratory include radial diffusion in gel and a commerciall,v available solid-phase radioimmunoassa~ (Pharmacia Laboratories). Each of these two techniques has been used in the analysis of IgE levels in more than 1,000 serum specimens in this laborator>-, and each has been found to be less sensitive and less reliable than the doublcantibody technique. Our experience in this regard is parallel to that, of Polmar, Waldmann, and Terrp,lG who have discussed some of the reasons for this advantage, MATERIALS AND Patient information

METHODS

Serum samples were obtained from cancer patients attending the UCLA Center for the Health Sciences Oncology Clinics and from control subjects as follows: (1) 67 untreated patients with advanced neoplasms of various histopathologic types but no evidence of clinical allergy, (2) 18 healthy donors with no clinical evidence of allergy or cancer, (3) 17 normal (nontumor) subjects with clinical allergy, and (4) 4 subjects with advanced untreated neoplasms who had clear evidence of clinical allergy. The 67 cancer patients were selected according to tumor types and the absence of clinical allergy but otherwise were randomly chosen for study. Healthy subjects included normal blood donors, hospital staff members, and laboratory personnel. Fourteen consecutive sera drawn from patients in a private allergy practice and 3 hospital workers with allergy constituted the allergy group. These included 6 subjects with predominant asthma, 6 with allergic rhinitis, 2 with atopic dermatitis, 2 with chronic urticaria, and 1 with gastrointestinal allergy. Blood specimens were obtained by venipuncture and serums were separated by centrifugation and stored at -20” C until tested.

Quantitative

measurement

of IgE

Double-antibody radioimmunoassays of serum IgE were performed in a manner similar to that described by Gleich, Averbeck, and Swedland,lo using anti-IgE (PS) and izsI-labeled IgE (ND). The IgE was radiolabeled with 1251 by the method of Hunter and Greenwoodir to a specific activity of 10 pCi/pg of IgE and when used in the assay had a final specific activity of 2.1. All solutions contained 0.01% sodium azide. In brief, 25 gl of serum were added to a test tube containing 100 hl of a 1:80,000 dilution of anti s-chain in 0.1 M sodium phosphate and 1:50 normal rabbit serum, pH 7.5. The mixture was incubated with agitation for 4 hr, after which 100 d (lo-15,000 epm) of radiolabeled IgE (ND) in phosphate-EDTA buffer containing 0.25% human serum albumin (HSA) were added. The tubes were gently agitated for 1 hr and incubated at room temperature overnight and 100 $1 of 1:5 dilution of goat antiserum to rabbit gammaglobulin were added (slight excess of goat antibody). The tubes mere incubated with agitation for an additional 24 hr. Each tube and its reactants was counted for 0.5 min in a gamma counter (total cpm) ; and then all tubes were centrifuged and supernatants mere removed. The precipitates were mashed twice, and the tubes containing the mashed precipitate again mere counted in an automatic gamma counter for 5 min (bound cpm). The rpm bound in the precipitate was inversely proportional to the IgE content of the specimen being analyzed. The serum value was determined from :I standard curve plotted on semi-log paper. Standards were prepared from dilutions of 2 sera of high IgE content (22,000 and 36,000 IU/ml, respectively) in phosphate-buffered 0.25v0 human serum albumin. These standards had been assayed both by Dr. S. G. 0. Johansson (Sweden) and by Dr. Robert Hamburger (La Jolla, Calif.) and were standardized tmice in our laboratory against the WHO IgE standard, 68/341. The technique used was reliably

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IgE

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patients

183

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SUBJECT

GROUPS

FIG. 1. Serum IgE values as measured by DA RIA for patients sarcoma, melanoma, carcinomas, and allergic subiects). The mean duplicate determinations of one person’s serum IgE concentration geometric mean value for the group.

in subgroups (controls, value of closely matched (IU/ml) represents the

sensitive to 1 IU/ml but was most precise between 5 and 500 IU/ml, a range including the majority of normal values. Levels above 500 IU/ml fell into proper sequence although the absolute values mere subject to increasing error due to the increasing slope of the standard curve at higher concentrations. Antiseruln to IgE (I’S) was prepared by injecting rabbits with E-myeloma protein purified by fractional ammonium sulfate precipitation, DEAE-Sephadex A-50 gradient elution, and Sephadex G-200 gel filtration. The most potent rabbit antisera mere pooled and absorbed twice with one-fourth volume of ethyl chloroformate-copolymerized pooled human serum from selected healthy adults (IgE level of pool = 18 III/ml). The absorbed antiserum

