Willem Den Otter

Second primary tumors in patients with hereditary retinoblastoma: A register-based follow-up study, 1945-1994

The aim of this register‐based follow‐up study was to evaluate the long‐term cumulative incidence of second primary tumors (SPT) among survivors of hereditary retinoblastoma, with special interest for the incidence of pineoblastoma in retinoblastoma patients born after 1970. The Dutch Retinoblastoma Register was completed and updated: in the period 1945–1994, 639 retinoblastoma patients were registered. The vital status of each patient was obtained from the municipal registries and the Central Office of Genealogy. SPT were traced and histopathologically confirmed. Survival curves and cumulative incidence of SPT were calculated by the Kaplan‐Meier method. The survival of patients with hereditary retinoblastoma was significantly shorter than that of patients with non‐hereditary retinoblastoma. The cumulative incidence of SPT in hereditary patients was 3.7 and 17.7% at the ages of 10 and 35 years, respectively. Long‐term follow‐up revealed a high proportion of melanomas (7 melanomas out of 28 SPT). In the sub‐cohort of the hereditary‐retinoblastoma patient group born after 1970, the cumulative incidence of pineoblastomas at the age of 5 years was 9.3%. Our results suggest that patients with hereditary retinoblastoma should have careful follow‐up, and procedures for diagnosing SPT and pineoblastomas at an early and potentially treatable stage should be developed.

Interleukin-2 in cancer treatment : disappointing or (still) promising ? a review

The central question to discuss in this review is whether the results of interleukin-2 (IL-2) treatment are still disappointing or again promising. Although in the (recent) past application of high doses of systemically applied rIL-2 has led to some success, the overall results are not as one had hoped. Considering these poor results it seems clear that the application of high systemic doses rIL-2 was not a good choice. IL-2 has been used more or less as a chemotherapeutic compound in the highest tolerable dose. This has led to a great number of unwanted toxic side-effects. In addition, these doses mainly stimulated nonspecific lymphokine-activated killer activity through low-affinity IL-2 receptors, which does not lead to systemic immunity. On the other hand, several groups have shown that application of intratumoral low doses of IL-2 can be highly effective against cancer and without toxic side-effects. Significant tumor loads constituting up to 6% of the total body weight of a mouse were eradicated after treatment with low-dose rIL-2 given locally. Furthermore local treatment can lead to eradication of a tumor at a distant site. This type of therapy is effective in many systems namely against different tumor types in mice, hepatocellular carcinoma in guinea-pigs and vulval papilloma and carcinoma and ocular carcinoma in cattle. Low-dose IL-2 is very effective in experimental animals if it is given relatively late after inoculation of the tumor cells. In other words, it seems necessary that some sort of immune reaction has started or is developing before low doses of rIL-2 effectively stimulate it. In fact there is strong evidence that T lymphocytes, both CD4+ and CD8+ cells, are directly involved in the process leading to induction of specific immunity. In our opinion rIL-2 therapy should therefore aim at the stimulation of such (originally weak) specific immune reaction. Under these conditions also systemic immunity can be induced. In conclusion, application of rIL-2 as a modality for cancer treatment is still promising. High priority should be given to a further delineation of the mechanisms involved after local application. The method of giving IL-2 systemically in the highest tolerable dose should be abandoned. Specific stimulation of the immune system by low-dose rIL-2 is a much more promising option.

