Unspecific cellular immunity before therapy in patients with squamous cell carcinoma of head and neck

J. max.-{ac.Surg. 6 (1978)75-93

Unspecific Cellular Immunity before Therapy in Patients with Squamous Cell Carcinoma of Head and Neck Jfirgen BIER, Klaus BITTER, Ulrike NICKL1SCH Department of Maxillo-facial and Plastic Surgery of the Face (Head: Prof. R, Stellmach, M.D., D.M.D.), Klinikum Steglitz, Free Univ. of Berlin, W.-Germany

Summary An introduction to the role of lymphocytes in immunological reactions is given. Two fundamental categories of immunological response are described which are mediated by two distinct subpopulations of lymphocytes: B-lymphocytes are responsible for humoral immune reactions and T-lymphocytes are involved in cell-mediated immunity. Information is given on the role of the immune system in generation of anti-tumour activities and of mechanisms leading to an acceleration of tumour growth. Several pathways of cytotoxic and blocking reactions against target cells are mentioned. Furthermore, methods are described for monitoring the non-specific immune reactivity of the host. These nonspecific cellular immune responses in 30 patients with squamous cell carcinoma of the head and neck were compared with those in 30 healthy controls. Assays were performed in vitro to evaluate the blastogenic response of lymphocytes to the mitogens PHA (phytohaemagglutinin) and PWM (pokeweed mitogen) and to quantify T-rosetteforming lymphocytes in the peripheral blood. The in vivo assays used were the delayed cutaneous hypersensitivity reaction to the primary stimulus of DNCB (dinitro-chloro-benzene) and the recall reaction to PPD (purified protein derivate). The carcinoma patients demonstrated significant impairment of lymphocyte blastogenesis reactions to PHA but not to PWM. The percentage and absolute counts of Trosettes was significantly reduced in cancer patients cmnpared with normal controls. Skin test reactivity to de-novo sensitation with DNCB was significantly abnormal in patients with head and neck cancer. However, delayed type hypersensitivity evaluated with PPD (recall antigen) was not significantly rcduccd. After subdividing the cancer patients according to their clinical stage of disease and subsequent analysis, they showed no correlation between clinical stage and immune reactivity. These data indicate that PHA induced lymphocyte blastogenesis, enumeration of Trosette levels and evaluation of delayed hypersensitivity reaction to DNCB are potentially useful for the study of squamous cell carcinoma of head and neck to monitor effects of tumour treatment and perhaps to evaluate a correlation between immunocompetence and prognosis.

0301-0503/78 1500-0075 $ 05.00 6 .]. max.-fac. Surg. 2/78

Key-Words: Squamous cell carcinoma of head and ne&; Nonspecific cellular immune reactivity; Lymphocytes and immune response; Tumour immunology.

Introduction Traditionally only morphological criteria were used to define lymphocytes which have a large nucleus in relation to cytoplasm contents. The cytoplasm contains a small golgi area, few lysosomes, a number of mitochondria and is rich in free ribosomes (Fig. 1). But in the last few years it has become possible to divide lymphocytes into several groups according to their immunological function. Moreover, for a long time, lymphocytes have been considered as ,,end cells" with no further differentiation and proliferation. Experiments, however, have now shown that specific antigens in vivo and in vitro (Oppenheimet al.

Fig. 1 Small lymphocyte (x15,000) (courtesy of Prof. Stolpmann).

@ 1978 Georg Thieme Publishers

76

]. Bier, K. Bitter, U. Nid~lisch

1968) and a variety of non-specific mitogens could stimulate lymphocytes to proliferate and differentiate into large ,,blast cells", and sometimes to plasma cells (Ling 1968).

Background of Cellular Immunity

Perhaps the most important discovery concerning lymphocytes during the last 15 years was that immunologically competent lymphocytes were not a homogenous population but consisted of two distinct populations with different functions and properties.

Lymphocytes and the immune response

Two types of immune response

Involvement of lymphocytes in immunological phenomena has long been suspected (Murphy 1926). Conclusive evidence for their definite role in the immune response was only produced in the late 1950s by showing that lymphocytes could transfer delayed hypersensitivity (Wesslen 1952), skin allograft rejection (Billingham and Silvers 1963) and tumour immunity (Mitehison 1955). Yet, the precise role of lymphocytes in these various immunological reactions still remained controversial. There was, in particular, considerable doubt as to whether they ever served as effector cells in many of these reactions since very few sensitized cells were found at the sites of delayed hypersensitivity or in the bed of allografts undergoing rejection (MeCluskey et al. 1963). Indeed, many of the infiltrating cells at these sites were non-lymphocytic (Waksman 1970).

Immunology as an experimental science evolved out of bacteriology as a study of the antimicrobial response to infective pathogenic organisms acquired by the host. Even during this early period it was apparent that the ,body has more than one way of expressing ,,protective" immunity. Considerable debate centred around the relative role of antibodies and phagocytic cells (Metchnihoff

The development of in vitro models using ,,purified" cell populations have subsequently provided convincing evidence for an active role of lymphocytes in various ,,cell-mediated" immunological reactions and have permitted analysis of reactions where lymphocytes may be initiators, but not necessarily effectors (Govaerts 1960). Similarly, classical plasma cells, at the time thought to be independent of lymphocytes and derived from primitive reticulum cells, were generally considered as the main producers of antibodies (Marshall and White 1950, Thiery

1960). However, it could be demonstrated that some lymphocytes can produce antibodies (MiikeIa and Nossal 1961). Lymphoid cells which could be observed in the lymphoid tissue of animals undergoing immunological reactions were shown to be intermediate in ultrastructural appearance between lymphocytes and plasma cells (Cunningham et al. 1966). This provides a strong argument for a lymphocytic origin of antibody-producing plasma cells.