184

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TABLE

I. Serum

J. ALLERGY

Heiner

IgE levels

by

double-antibody

I Subject

category

Nonallergic: Control group A* Control group B* Sarcoma group Melanoma group Carcinoma group Breast? Colont Lund Othert Allergic. Control Cancer

group$ prow

RIA

I No.

of patients

:: 1: 31 : ; 17 4

CLIN. IMMUNOL. FEBRUARY 1976

t

Serum Range

25IO92IO25ll-

IaE

(IlJ/ml) Geometric

750 2,800 1,400 180 6,000 455 6,000 3,000 276

42.9 41.9 72.3 34.2 61.4 43.6 116.1 81.3 38.1

12-10,000 13- 2.500

274.1 249.4

mean

*Normal, nonallergic subjects from UCLA (Group A) and Harbor General Hospital (Group B). tCarcinoma patient subgroups by cancer site; ‘(other” group composed of 3 squamous w11 carcinomas, 2 pancreatic, 2 renal cell, and 2 head and neck carcinomas. $Normal, allergic patients from Harbor General Hospital.

produced a single precipitate line when diffused against sera of high IgE content (522,000 IU/ml) on Ouchterlony tests but produced no line with normal human sera. It also produced a line of identity with that of anti-FcND kindly supplied by Dr. S. G. 0. Johansson when tested by Ouchterlony analysis against sera from allergic individuals with very high IgE levels or against appropriately diluted E-myeloma serum (PS). It had a precipitate-in-gel titer of 1:32.

Statistical

methods

It

has previously been observed that IgE serum levels and the log,, of serum IgE levels have a multimodal rather than a Gaussian distribution in large samples, thus suggesting that the use of parametric tests for establishing statistical significance on such data would not be permissible.% 19 With this information in mind, even though our data did not reflect a strong hi- or multimodality, the IgE concentrations of the serum specimens were transformed to log,, and 2 methods were used to establish statistical significance. Computations were first performed with a parametric analysis, ie, t test. Second, Mann-Whitney tests were performed on the same data to check the relationships between group means and to take into account any non-Gaussian distribution in the data. The same results lvere obtained with either test, thus lending confidence in the conclusions drawn from the statistical analyses.

RESULTS

Double-antibody RIA of serum IgE concent,rations in normal donors and the cancer patient diagnostic subgroups are presented in Fig 1. There were no significant differences between the mean serum IgE levels of healthy controls, the mean for the total cancer group, or for the three major cancer subgroupssarcoma, melanoma, and carcinoma. There was a significantly increased mean level in the subjects with allergy, however. Five of the 67 cancer subjects (7.5% ) had a level in excess of normal for this laboratory (90% of healthy adults’ values fall between 10 and 500 IV/ml). Using these criteria, one of 17 healthy controls (5.892 ) and seven of 17 (11%) of the allergic group had high levels by doubleantibody RIA (DA RIA) .

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Statistical analyses demonstrated no significant diffcrcnce hetwecn health? control subjects and any cancer subgroup or the total group of untreated cancer patients. There was, however, a significant difference bctwcen geometric* mean serum IgE levels in healthy control subjects and in consecutively studied subjects with active clinical allergy (Fig. 1). A breakdown of the cancer subgroups tested is given in Table I. DISCUSSION

