Biomedical subjects
H B Hewitt
Publications and source records attributed to H B Hewitt.
A critical examination of the foundations of immunotherapy for cancer.
It is argued that immunotherapy (IMTH) for cancer, as well as the theory of cancer immunogenicity on which it rests, derives no secure foundation either from clinical observations or from experimental study of valid animal tumour models. Discouraging clinical observations include: the long history of failure of IMTH; the rejection of IMTH for choriocarcinoma--a true allograft in the patient; the extreme rarity of spontaneous regression, and progressive discrediting of the theory of immunosurveillance; the high frequency of nodal metastasis; and the failure to demonstrate a tumour-specific antigen in man. The great majority of animal tumours used for experimental studies of IMTH display artefactual immunogenicity associated with their mode of induction or conditions of transplantation and cannot be accepted as valid models of clinical cancer. The author reviews his total failure to demonstrate immunogenicity or successful IMTH using a wide range of animal tumours from which known laboratory artefacts have been strictly excluded. The inordinate promotion of tumour immunology and IMTH in recent years is attributed to unfortunate sociological influences encouraging premature assertion of clinical relevance from experimental research.
Nutrition and the cancer patient.
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The choice of animal tumors for experimental studies of cancer therapy.
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Stability of transplanted murine tumour systems after storage of cells at -196 degrees C for up to 13 years.
Two murine lymphomas of spontaneous origin were stored in liquid N2 as cell suspensions in DMSO for 8 and 13 years respectively. Isogenic transplantation assays done some years before freezing and immediately after thawing indicated no measurable loss of clonogenic cells during freezing, storage or thawing; the number of cells required for 50% successful transplantation remained close to unity in both cases. The overall data revealed no evidence of an alteration of the receptivity of the mouse colonies over a period of 13 or 20 years. We attribute this remarkable stability to close supervision of the systems within a single laboratory.
Failure of preoperative C. parvum vaccine to modify secondary disease following excision of two non-immunogenic murine carcinomas.
Sadler and Castro (1976) reported that a single dose of C. parvum vaccine given i.p. or i.v. to mice 4 days before excision of subcutaneous transplants of Lewis lung carcinoma significantly reduced the incidence of lung metastases in the operated mice. In similarly designed experiments, using 2 different carcinomas of spontaneous origin in our own inbred mouse colonies, we were unable to demonstrate any influence of C. parvum vaccine on the incidence or latent period of secondary disease in operated mice. We discuss possible reasons for our failure to reproduce the findings of Sadler and Castro.
Immunotherapy trials.
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Facilitation of nodal metastasis from a non-immunogenic murine carcinoma by previous whole-body irradiation of tumour recipients.
Of 193 CBA mice kept under prolonged observation after excision of small intradermal transplants of a non-immunogenic tumour (CBA Carcinoma NT), 27 (14%) presented with local recurrence, 19 (10%) with regional lymphnodal metastasis (RNM) and 72 (37%), with pulmonary metastasis +/- other systemic metastases. When mice were exposed to sublethal whole-body irradiation (WBI) before tumour transplantation, the incidence of RNM rose to approximately 80% and the latent period was reduced from approximately 60 days to approximately 40 days after tumour transplantation. This enhancement of RNM by WBI was undiminished when the interval between WBI and tumour transplantation was increased from 1 to 90 days. An explanation for this effect in terms of immunosuppression by the WBI is unlikely for the following reasons: the tumour was non-immunogenic by standard quantitative tests; the effect persisted long after the expected time for recovery of immune reactivity; and i.v. injection of normal marrow and lymphoid cells after WBI failed to reduce the effect. That the effect was systemic was proved by failure of local pre-irradiation of the tumour bed or regional node to enhance RNM. The effect was not observed when WBI was given 4 days after excision of tumours. These and other experiments failed to indicate the mechanism of the effect of WBI, but its long persistence suggests that it may relate to stored lethal radiation damage in migrating cells of slow turnover tissues.
Further studies of the relationship between lymphatic dissemination and lymphnodal metastasis in non-immunogenic murine tumours.
In all 6 different murine tumours of spontaneous origin, a high proportion (22-95%) of the regional lympgh nodes draining small intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. In separate experiments with 4 of these tumours, equivalent tumour-bearing mice had their tumours surgically excised and were observed for the development of regional nodal corresponding frequency of tumour formation by transplanted nodes. After high-dose radiotherapy of intradermal carcinomas, there was a progressive fall in the incidence of positive regional node transplants from 48 to 96 h after irradiation. It is concluded that continual lymphatic dissemination of viable cancer cells is characteristic of malignant tumours, but that there is a relatively small chance of such cells giving rise to nodal metastatic growth. Related studies showed that the ability of a small number of cancer cells to give rise to tumours was very much greater if they were incorporated in a lymph node at transplantation than if they were transplanted directly as a suspension.
A critique of the evidence for active host defence against cancer, based on personal studies of 27 murine tumours of spontaneous origin.