1905). When Denys and Leclef (1971) demonstrated the opsonizing (enhanced phagocytosis) effect of serum antibodies, a link was found between antibodies and phagocytic cells in the immune response, thus illustrating the interaction and synergism between ,,cell-mediated" and ,,humoral" immunity. Those immunological reactions transferable by cells and not by serum are defined as ,,cellmediated".They include such diverse manifestations as allograft rejection, allogenic (graft versus host) disease and delayed hypersensitivity in addition to the classical cell mediated protective immunity to pathogenic organisms. Immunological responses transferable by serum are now defined as ,,humoral" immunity. Manifestations of humoral immunity include immediate hypersensitivity or allergic response and Arthus reactions in addition to the classical antibodymediated protective immunity. However, the most commonly used parameter of ,,humoral" immunity is the presence of serum antibodies as revealed by a great variety of chemical and biological test systems. The molecular and cellular events involved in the diochotomy of ,,humoral" and ,,cell-mediated" immunity are now sufficiently understood to justify some characterization of immunological phenomena (Coombs and Gell 1968). The most significant advance at the cellular level has been the realization that two different types of immune response reflect differences in two distinct types

Unspecific Cellular Immunity before Therapy in Patients with Squamous

77

STEM CELLS ( FOETAL LIVER and BONE MARROW)

|

r

1

THYMUS PROCESSING

EQUIVALENT PROCESSING

i T . LYMPHOCYTE

l

~

/~'~

%2/.

ANTIGEN

2-~.

LYMPHOBLASTS @

~

=

A

.......COOPERATION..... ......~'~. @

CELL MEDIATED IMMUNE REACTIONS

g - LYMPHOCYTE

PLASMAcELLS

HUMORAL IMMUNE REACTIONS

Fig. 2 Processing of stem cells by thymus and Bursa Fabricius- equivalent lymphoid tissue into immune competent T- and B-lymphocytes. Proliferation and transformation to lymphoblasts and plasma cells occurs on antigenic stimulation, of lymphocytes which mediate them. The development of immunology also brought more significant advances in respect of the molecular basis of antibody and cellular specificity.

Two types of lymphocytes There now follows a description of experimental observations which resulted in the concept of two different types of lymphocytes referred to as T- and B-cells. Glick et al. (1956) reported that surgical removal of the Bursa Fabricius - a cloacal lymphoid organ - in birds at hatching impaired their capacity to produce serum antibodies following subsequent challenge with antigen. In addition, it was shown that bursectomy did not affect homograft and graft versus host reaction (Mueller et al. 1960, Warner et al. 1962). On the other hand, it was demonstrated that homograft rejection in chickens was considerably retarded by neonatal thymectomy (Aspinall et al. 1963).

These observations suggested an essential subdivision of the immune response controlled by two distinct lymphoid organs. Parallel experiments were carried out in rodents and complemented by clinical observations (Miller 1961, Good et al. 1971). Efforts to locate a bursa-equivalent organ or site in mammals have not yet brought definite results. But Good et al. (1971) have provided some evidence that the gut-associated lymphoid tissue may serve the role of the Bursa Fabricius in rabbits. Now the bipolar organisation of the immune system was emerging with two ,,central" lymphoid organs. On the one hand, there is the bursa or its hypothetical mammalian equivalent, on the other the thymus which contains immature lymphoid cells, seeding them out to the periphery where they change into immunologically competent cells (Cooper et al. 1968). Additional experiments led to the suggestion that thymus-derived lymphocytes collaborate with

78

]. Bier, K. Bitter, U. Nichlisdt

bone marrow-derived lymphocytes (bursa equivalent-derived) in the antibody response. Cells from the bone marrow and thymus of mice injected together into irradiated recipients react with a high output of antibodies against antigen challenge, whereas each kind injected independently was only capable of very low antibody production (Claman et al. 1966). In complementary experiments it could be proved that antibodies are produced by cells of the ,bone marrow, yet this production can only be effected by the stimulus of the thymus cells injected simultaneously (Davis et al. t967).

Roitt et al. (1969), in an analysis of the ,,two-cellconcept", suggested the terminology ,,T-cell" for thymus-derived or thymus-dependent lymphocytes, and ,,B-cell" for bursa-equivalent or thymus-independent-derived lymphocytes (Fig. 2). However, it should not be assumed that T- and B-cells are homogeneous populations as there is considerable evidence for the existence of subpopulations (memory-, effector-, helper- and suppressor-cells) (Roberts et al. 1957, Gershon et al. 1972, Hiiyry and Anderson 1974, Wagner et al. 1974, Cantor and Boyse 1975).

Tumour immunology Ehrlich (1909) was one of the first of many investigators who have given emphasis to the general role of the immune system in preventing or limiting turnout growth. Burnet (1957) stated ,,it is by no means inconceivable that small accumulations of tumour cells may develop and because of the possession of new antigenic potentialities provoke an effective immunological reaction with regression of the tumour and no clinical hint of its existence". Thomas's postulation (1959) that the mechanisms for homograft rejection have evolved primarily as a natural defence against neoplasia meant a further extension of this concept. The theory of immune surveillance was made possible after the demonstration that many tumours of animals (Prehn and Main 1957, Klein et al. 1960) and also of man (HellstrSm 1967, Hellstrdm and HellstrOm 1969) have tumourassociated antigens. The immunological response of the individual to some of these antigens can result in protection against tumour growth (Prehn 1960, Klein et al. 1960). However, the generation

of anti-tumour immunity was found to be not always effective but could, at times, even lead to an acceleration of tumour growth (Hellstr6m et al. 1971, Baldwin et al. 1972).