At least two retrospective studies of allergy in cancer patient populations suggested that a history of allergy was more common in noncancer patients than in cancer patients.“> 7 Others even suggested that allergy and malignant>may be mutually exclusive. 8, I2 Additional reports indicated that there was no difference in the incidence of allergy in patients without cancer when compared to those with neoplastic disease,l”’ l* suggesting that patients with tumors may be even more likely to have allergic disorders. Jacobs and associates”tested the sera of healthy donors and a large group of untreated and treated patients with advanced cancers for IgE levels using a commercial solid phase radioimmunoassay (Pharmacia Laboratories) and radial immunodiffusion in gel. Their results suggested that most untreated patients with advanced bronchial and miscellaneous tumors had low or undetectable IgE serum levels by radial immunodiffusion. In about half of the casrs, the radioimmunoassay confirmed the low IgE levels; however, in others the serum TgE levels were extremely high by the radioimmunoassay though not 1~7 radial immunodiffusion. This result was ascribed to a possible inhibitor substance in cancer sera that interfered with the IgE binding. Our findings are in general agreement with the study by Waldmann and co-workers,1’ which appeared after our analyses had been completed. Each 1aborator.v after extensive testing found the double-antibody liquid phase radioimmunoassap procedure to be superior to the currently available commercial solid-phase assay and to one or more additional methods in use for the quantitation of serum levels of IgE. Neither laboratory found inhibitory substances in cancer sera which interfered with the double-antibody assay. Subjects with most types of cancer have normal serum IgE levels. The cancer types represented in the study by 1Valdmann and associates” were somewhat different from those of the present study since subjects with melanomas and sarcomas were not included in the former study. Thus, our report confirms and extends Waldmann‘s study and provitles an opportunity to emphasize that in our hands the double-antibod- technique employing radiolabeled IgE (ND) and rabbit anti-IgE (T’S) is presently the method of choice for measuring serum levels of IgE. All of the currently available commercial techniques have proved to be less satisfactory in our hands. REFERENCES 1 Ishizaks, K., and Ishizaka, T.: Identification of E antibodies as a carrier of reaginic activity, J. Immunol. 99: 1187, 1967. 2 Johansson, S. G. O., Mellbin, T., and Vahlqvist, B.: Immunoglobulin levels in Ethiopian preschool children with special reference to high concentrations of immunoglobulin E (IgND), Lancet 1: 1118, 1968. 3 Waldmann, T. A., Polmar, S. H., Balestra, S. T., Jost, M. C., Bruce, R. M., and Terry, W.

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D.: Immunoglobulin E in immunologic deficiency diseases. II. H(Lrum 1gE concentration of patients with acquired l~ypogammaglobulinemia, thymoma and hypogammaglobulinemia, myotonic dystrophy, intestinal lymphangiectasia and Wiskott-Aldrich syndrome, J. Immunol. 109: 304, 1972. Heiner, D. C., and Rose, B.: Elevated levels of IgE in conditions other than clinical allergy, J. ALLERGY 45: 30,197O. Serum immunoglobulin lcvrls Nordbring, F., Hogman, C. F., and Johansson, S. G. 0.: in the course of acute pneumonia, Stand. J. Infect. Dis. 1: 00, 1969. MacKay, W. D.: The incidence of allergic disorder and cancer, Br. J. Cancer 20: 434, 1966. Fisherman, E. W.: Does the allergic diathesis influence malignancyB J. ALLERGY 31: 74,

1960. 8 Gabriel, R., Dudley, B. M., and Alexander, W. D.: Lung cancer and allergy, Br. J. Clin. Pratt. 26: 202, 1972. 9 Jacobs, D., Landon, J., Houri, M., and Merrett, T. G.: Circulating levels of immunoglobulin E in patients with cancer, Lancet 2: 1059, 1972. 10 Gleich, G. J., Averbeck, A. K., and Swedland, H. A.: Measurement of IgE in normal and allergic serum by radioimmunoassay, J. Lab. Clin. Med. 77: 690, 19il. 11 Hunter, W. M., and Greenwood, F. C.: Preparation of iodine labeled human growth hormone of high specific activity Nature 194: 495, 1962. 12 Ure, D. M. J.: Negative association between allergy and cancer, Scott. Med. J. 14: 51, 1969. 13 Augustin, R., and Chandradasa, K. D.: IgE levels and allergic skin reactions in cancer and non-cancer patients, Int. Arch. Allergy 41: 1414, 1972. 14 McKee, W. D., Arnold, C. A., and Perlman, M. D.: A double-blind study of the comparative incidence of malignancy and allergy, J. ALLERGY 39: 294, 1967. 15 Logan, J. and Sacker, D.: The incidence of allergic disorders in cancer, PT. Z. Med. J.

52: 210,1953. 16 Polmar, S. H., Waldmann, T. A., and Terry, W. D.: A comparison of three radioim munoassay techniques for the measurement of serum IgE, J. Immunol. 110: 1253, 1973. 17 Waldmann, T. A., Bull, J. M., Bruce, R. M., Broder, S., Jose, M. C., Balestra, 8. T., and Suer, M. E.: Serum immunoglobulin E levels in patients with neoplastic disease, J. Immunol. 113: 379, 1974. 18 Orgel, H. A., Lenoir, M. A., and Bazaral, M.: Serum IgE, IgA, IgM and IgE levels and allergy in Filipino children in the United States, J. ALLERGY CLIN. IMMVNOL. 53: 213,

1974. 19 Bazaral, M., Orgel, IgE levels in twins,

H. A., and Hamburger, R. N.: J. ALLERGY CLIN. TMMUNOL. 54:

Genetics

288,1974.

of IgE

and

allergy:

serum