Intratumoral low-dose interleukin-2 induces rejection of distant solid tumour

SummaryThis study shows that local tumour treatment with low-dose recombinant interleukin-2 (IL-2) can mediate rejection of a large distant solid tumour. When SL2 lymphoma cells were injected intraperitoneally (i.p.) in syngeneic DBA/2 mice on day 0, 70% of these mice were cured by daily i. p. injections with 20 000 units IL-2 on days 10–14. After injecting mice with SL2 both i.p. and subcutaneously (s. c.) on the flank, 50% of the mice treated i.p. with low-dose IL-2 rejected both the i.p. tumour and the large distant s.c. tumour. In contrast, i.p. IL-2 treatment on days 10–14 cured fewer than 10% of the mice bearing only a s. c. SL2 tumour. The described IL-2 immunotherapy also caused systemic tumour rejection in mice bearing both ascitic and solid P815 mastocytoma. Thus it was shown that low-dose IL-2 can induce systemic tumour rejection, when injected at a site of tumour growth. Interleukin-2-induced rejection of s. c. SL2 tumour was highly specific, as mice that were rejecting i.p. and solid s. c. SL2 lymphoma did not reject solid P815 mastocytoma, which was injected s.c. simultaneously on the other flank. Furthermore, solid s.c. tumours consisting of mixtures of SL2 and P815 were not rejected in mice that rejected i.p. SL2 or P815. We conclude that intratumoral injections of low-dose IL-2 can enhance an ongoing weak immune reaction against the tumour resulting in systemic tumour rejection.

Mechanisms of macrophage cytotoxicity in IL-2 and IL-12 mediated tumour regression

IL-2 and IL-12 are promising anti-tumour agents. However, little attention has been paid to the role of macrophages during IL-2/IL-12 mediated tumour rejection. We studied the role of macrophages during IL-2/IL-12 mediated tumour rejection in DBA/2 mice bearing syngeneic SL2 lymphoma. Local treatment with IL-2 and IL-12 cured 85% of mice with severe metastasised tumour load. In vivo depletion studies showed that macrophages were required for the anti-tumour effect of IL-2 and IL-12. Macrophages could kill tumour cells both non-specifically and by antibody-dependent cellular cytotoxicity (ADCC). Treatment with IL-2, IL-12 or IL-2/IL-12 enhanced production of specific IgG1 immunoglobulins, while treatment with IL-12 and IL-2/IL-12 additionally induced IgG2a production. FcγRII and/or III were essential for ADCC expression after treatment with IL-2 and IL-12. These data show for the first time the essential role of macrophages during IL-2/IL-12 mediated tumour rejection and also suggest that IL-2 and IL-12 act via different mechanisms.

Induction of an antibody response in mice against human papillomavirus (HPV) type 16 after immunization with HPV recombinant Salmonella strains

Abstract Human papillomaviruses (HPV) are present in approximately 95% of all cervical carcinomas and the HPV E6 and E7 genes are continuously expressed in these lesions. There is also circumstantial evidence that often natural immunity against HPV is generated and that this is of influence on HPV-induced lesions. Stimulation of the immune system by proper presentation of relevant HPV antigens might, therefore, lead to a prophylactic or therapeutic immunological intervention for HPV-induced lesions. For this purpose we have expressed the E6 and E7 protein of HPV 16 in an attenuated strain of Salmonella typhimurium (SL3261, aroA mutation), which has been used extensively as a live vector. Live recombinant Salmonella vaccines have the ability to elicit humoral, secretory and cell-mediated immune responses, including cytotoxic T cells, against the heterologous antigens they express. This report describes the construction of recombinant Salmonella strains expressing the HPV 16 E6 and E7 proteins, and the induction of an HPV-16-specific immune response in mice after immunization with these live vectors.

Immune surveillance and natural resistance: an evaluation.

SummaryConcepts in tumour immunology are changing fundamentally.Around 1970 tumour immunology contained the following related concepts: 1. Thousands of tumour cells arise de novo each day. 2. Tumour cells are antigenic in their host. 3. All these antigenic tumour cells are killed by a strong immune surveillance system.A more likely set of concepts looks as follows: 1. Tumour cells do not arise frequently. 2. Tumour cells may be antigenic or not. 3. There is no need to postulate a very strong immune surveillance or natural resistance system.In this paper I am reviewing our present knowledge of immune surveillance and natural resistance. Only scanty information appears to be available. This information suggests that virally induced tumours are usually killed by cytotoxic T lymphocytes, and natural killer cells, whereas immune surveillance and natural resistance against other tumours may be quite weak.

Interleukin-2-containing liposomes: interaction of interleukin-2 with liposomal bilayers and preliminary studies on application in cancer vaccines.