Immune reactivity to tumour cells It has been generally accepted that cell populations including thymus-derived lymphocyte,s (Tcells), bone marrow-derived lympocytes (B-cells) and macrophages are responsible for cell-mediated and humoral reactions against turnouts. They are able to transfer immunity against tumour cells and neutralize tumour cells in vivo (Mitchison 1954, Billingham et al. 1954, Old and Boyse 1964). However, there are a number of pathways of tumour cell destruction as shown in Fig. 3. Evidence has been o.btained that T-cells may play the main role in the immune cell category (Brunher and Cerottini 1971). Decreased resistance to tumour growth in thymectomized animals (Payne 1972) or in animals treated with anti-lymphocyte serum (Allison and Law 1968) strongly implicated T-cells as effector cells in tumour immunity. As pointed out by Bloom (1973), these studies indicated only that T-cells are essential in this process, not that they are necessarily the cytotoxic cells. However, in in vitro assays immune T-cells destroy specific target cells as a result of direct interaction (Ginsburg 1970). This requires specific sensitized immune cells with receptors for the target cell antigen as shown by Goldstein and co-workers (1971). B-cells are also involved in target cell killing by their capacity to release antibodies. On the one hand target cells, sensitized and coated by antibodies (MacLennan et al. 1969, 1970, Wunderlich et al 1971) or antibodies and complement (Perlmann et al. 1969), are susceptible to cytotoxicity by different types of immune cells. On the other hand, antibodies and complement can directly destroy target cells (Green et al. 1959). As pointed out by Katz and Benacerraf (1972) ,,helper" Tcells are involved in these B-cell dependent immune reactions. Since we have nowadays ways and means for separating human T- and B-cells, studies on the nature of cells reactive against human tumours have become feasible. O'Toole et al. (1973) reported that cells reactive with target cells from

Unspecific Cellular Immunity before Therapy in Patients with Squamous

79

ANTIGEN

MEMORY CELL

EFFECTOR CELL :

RELEASE of SPECIFIC MACROPHAGE ARMING FACTORS

DIRECT T - CELL CYTOTOXIClTY

SPECIFIC MACROPHAGE CYTOTOXICtTY

:

~

~

EFFECTOR CELL

- ' ~ ' ~

RELEASE of UNSPECIFIC MACROPHAGE ACTIVATING FACTORS

RELEASE of ANTIBODYS

RELEA E of ANTIB( PDYS

RELEASE of ANTIBODYS

UNSPECIFIC MACROPHAGE CYTOTOXICITY

DIRECT ANTIBODY and COMPLEMENT DEPENDENT CYTOTOXICITY

ANTIBODY DEPENDENT CELLULAR CYTOTOX~CtTY

BLOCKING of CYTOTOXICITY

RELEASE of ANTIBODYS

t

DE BLOCKING of BLOCKING REACTIONS

1

W Fig. 3 Cytotoxic pathways of T- and B-lymphocytes after antigen stimulation.

bladder cancer could be removed by passage over an anti-immunoglobulin column. From this it could be deduced that B-cells are the effectors in this system. Contrary to this, it was demonstrated by Wybran et al. (197,8) that rosette-forming T-cells were responsible for much of the reactivity against turnout cells in vitro. Apart from this, macrophages - either specific or nonspecific - can mediate cytotoxic effects in collaboration with lymphocytes (Evans and Alexander 1972 a, b, Feldma~zn 1972). Also granulocytes should be mentioned at this point as they can have cytotoxic effects against antibody-coated target cells (Perlmann et al., 1969), but here again lymphocytes seemed to be the cells principally involved. There are other studies in which the active cells are regarded as ,,null" lymphoid cells, ,,real null': cells and ,,K" cells, i. e. cells without T- or B-cell characteristics (Greenberg et al. 1973).

Enhancement of turnout growth One of the main points concerning the growth of antigenic tumours is the immunological enhancement by humoral antibodies or antigen-antibody complexes (blo&ing factors) (HellstriJm and HellstrSrn 1970). Sera from immune animals can cause an increased growth of turnout cells (Bansal and SjSgren 1972). Three possible types of mechanisms of enhancement have been thoroughly studied. Afferent enhancement: antibodies bound to the tumour cells interfere with tumour immunogenieity (Snell et al. 1960). Central enhancement: humoral factors cause suppression of reactivity of immune cells (Mitchell

1972). Efferent enhancement: antibo,dies bound to antigenie sites protect the tumour from attack, presumably from immune cells (HellstrSm and HellstrSm 1974).

80

J. Bier, K. Bitter, U. Niddisch

@

Greaves et al. 1974, Mellstedt 1975, Weksler and Kuntz 1976). Consequently the response of human lymphocytes to P H A has been employed for the clinical evaluation of T-lymphocyte functions. Because there is no substance available with selective biological properties to stimulate human B-cells, P W M is generally accepted for the evaluation of B-lymphocyte reactivity in clinical research. The quantitation of T-cells seems to be necessary because these cells are essential for the host's reactivity against tumour cells as mentioned above.

Fig. 4 Rosette-forming cell (xl,400): Central thymus derived lymphocyte with surrounding sheep red Mood cells.

An additional type of humoral factor termed ,,unblocking factor" has also been described. Sera with unblocking factors are able to interfere with the effects of blo&ing sera (Hellstr6m and HellstrSm 1970, Bansal and SjSgren 1972).

Measuring of nonspecific immune reactivity Cell mediated immunity can be evaluated by a considerable number of methods in order to monitor immunological deficiency states.

Stimulation of lymphocytes A variety of in vitro assays of cell-mediated immunity have been developed since the discovery by Nowell in 1960 that phytohaemagglutinin (PHA), a lectin extract of Phaseolus vulgaris, transforms normally non-growing peripheral blood lymphocytes. The cells enlarge progressively, begin to replicate D N A and finally undergo mitosis. These events closely parallel the physiological blastogenesis which occurs in vivo in response to immunological challenge. The plant lectins phytohaemagglutinin and pokeweed (PWM) stimulate human lymphocytes to divide. Numerous experimental data indicate that PHA stimulates only T-cell proliferation, while human B- and T-lymphocytes are both stimulated by PWM (Geha and Merler 1974,

During the past few years a number of receptors and differentiation antigens have .been identified on different immunologically competent cells. The presence or absence of these receptors on mononuclear cells of unknown type may provide information as to their origin. It has thus been possible to identify T-cells by different methods. In the mouse thymus-derived lymphocytes can be identiffed by the presence of the theta-iso antigen (Raft 1969). In man the use of heterologous antithymocyte sera (Aiuti and Wigzell 1973), which has been rendered specific of T-cells ,by absorption with a pure population of B-cells, can be useful for detection of T-lymphocytes. But an easier method to identify human thymus-derived lymphocytes is to show their capacity to form nonimmune rosettes (E-rosettes) with sheep red blood cells (SRBC) by as yet unknown receptors (Jondal et al. 1972, Wybran et al. 1972) (Fig. 4).