Extensive experience with isotransplants of 27 different tumours (leukaemias, sarcomata, carcinomata), all of strictly spontaneous origin in laboratory bred mice of low cancer strains CBA/Ht and WHT/Ht, has revealed no evidence of tumour immunogenicity. Of approximately 20,000 maintenance transplants, none failed and none regressed; of almost 10,000 carefully observed tumours arising from small or minimal inocula of tumour cells, none spontaneously regressed. The number of injected viable tumour cells required to give a 50% probability of successful transplantation (the TD50) ranged from approximately 1 cell to greater than 10,000 cells among the 27 tumours; high TD50 values, which were dramatically reduced by various procedures having no immunological significance, did not signify active "resistance" of the hosts. In the case of all of 7 randomly selected tumours, prior "immunization" of recipients with homologous lethally irradiated cells increased their tumour receptivity. Several experiments using various tumours failed to give evidence that immunity could be non-specifically induced or that a massive preponderance of lymphocytes from specifically sensitized mice could inhibit tumour transplantation or growth in vivo; no trace of "resistance" to tumour was adopted by isogeneic recipients of lymphocytes from regional nodes of tumour bearers. A limited review of the recent literature on tumour immunity shows that practically all the animal data presented in support of a general theory of tumour immunogenicity or to provide a basis for active clinical immunotherapy have been obtained from transplanted tumour systems which entail artefactual immunity associated with viral or chemical induction of the tumours or their allogeneic transplantation. It is suggested that isotransplants of spontaneously arising tumours are the only appropriate models of human cancer and that any genuine rapport between the animal laboratory and the clinic requires their exclusive use.
Quantitative studies of translymphnodal passage of tumour cells naturally disseminated from a non immunogenic murine squamous carcinoma.
A squamous cell carcinoma of spontaneous orgin in a WHT/Ht mouse was used to study the frequency with which the regional axillary lymph nodes draining subcutaneous or intradermal tumours gave rise to tumours after their isogeneic transplantation as whole nodes. This frequency (similar to 40%) was found not to vary significantly with the size or duration of the tumour drained and not to be increased by coincident infective, traumatic or antigenic stimuli acting at the tumour site or in adjacent tissue. Because tumour growth occurred in only 2/55 (4%) nodes which were left in situ in mice whose tumours were radically excised, it was concluded that tumour forming node transplants reflected a small and limited content (estimated to be about 13) of transnodally passing tumour cells destined to pass on to the blood; separate experiments showed that tumour cells reaching the blood survived for only a few hours. Nodes from tumour-excised mice gave rise to tumours as frequently when autografted as when isografted to mice with no previous expose to the tumour. A review of the finding reported here and of previous quantitative data for this system enabled us to exclude any implication of anti-tumour immunity from our interpretation of the results of the experiments.
Proceedings: Some personal experience of techniques for examining sensitizers.
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The influence of fibrin formation on the transplantability of murine tumour cells: implications for the mechanism of the Révész effect.
Experiments were undertaken to test a new hypothesis for the mechanism underlying the Révész effect. The hypothesis proposes that lethally irradiated (LI) tumour cells enhance the take probability of a small number of transplanted viable (V) tumour cells mixed with them by exerting a thromboplastic effect at the site of injection; local fibrin formation prevents emigration of V cells from the site or secures their survival there. The evidence presented to support this hypothesis is as follows: in the case of 3 isogeneically transplanted tumours, admixed particulate brain extract simulated the effect of LI cells in increasing the take probability of V cells; brain extract simulated the effect of LI cells in greatly delaying the disappearance of (125)IUdR-labelled viable carcinoma cells from the injection site; V cells acquired a raised take probability by their incorporation in fibrin clots; it was confirmed that admixed erythrocytes increased the take probability of V cells; using a newly devised microscopical test for detection of the thromboplastic activity of individual cells, it was found that cell death was almost always required for the display of such activity; lymphocytes and bone marrow cells, ineffective in enhancing the take of V cells, were almost totally devoid of thromboplastic activity. Possible explanations are given for failure of a fibrinogen depleting agent, ancrod (Arvin) to inhibit the Révész effect when administered to recipients. It is concluded that the evidence strongly supports the hypothesis presented whilst seriously weakening the long-standing theories that admixed LI cells act by provision of nutrients or by local quenching of postulated immune reactivity.
Studies of the mechanism of the Révész effect.
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The effect of lethally irradiated cells on the transplantability of murine tumours.
Fully quantitative isogeneic transplantation assays of viable (V) cells of a CBA carcinoma showed that the relationship between log inoculum and frequency of tumour "takes" accorded strictly with a Poisson distribution and indicated that 6900 cells were required for 50% takes (TD50). Addition of 10(5) lethally irradiated (LI) cells of the same tumour to the inocula reduced the TD50 to about 4 cells, yet the Poisson relationship was retained. From this and other data it is concluded that LI cells act by increasing the proportion of viable cells which contribute to tumour initiation; there was no evidence that LI cells affected the rate of proliferation of viable cells. The ability of non-homologous LI cells to reduce the TD50 was widely variable, but LI cells of one allografted tumour were almost as effective as homologous LI cells. Lethally irradiated cells did not assist the "take" of allografted viable tumour cells. Histological comparison revealed no difference of the tissue reaction to inocula of viable and LI cells, and it is questioned whether radiation induced lysis of these latter cells is required for their effect on viable cells. Evidence relating to a hypothesis that viable cells interact with one another as they do with lethally irradiated cells was conflicting.
The transplantation kinetics of tumour cells.
The data from a dilution assay can be used not only to form an estimate of the TD50 (or log TD50), but also to throw light on the transplantation kinetics of tumours. Transplantation "by single cells" is the simplest sort of kinetics, and some tumours, of which we have given two examples, will transplant by single cells. Other tumours consistently transplant with anomalous kinetics-i.e. non-single-cell. A sensitive statistical test for single-cell behaviour has been developed, and we give three examples of tumours that clearly fail the test. The mechanism by which these anomalous tumours depart from single-cell behaviour is unknown, but we suggest an approximate statistical analysis for their assay.
Rationalizing radiotherapy: some historical aspects of the endeavour.
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