Interleukin-2 (IL-2) incorporation in liposomes was studied under different conditions. Information was obtained on the mechanism of interaction of glycosylated recombinant IL-2 with liposomal bilayers. This information was utilized to formulate liposomes with high levels of incorporated IL-2. Multilamellar vesicles were prepared by hydration of a lipid film with an IL-2 solution. The incorporation efficiency, measured with a bioassay after forced release of IL-2 from the vesicles, was strongly dependent on the charge of the liposomes and the pH and ionic strength of the hydration medium. Negatively charged liposomes composed of phosphatidylcholine/ phosphatidylglycerol (9:1) and prepared with IL-2 dissolved in 10 mM NaAc/270 mM glycerol, 0.1% BSA, pH 5, showed the highest incorporation efficiency (81%) among the investigated preparations. This type of liposome was selected for further study. Electrostatics play a crucial role in the process of IL-2 association with this type of liposome. Initial studies concerning induction of protective tumor immunity by immunization with reconstituted membranes with mu-ramyl tripeptide phosphatidylethanolamine indicate that coinjection of IL-2-containing liposomes provided a significant enhancement of the immune response.

Therapeutic efficacy of IL-2-loaded hydrogels in a mouse tumor model.

Interleukin‐2 (IL‐2) is a highly effective anticancer drug if it is applied locally for 5 consecutive days. In most cases this requires 5 invasive treatments, which is not usually acceptable for either the patient or the clinician. For this reason we have developed dextran‐based hydrogels from which the required amount of encapsulated IL‐2 (1–4 × 106 IU of IL‐2) is gradually released during 5–10 days. Initially IL‐2‐containing macroscopic cylinder‐shaped gels (implants), and later IL‐2‐containing injectable microspheres, were developed. These preparations were characterized in vitro, and the therapeutic activity was tested in DBA/2 mice with SL2 lymphosarcoma. The therapy was given to mice with a large and extensively metastasized tumor load (at least 5% of the body weight). If 1–4 × 106 IU of IL‐2 was slowly released from the hydrogels over a period of 5–10 days, the therapeutic effects were very good and comparable to the effects of free IL‐2 injections for 5 consecutive days. In conclusion, dextran‐based hydrogels are promising systems for the controlled release of IL‐2.

Parental Age in Sporadic Hereditary Retinoblastoma

Of 104 children with sporadic hereditary retinoblastoma born between 1945 and 1970, we studied the age of their parents at the birth and compared this age with the mean age of parents at the birth of their children during the same period in The Netherlands. The mean age of fathers at the birth of their children with sporadic hereditary retinoblastoma (33.7 years) was significantly higher than the mean age of fathers at the birth of their children in the general population (32.5 years) (P less than .05, one sided). Similarly, the mean age of mothers at the birth of their children with sporadic hereditary retinoblastoma (31.2 years) was significantly higher than the mean age of mothers at the birth of their children in the general population (29.5 years) (P less than .05, one sided). We further analyzed this parental age factor by measuring the relative risk of age groups and comparing the incidence of sporadic hereditary retinoblastoma in the various parental age groups with the incidence of sporadic hereditary retinoblastoma in the total population. Mothers 35 years of age or older had a relative risk of 1.7 to have a child with sporadic hereditary retinoblastoma compared with mothers in the population in general (P = .006, one sided). Similarly, fathers 50 years of age or older had a relative risk of 5.0 to have a child with sporadic hereditary retinoblastoma compared with fathers in the population in general (P = .04, one sided). No parental age effect was found in children with nonhereditary retinoblastoma. We conclude that a high paternal and a high maternal age are significant risk factors for sporadic hereditary retinoblastoma.

Effective cancer therapy with a single injection of interleukin-2 at the site of the tumour

Abstract Mice with a severe metastasized tumour burden can be cured with a single local injection of interleukin-2. Such a treatment can also be effective against ocular squamous cell carcinoma in cows and transmissible venereal tumours in dogs. We did not notice any toxic effects of this treatment.