Delayed type hypersensitivity In contrast to the in vitro systems of lymphocyte stimulation and determination of T-cell levels, the elicitation of delayed cutaneous hypersensitivity reactions has been employed widely to measure cell-mediated immunity in vivo. Landsteiner and Chase (1942) laid the groundwork for the current understanding of delayed hypersensitivity reaction as a part of cellular immunity. They showed that sensitivity to a chemical antigen could be transferred by means of cells. Normally skin sensitization with intradermally injected antigens develops a reaction in healthy people characterized by erythema and induration at the site of application (Fig. 5). This means that the afferent, central

Unspecific Cellular Immunity before Therapy in Patients with Squamous

81

and efferent limbs of the immune response are intact. In patients with diminished cellular immunological reactions skin tests can be impaired and are useful for .determination of immunological defects. The antigens selected for this purpose are on the one hand those to which the patients are commonly exposed like purified protein derivate (PPD) to prove the anamnestic response. On the other hand, it is possible to gain more information b y sensitizing patients to an unknown agent such as dinitro-chlor-benzene (DNCB) with subsequent testing of reactivity by applying test doses of the same antigen,

Immune Reactivity before Treatment A n important contribution in clinical oncology has been the demonstration that cancer patients with different types of malignancy have immune defects in every clinical stage of their disease and that this deficiency correlates with both tumour stage and prognosis (Hersh et al. 1976). These reports suggest that measurement of immune reactivity in patients with squamous cell carcinoma of the head and neck may be useful in determining mechanisms of tumour development and progress, detecting early p r i m a r y or recurrent malignancy, and in guiding conventional and immunotherapy. W i t h the development of methods for quantitating nonspecific lymphocyte reactivity in vitro and in rive, such studies appear feasible. As an initial step in discerning the usefulness of these assays, we measured nonspecific cellular immune reactivity with various methods in patients with squamous carcinoma of the head and neck before therapy and compared them with normal controls. On the one h a n d we were interested in looking for possible immunological differences in our patients compared with the controls, and on the other we hoped to find a correlation ,between the immune status and clinical stage of their disease.

Material and Methods

Patients 30 patients - 15 :females, 15 males - with a definite histological diagnosis of squamous ceil carcinoma of the head and he& were studied before therapy and compared with 80 age and sex matched normal controls. Cancer patients ranged in age from 27 to 91 years, i. e. on average 59 years of age. Patients were

Fig. 5 Delayed cutaneous hypersensitivity reaction after sensitisatien with DNCB.

classified according to the TNM system as described by the UICC (1973). 5 cancer patients ranged in the clinical stage of TINo_2Mo, 10 in T2N0_2M0, and 15 in T3No- 2Mo.

Separation of lymphocytes The lymphocyte suspension was prepared according to the method of B6ym (1964). 50ml of venous blood were drawn into a plastic syringe containing 10 units of heparirdml (Liquemin ®, Hoffmann La Roche, Grenzach, W-Germany). The blood was diluted under sterile conditions with an equal volume of physiological saIine solution (NaCt, 0.15M, Braun, Melsungen, W-Germany). 20 ml of a mixture of Ficoll (Pharmacia, Uppsala, Sweden) and Isopaque (Ronpacon ® 75 °/0, Cilag-Chemie, Alsbach, W-Germany) in sterile 25 X 150 mm glass tubes. Ficoll-Hypaque solution was composed of 20 ml Isopaque with 9.556 g Ficoll in 130.4 ml distilled water. The density of the Ficoll-Hypaque mixture was adjusted to a specific gravity of 1.077 with distilled water and passed through a 0.45 ~m Millipore filter (Millipore, Bue France). Tubes containing the diluted blood layered over Ficoll-ttypaque were centrifuged at 400 g for 40 rain at 4°C. Mononuclear ceils, removed from the interface, were washed three times with RPMI (Roswell Park Memorial Institute) 1640 medium (Seromed, Munich, W-Germany) and collected by centrifugation at 150 g for 10rain at 4°C. The cells were then resuspended in RPMI 1640 medium.

82

J. Bier, K. Bitter, U. Nicklisch

Lymphocyte stimulation with phyto-mitogens The stimulation experiments were prepared according to the method of Penhale et at. (1974). The lymphocyte suspension was adjusted at 1 × 106 viable cells/ml containing 2 mM/ml L-glutamine (Gibco Bio Cult, Glasgow, England), 100 E penicillin/ml (Gibco Bio Cult), 100,ug Streptomycin/ml (Gibco Bio Cult) and 1 0 % pooled human sterile filtered (0.22 ~tm filter, Millipore), inactivated (30 min in 56°C water bath) ABO serum. 2×105 ( ~ 0.2 ml) cells of this suspension were incubated in quadruplicate in microtitre plates (Greiher, Nfirtingen, W-Germany) with the mitogens phytohaemagglutinin - P (P-PHA, Difco Lab, Detroit, USA), pokeweed mitogen (PWM, Grand Island Biological Comp., Grand Island, USA) and without mitogen as unstimulated control. The mitogen concentration was evaluated in dose-response experiments as shown in Figs. 6 + 7. The optimal concentration was 1 ,t~l for P H A and 10 ,ul for PWM. These cultures were then incubated at 37°C in an atmosphere of 5 °/0 CO 2 in humified air. By means of kinetic studies an optimal incubation time of 3 days was determined for PHA stimulated cultures and of 6 days for P W M cultures (Figs. 8+ 9). 18 hours before harvesting I ~tl of tritiated thymidine (3H-TdR, specific activity 5 Ci/ mM ;Radiochemical Centre, Amersham, England) was added to each culture. Then the cultures were harvested on 0.45 ,am Millipore filters and washed twice with distilled water. The filters were dried for 60 rain in 56°C and separately dissolved in plastic tubes (Packard Instr. Comp., Inc., USA) with 5 ml scintillation fluid (Toluene Scintillation Grade containing 5g/l PPO and 0.3 g/1 POPOP, Merck, Darmstadt, W-Germany). In a Tricarb spectrometer model 3380 (Packard) the thymidine incorporation of each culture was measured. The arithmetical mean of the cultures in quadruplicate was determined, subsequently the average value of the unstimulated control cultures was deducted. The results were expressed as A counts per minute per culture. (Counts of mltogen stimulated cultures minus counts of nnstimulated control cultures = A cpm).

E-rosettes assay The method used in this study has been described previously by Seiler et al. (1972). The Ficoll-Hypaque separated lymphocytes were adjusted to a final concentration of approximately 2.5× 10%ells/ml. Sheep red blood cells (SRBC) (Animal Farm, Robert Koch Institute, Berlin, Germany) were washed three times with RPMI 1640 medium and adjusted to 8×107cells/ml. 0.25 ml of each cell suspension was incubated with 0.1 ml human pooled and heat-inactivated (30 rain at 56°C) AB0 serum. The serum was absorbed three times for 30 rain in 4 °C with SRBC. Preparations were incubated at 37 °C for 5 rain, centrifuged at 100 g for 5 rain and maintained at 4 ° C for 120 rain. The supernatant was then discharged and the cell pellet fixed with glutaraldehyde solution (3.2 ml glutaraldehyde with 96.8 ml RPMI

1640 medium) and incubated again for 20 min at 4 °C. The pellet was resuspended and one drop of methylene blue (Merck) was added for staining. 400 cells from each sample were counted and the percentage of E-rosette forming cells was determined. Lymphocytes with more than 3 adherent SRBC were counted as rosettes. Absolute T-cell levels were evaluated by percentage of E-rosette forming cells and lymphocyte levels were determined by differential blood picture.

Delayed-type hypersensitivity assays Tests were performed in cancer patients and healthy subjects using the Tuberculin Tine Test (Cyanamid, Muni&, W-Germany) for determination of ,,preexisting immunity" by PPD (purified protein derivate). This is a multiple puncture test introduced by Rosenthal (1961) and carri.ed out with disposable units. Each unit carries four prongs or ,,tines" of 2ram length. The tines have been dipped in old tuberculin and were subsequently sterilized. The tines are pressed on the cleaned skin on the flexor surface of the forearm to form four puncture sites. The test is read in 48 to 72 hours. A palpable induration of 2 mm or more around one or several of the puncture sites is called positive, and approximately equivalent to a 5 mm reaction of the 5 T U (tuberculin unit) Mantoux test (Maha, 1962, Badger et al., 1963). Delayed-type hypersensitivity tests for ,,primary immune response" were prepared with DNCB (dinitrochloro-benzene) according to the method of Epstein and Klingman (1958). 2000 ,ag DNCB were dissolved in 0.1 ml absolute acetone and applied on a cleaned area of the flexor aspect of the arm in a diameter of 2 era. After drying by air, tile site of application was covered with a bandage for 24 hours. 10 to 14 days after sensitisation the site was examined for spontaneous flare reaction with erythema, with or without induration and vesicle formation. Patients who showed these reactions were considered positive in de-novo sensiation. Two weeks after primary sensitization all patients were rechallenged on the other arm with 200, 100, 50 and 25 ~tg of DNCB. Reactions were recorded 48 hours after application. Positive skin reaction to the challenging dose appeared as an erythematous plaque showing papular, vesicular or bullous eruption, according to the degree of response. A patient was considered to be positive, if a delayed hypersensitivity reaction was present at the 50 [xg site.

Statistics Experiments with P H A and P W M and determination of E-rosettes on normal people and tumour patients were tested with the zero-hypothesis (H0) by means of the Wilcoxon-test, and from two angles pertaining to random samples. Significant differences were evaluated as 2 a < 5 °/0. In addition the median value (X), the 10 (xt0) and the 90 (xg0) percentage were determined. The DNCB and PPA skin tests were analyzed as positive or negative by the use of the chi-square test.

Unspecific Cellular Immunity before Therapy in Patients with Squamous

83

Table 1 In vitro immune reactivity according to clinical stage.

PHA

PWM

E°/0

E.ABS.

Distribution differs from T1 T2

T1N0_~M o

60.44P

53.7861

482

697 ~

T~No_.~Mo

102.849

92.237

38

649

ns.

T~N0_~Mo

79.620

67.714

36

810

ns.

' = x in A cpm 2 = x i n 0/0

ns. 4

T3 ns, ns.

ns.

= x in cells/ram 3 4 = not significant by Wilcoxon test

T a b l e 2 DNCB reactivity. DNCB positive

DNCB negative

Normal controls

27/30

900/0

3/30

10°/0

Cancer patients

17/80

570/0

13/30

430/0

Total

44/30

730/0

16/30

270/0

Distribution differs from normals

significant (2 ct <0.0l)':"

;:" 7:2 analysis

Results

PHA lymphocyte stimulation Fig. 10 shows the overall results of P H A induced lymphocyte stimulation in 30 patients and 30 normal controls. It should be noted that in both groups a wide scattering of responses was observed with remarkable overlapping between these populations. For one half of the tumour patients, the aH-thymidine uptake is lower than the lowest value of the controls. T h e mean P H A response of the normal controls after a 3 day incubation time was x = 138.987 A cpm; the mean response in cultures from cancer patients was x=91.866 zl cpm. The difference between the mean response of the controls and the group of tumour patients is significant (2 a < 0.00l). After subdividing the cancer patients according to their clinical stage into groups of T,N0-2Mo (x= 60.441 A cpm), T2N0_2Mo (x=102.849 A cpm) and TaNo_2M0 (x= 79.620 A cpm) and subsequent analysis they showed no correlation between clinical stages of the disease and lymphocyte stimulation as shown in

Table 1. PWM lymphocyte stimulation In the lymphocyte response to P W M of tumour bearing individuals compared with the control group there were neither major differences nor was there a progressive depression of lymphocyte response proportional to the size of the primary tumour and lymph node involvement (Fig. 11 and Table 1). The mean thymidine uptake for normal controls was

x = 86.061 A cpra, for the group of cancer patients x = 69.958 cpm (2 a > 0.05). Lymphocytes of patients with stage of disease T1N0_2M0 were stimulated with x = 53.786 A cpm, T2No_2M0 with x = 92.237 d cpm and T3N0.2M0 with x - 67.714 A cpm.

T-lymphocyte levels E-rosctte levels enumerated as percentage counts are shown in Fig. 12. Rosette forming lymphocytes are decreased in patients with head and neck cancer with a median count of x =390/o compared with normal controls with x = 52 °/0. W i t h 2 a < 0.001 the difference between both groups is significant. In order to demonstrate that the percentage of T-cell depression is not an artefact phenomenon with a simultaneous absolute increase in the number of lymphocytes in the peripheral blood, we additionally determined the absolute T-cell values from the differential blood count and the T-cell percentage as shown in Fig. 13. Normal volunteers had a range from 592 to 2028 T-lymphocytes per mm 3 with a mean of x = 1065 cells per mm a. Cancer patients showed significantly decreased levels with a mean of x = 699 T-cells/mm a and a range from 234 to 1530 cells/ram 3. The total range in both, percentage and absolute Tcell levels, in the cancer patient is similar to that of the controls and in a wide section there is again an overlapping of values in these two groups. In patients with different stages of disease there was neither a significant difference detectable for E-rosettes enumerated as percentage nor for absolute T-cell levels (Table 1).

84

]. Bier, K. Bitter, U. Nicklisch

Table 3 DNCB reactivity according to clinical stage. DNCB positive

DNCB negative

Distribution differs from

T1 T1No_2M 0

5/ 5

100%

0/ 5

0%

TeNo_ 2M0

5/10

50°/0

5 / 10

500/0

ns.

T3N o _.oMo

7/15

47%

8/15

53°/o

ns.

17/30

570/0

13/43

430/0

Total

T~

T3

ns.':"

ns. ns.

ns.

~:"Not significant by %2analysis.

Table 4 PPD reactivity. PPD positive

PPD negative

Distribution differs from normals

Normal controls

24/30

80%

6/30

20O/o

Cancer patients

18/30

600/0

12/30

400/0

Total

42/60

70

18/60

30%

not significant':

':" )~ analysis

]'able 5 PPD reactivity according to clinical stage. PPD positive

PPD negative

Distribution differs from T1 T2 T~

T1No__.M o

3/ :5

600/0

2/ 5

400/0

T2No .oMo

7/10

700/0

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Skin test reactivity

Discussion

Delayed hypersensitivity reactions to DNCB at~d PPD are shown in table 2-5. 27 of 30 normal controls (900/0) responded to DNCB. However, only 17 of 30 eanc.er patients (570/0) showed positive reactions (Table 2). After subdividing the patients according to their clinical stage of disease 100°/0 with T~No-~Mo, 500/0 with T2No-2Mo and only 47 0/0 with T3No_2M o showed delayed hypersensitivity reactions (Table 3). PPD reactivity showed 80°/o positive tests in normal controls compared to 60 °/o in cancer patients (Table 4). According to the clinical stage of disease there were no significant differences between the three groups of cancer patients as shown in Table 5.

T h e most significant result in this study is the decreased i m m u n e r e a c t i v i t y in patients with squamous cell c a r c i n o m a of the head and n e & before t h e r a p y c o m p a r e d with n o r m a l controls. These findings indicate - with respect to the t h e o r y of Thomas (1959) and Burnet (1970) that our cancer patients suffer f r o m a defect in i m m u n e surveillance. H o w e v e r , a correlation between i m m u n e r e a c t i v i t y and clinical stage of disease could not be detected.

Unspecific Cellular Immunity before Therapy in Patients with Squamous DOSERESPONSE LYMPHOCYTESTIMULATION: PHA CONCENTRATION 180

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Fig. 6 PHA dose-response: incubation of 2 x 102 peripheral blood lymphocytes of a different normal subjects with varying PHA-concentrations for a period of 3 days, The highest mean of SH-thymidine incorporation occurs after stimulation with 1 FI PHA.

Fig. 7 PWM dose-response: incubation of 2 x 105 peripheral blood lymphocytes of 6 different normal subjects with varying PWM-concentrations for a period of 6 days. The highest mean of ~H-thymidineincorporation occurs after stimulation with 10 ul PWM.

In the in vitro assay, cancer patients showed a striking depression of T-cell function measured by PHA lymphocyte stimulation (Fig. 5) and determination of E-rosette levels (Fig. 7 + 8). In contrast, B-cell functions evaluated by cell-culture stimulation with PWM were slightly but not significantly reduced (Fig. 6). These results correlate with findings of other investigators in patients with lympho-proliferative disease as well as in patients with solid malignant tumours. Impaired lymphocyte reactivity to PHA and depressed T-cell levels are described in patients with Hodgkin's disease (Hersh and Oppenheimer 1965, Hart and Sokal 1970, Fuks et al. 1976, Many et al. 1976), chronic lymphatic leukemia (Han 1973, Catovsky et al. 1974, De Vaan et al. 1975, Hunyadi et al. 1976) and lymphosarcomas (Trubowitz et al. 1966, Al-Sarraf et al. 1972).

(1975), Turner et al. (1976), Keller et al. (1976) for cancer of the breast. Similar findings are reported by Catalona et al. (1973), Chretien and Ketchan (1973), Golub et al. 1974) and Bichhardt and Kunze (1974) for malignant melanoma, and by dl-Sarraf et al. (1972), Tarpley et al. (1975), Wanebo et al. (1975) and Thomas et al. (1976) for kidney-, ovary-, colon-, lung-, head and ned{-, prostate-, and laryngopharyngeal cancers. Other laboratories, however, have been unable to demonstrate any defects in lymphocyte functions in similar studies (Nelson 1969, Paty and Bone

Reports of depressed cellular immunity measured in vitro in patients with different types of solid turnouts are given by Knight and Davidson

1973). The data about PWM lymphocyte stimulation are also controversial. On the one hand Smith et al. (1973) reported abnormal low thymidine uptake in P W M cultures of patients with chronic lymphatic leukemia, on the other, Golub et al. (1974) described slight but not significantly decreased stimulation val,ues for carcinomas and sarcomas. Only melanomas showed impaired PWM reactivity in his reports.

]. Bier, K. Bitter, U. Nicklisch

86

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Fig. 8 PHA-kinetics: 3H-thymidine-incorporation in 2 x 105 peripheral blood lymphoeytes of 6 different normal subjects after incubation with 1 al PHA measured by day 1-8. The highest mean stimulation values are reached on day 3.

Our patients with squamous cell carcinoma of the head and ne& have shown frequent depression of skin reactions to DNCB (de-nero sensitisation) (Table 2) and slight but not significant reactions to PPD (recall antigen) (Table 4). Many studies have included skin tests with one or more recall antigens, to examine delayed-type hypersensitivity reactions. In patients with carcinomas, impaired reactivity to these antigens has been mainly with advanced disease (Lamb et al. 1962, Solowey and Rapoport 1965, Hughes and Mackay 1965, Hettler and Amos 1965). Reports that patients with Hodgkin's disease have depressed delayed hypersensitivity to recall antigens have been made by Sehier et al. (1956) and Lamb et al. (1962). Findings in patients with acute leukemia demonstrated an increased reactivity to these skin tests (Hersh et al. 1971, Santos et al. 1973). Tests for delayed hypersensitivity which involve sensitization to an antigen to which the patient had not been previously exposed and then subsequently challenged appear to be more sensitive to immune suppression (Southam 1968). Levin

t

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in 2 x 10'~ peripheral blood lymphocytes of 6 different normal subjects after incubation with 10 FI PWM measured by day 1-8. The highest mean stimulation values are reached on day 6,

et al. (1964), using DNCB for primary sensitization, found that only 11% of lymphoma patients and 23o/o of carcinoma patients were reactive compared with 75 % of the controls. For different types of malignancies including squamous cell carcinoma, adenocarcinoma, malignant melanoma and sarcomas, Eilber and Morten (1970) reported 60 % with no reactivity after DNCB sensitization. In the control group, 95 °/o were positive. Similar results are reported by Catalona and Chretien (1973). DNCB reactivity was most abnormal in patients with squamous cell carcinoma (43o/0 anergic), less in patients with malignant melanomas (25 °/o anergic) and adenocarcinomas (26 0/0 anergic), and least in patients with sarcomas (13 °/o anergic). These experimental findings raise the question whether squamous cell carcinomas of the head and ne& and, possi`bly, other malignant diseases occur due to already existing genetic, organic or induced immune defects. If one assumes that tumour patients belong to a group with abnormal thymus-dependent immune reactivity, a reduced cellular immune status should ,be already demonstrable prior to the tumour formation. So far there are no controlled investigations supporting this hypothesis. There are, however, some indirect indications which make this assump-

Unspecific Cellular Immunity bcfore Therapy in Patients with Squamous PHA STIMULATION

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tion probable. Fraumeni (1969) and Doll and Kinlen (1970) detected a distinct connection between immune defect diseases - some with genetical cause, others were acquired - and malignant lympho-proliferative diseases. Gatti and Good (1971) reported in addition a 10,000fold increase of malignant lymphomas and leukemias in patients with reduced immune reactivity in comparison with healthy controls. Similar findings are mentioned in the papers of Penn and Starzl (1972) and Westburg and Stone (1973), who report a significantly increased formation of malignant diseases with frequent occurrence of tumours of epithelial origin, pertaining to immune-suppressed transplant carriers. Further indications for a predisposed immuneinsufficiency in tumour patients result on the one hand from studies proving a correlation between extensive alcohol abuse and the occurrence of

C.P.

Fig. 11 PWM-induced lymphocyte stimulation in normal persons (NP) and cancer patients (CP). Each point represents the mean value of four culture preparations.

malignant tumours of different histology (Hahulinen et al., 1974). From retrospective studies of Lyndy et al. (1974) and Hahulinen et al. (1974) it can be concluded that alcohol abuse mainly involves an increased risk pertaining to the formation of squamo.us cell carcinomas of the head and neck, pharynx, larynx, oesophagus and lungs. On the other hand for increased alcohol consumption a changed immune status has been detected. Tennenbaum et al. (1969) found a reduced cellular immune reactivity together with a thymus degeneration in rats, which had been fed for three months with an alcohol solution. Investigations on alcoholics demonstrated in addition a reduced stimulation of lymphocytes to PHA, and a decrease of circulating T-lymphocytes in the peripheral blood (Lyndy et al. 1975). As a further cause for a reduced cellular immune reactivity of the cancer patients examined it must be assumed that the tumours occurred despite a sufficient immune system, and that the immune status changed only in the course of the disease. In this context, arguments were presented against the immuno-surveillance-theory by Stutman (1974) based on experiments which did not show a varying tumour frequency between mice with

]. Bier, K. Bitter, U. NicMisch

88

ABSOLUTET-CELL LEVEL

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and without a thymus after feeding them with the carcinogen methyl-cholanthrene. For some patients a connection has been confirmed between tumour growth and the immune mechanism. With progressive tumour growth the cellular immune reactivity grows less (own unpublished observations). These findings agree with those of other authors (Catalona et al. 1973, Dellon et al. 1975, Wanebo et al. 1975). Conclusions

Concerning the evaluation of the immune status with nonspecific methods in patients with tumours of different sites and histology, there are a number of investigations of other authors who achieved results similar to those demonstrated in this paper. In all papers it is stated that a ,,normal" immune status does not exclude a malignant tumour and, vice versa, a reduced immune competence does not permit the conclu-

--1---

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sion that a malignant growth exists. The findings show a too distinct overlapping (Figs. 5-8, Tables 2 + 4). The method therefore is not suitable for use for preventive investigations. Possibly consequences might be drawn from our findings for therapeutic measures. As irradiation therapy has an immune-suppressive effect (McCredie et al., 1972), patients with reduced immune-reactivity should rather be treated surgically, while patients with normal immune parameters appear more suitable for radiotherapy or combined methods of treatment. As the immune system can also be influenced by cytostatics (Gabrielson and Good, 1967), the choice

Unspecific Cellular Immunity before Therapy in Patients with Squamous of drugs as well as their dosage ought to be seen in correlation with the immunological effect. There are, in addition, indications that accomp a n y i n g immunological investigations are of special importance with tumour-immuno-therapeutic measures. Immune therapy can lead either to a general (Gutterman et al. 1973) or to a tumourspecific (Zbar et al. 1976), immune stimulation linked to increased remission as well as with a longer survival chance. The continuous control of our patients should provide more evidence concerning the matter. T h e introduction of immunological investigations for our patients with squamous cell carcinomas of the head and neck is also aimed at o~btaining valid information as to the patients' prognosis. A relation between immune parameters and prognosis for some human malignant diseases as well as for different animal-models has been confirmed (Hersh et al. 1975). Tumour patients with normal immune status - compared with healthy controls - have a better chance of survival than patients with a disturbed immune competence (Hersh et at. 1976). Immunological and clinical case controls performed in our clinic should help to define the value of such prognostic statements. References

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89

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91

imnmnic system. Surg. Gynec. Obstet. 141 (1975) 212 MacLennan, 1. C. M., G. Loewi, A. Howard: A human serum immunoglobulin with specificity for certain homologous target cells which induces damage by normal human lymphocytes. Immunology 17 (1969) 897 MaeLennan, L C. M., G. Loeze~i, B. Harding: The role of immnnoglobulins in lymphocyte-mediated cell damage in vitro. I. Comparison of the effects of target cell specific antibody and normal serum factors on cellular damage by immune and nonimmune lymphocytes. Inmmnology 18 (1970) 397 M{ikela, 0., G. ]. V. Nossal: Bacterial adherance: a method for detecting antibody production by single cells. J. Immunol. 87 (1961) 447 Maha, G. E.: Comparative study of tuberculine tine and Mantoux test in 676 colleg students. J. Amer. reed. Ass. 182 (1962) 304 Many, A., M. Biniaminov, E. Rosenthal, E. Aghal, B. Pamot: The distribution of B and T lymphocytes in the peripheral blood of patients with Hodgkin's disease. Acta haematol. 56 (1976) t Marshall, A. H. E., R. G. White: Reactions of reticular tissues to antigens. Brit. J. exp. Path. 31 (1950) 157 MeCluskey, R. T., B. Benecerraf, ]. W. McCluskey: Studies on the specificity of the cellular infiltrate in delayed hypersensitivity reactions. J. Immunol. 90 (1963) 466 McCredie, ]. A., W. R. Inch, R. M. Sutherland: Effect of postoperative radiotherapy on peripheral blood lymphocytes in pati.ents with carcinoma of the breast. Cancer 29 (1972) 349 Mellstedt, H.: In vitro activation of hmnan T and B lymphocytes by pokeweed mitogen. Clin, exp. Immunol. 19 (1975) 75 Metchnikoff, E.: Immunity in infections Disease. Cambridge University Press, London 1905 Miller, ]. F. A. P.: hnmunological functions of the thymus. Lancet, 2 (1961) 748 Mitchell, M. D.: Central inhibition of cellular immunity to leukemia L 1210 by isoantibody. Cancer Res. 32 (1972) 825 Mitchison, N. A.: Passive transfer of transplantation immunity. Proc. roy. Soc. (London) B 142 (1954) 72 Mitehison, N. A.: Studies on the immunological response to foreign tumor transplants in the nmuse. I. The role of lymph node cells in conferring immunity by adoptive transfer. J. exp. Med. 102 (19:55) 157 Mueller, A. P., H. R. Wolfe, R. K. Meyer: Preeipitin production in chi&ens. XXI. Antibody production in bursectomised chi&ens and chi&ens injected with 19-nortestosterone on the fifth day of incubation. J. Immunol. 85 (1960) 172 Murphy, ]. B.: The lymphocyte in resistance to tissue grafting, malignant disease and tuberculous infection. Med. Res. Monograph, No. 21. Ro&efeller Inst. 1926. Nelson, H. S.: Delayed hypersensitivity in cancer patients: cutaneous and in vitro lymphocyte

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J. Bier et al.: Unspecific Cellular Immunity before Therapy in Patients with Squamous

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J. max.-fac. Surg. 6 (1978) 93 97

Experiences with 19'Hg Oh in the Diagnosis of Head and Neck Malignancies Carlo AVERSA, Luigino EIAMMINGHI, P. Umberto GENNARI, Aldo BERTANI

Clinic of Maxillo-Facial Surgery (Head: Prof. L. Rusconi, M.D.), Medical Faculty, University of Parma, Italy

Summary l'~VHg C12 (mercuric dichloride) was used in the diagnosis of 30 cases of epidermoid carcinoma of the head and the neck, and in 5 cases of benign tumours and non-neoplastic lesions. The results show that radioisotope scanning is not a certain method of diagnosis but that it affords supplementary information essential to correct therapy.

Key-Words: Carcinoma; Mercuric dichloride; Scan.

Introduction Scanning with direct imaging of neoplastic foci through the selective uptake of gamma rays emitted by radioisotopes is so safe, simple and repeatable as to be of obvious interest in the diagnosis of cancer. Extensively used in all regions of the body, especially for parenchymal tumours, radioisotopes have been little used in skin neoplasias because the lesions are readily accessible to direct inspection, palpation and 0301-0503/78

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biopsy. During the past ten years, however, attention has been directed to the identification of a tracer that would be taken up selectively by skin neoplastic tissue in such a way as to demarcate the neoplasia cIearly from the surrounding tissue. The following have been used: 14Co tyrosine (Grupper et al. 1954), 32p (Midana 1964), 125I quinoline (Beierwaltes et al. 1968), ssS (Thiers et al. 1970) and 57Co bleomycin (Robert et al. 1972) in skin lesions, though nearly always in malignant melanomas.

M o r e t e t al. (1974) scanned 7 patients with squanmus cell carcinoma and 2 with basal cell carcinoma with 57Co bleomycin, obtaining selective intralesional uptake of the tracer in 3 cases. The series was, however, too small to permit de-finite conclusions. Mercurial compounds afford far more meaningful information than previous tracers. It was Sodee (1964), Sodee and Clifton (1964) and W o l f and Fischer (1964) who proposed their use,

@ 1978 Georg Thieme Publishers