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Notes: Summary: This article reviews our immunotherapy research with natural killer (NK) cells in a syngeneic rat colorectal cancer liver and lung metastasis model. Using adoptive transfer of interleukin (IL)-2-activated NK cells, NK cells were shown to selectively infiltrate the tumors. More NK cells were found in tumors when the NK cells were directly injected into tumor-draining blood vessels than when the cells were injected in systemic blood vessels. Under optimal conditions, a limited, though significant, effect of adoptively transferred NK cells on tumor growth was shown. We observed that both endogenous and adoptively transferred NK cells were predominantly present in the stroma surrounding the tumor cell nodules. It is possible that they did not penetrate the nodules containing the tumor cells because of the presence of a basal membrane-like structure around these nodules. Adoptively transferred NK cells may initiate elimination of tumor cells by activating other effector cells, whereas some may eliminate tumor cells by direct cell–cell contact. A diverse array of molecules was shown to be involved in this process. CD45 on NK cells was found to be important in initiating the lysis-inhibitory signal upon binding of ‘self’ major histocompatibility complex (MHC) class I on potential target cells. Our results indicate that NK-cell cancer therapy is still promising and needs improvement.

Notes: Abstract Background: The morbidity of thyroid surgery is low. Despite this, some authors advocate a subtotal thyroidectomy instead of a total thyroidectomy, to avoid the higher morbidity associated with a total thyroidectomy. Methods: We retrospectively evaluated the complications of thyroid surgery in Leiden between January 1, 1982 and October 1, 1990. Three hundred forty-one patients—261 women and 80 men—had 356 operations; 15 patients were operated on twice; there were 152 total hemithyroidectomies, 3 subtotal hemithyroidectomies, 33 total thyroidectomies, 122 bilateral subtotal hemithyroidectomies, 12 combinations of total and subtotal hemithyroidectomies, and 34 other operations. Results: Calculated for the nerves at risk (n=489), the percentage of permanent recurrent nerve lesions was 3.1 (in the 5 most recent years it was 1.2%). There was no significant difference between total or subtotal (hemi)thyroidectomies. Initial symptomatic hypocalcemia necessitating supplementation was encountered 42 times (12.5%). The occurrence of permanent symptomatic hypocalcemia (6%) was not significantly different between total and subtotal (hemi)thyroidectomies (p=0.06). The duration of surgery was 137.8 min for bilateral subtotal thyroidectomies and 182.9 min for bilateral total thyroidectomies (p〈0.0001). There was no difference in blood loss between total and subtotal (hemi)thyroidectomies. Conclusions: Because total thyroidectomy carries a risk of complications similar to that for subtotal thyroidectomy, it is not logical to avoid total resections. If the number of total resections were increased, it is anticipated that fewer reoperations, which involve a relatively high morbidity rate, would have to be performed.

Notes: Abstract Although the availability and acceptance of fine-needle aspiration biopsy (FNAB) of thyroid nodules has increased, many physicians still use thyroid scintigraphy for distinguishing benign from malignant lesions. We evaluated these diagnostic tests in 350 patients who had thyroid surgery in our institution between 1977 and 1990. Histologic confirmation of FNAB was obtained in 265 patients. In the group of patients having surgery, 247 thyroid scintigraphies were performed. Our patients were divided into two groups (1977–1986 and 1986–1990). The first group comprised 173 patients with 173 FNABs and 126 scintigrams. The second group consisted of 177 patients having 92 FNABs and 121 scintigrams. Results of scintigrams were analyzed in the second group only. In 5 out of 120 cases where the FNAB result was “benign or probably benign” the lesion appeared to be malignant postoperatively. If the FNAB result was “malignant or probably malignant” (n=83) the pathology report confirmed a malignancy in 68 cases (81.9%). In 56 instances of all 265 FNABs the cytology report was not conclusive (“uncertain”); in 21.4% of these cases a malignancy was found postoperatively. An FNAB-result “(probably) malignant” had a positive predictive value of 0.819 while the negative predictive value of a result “(probably) benign” is 0.950. An “uncertain” result does not take away our concern so this result should have the same consequences as those of a result “(probably) malignant”. In that case, FNAB-sensitivity is 93.0% and specificity 66.1%. Eighty-five of the last 116 scintigraphies showed a solitary node. Eleven of these nodes were hot while 74 were cold. In 2 out of 11 hot nodules cancer was diagnosed (18.2%). Twenty-six of the cold nodules contained a malignancy (35%). Evaluation of 116 99mTc scintigrams yielded a positive predictive value of finding a malignancy in the thyroid if the scan showed a solitary cold nodule of 0.351. If a cold nodule is supposed to be an indicator of malignancy this test has a sensitivity of 72.2% and a specificity of 43.5%. The accuracy is as low as 52.1%. Our data indicate that the accuracy rate of nuclear imaging techniques in diagnosing thyroid nodules is low. There is no place for routine scintigraphy in preoperative diagnosis of a thyroid lesion. Although observer dependency exists with FNAB, the overall accuracy of this test is high, while this low-cost test is easy to perform and increasingly available. It enables cancer detection in a cost-effective manner. We recommend surgery for a clinically suspicious lesion and if the FNAB result is uncertain, probably malignant, or malignant. We advocate direct total thyroidectomy if the FNAB result is malignant, peroperative frozen section after hemithyroidectomy if the FNAB result was probably malignant or in a clinically suspicious lesion regardless of the FNAB result (unless malignant). A diagnostic hemithyroidectomy only, without frozen section, is performed if the FNAB result was uncertain.

Notes: Summary The role of 2-mercaptoethanol and indomethacin in the induction of lymphokine-activated killer (LAK) activity by interleukin-2 (IL-2) in rat splenocyte cultures was investigated. Spleens from 4-month-old male rats of five different strains were tested. Splenocytes were cultured for 3–5 days in the presence of IL-2 (1000 U/ml) and LAK activity was assessed by 4-h51Cr release assays with P815 and YAC-1 cells as targets. LAK activity could be induced by IL-2 in splenocytes from all rat strains, but only when 2-mercaptoethanol was present in the culture medium. Optimal LAK activity was induced when the 2-mercaptoethanol concentration in splenocyte cultures was at least 5 µM. Different rat strains showed differences in levels of in vitro induction of LAK activity. In the presence of 2-mercaptoethanol the level of LAK activity induced by IL-2 was high in BN and Lewis rats, intermediate in Wistar and Wag rats, and low in DZB rats. In the absence of 2-mercaptoethanol no or minimal LAK activity was induced. Furthermore we observed that addition of 50 µm indomethacin to the culture medium in the presence of 2-mercaptoethanol augmented the induction of LAK activity to some extent. In the absence of 2-mercaptoethanol, addition of indomethacin resulted only in low levels or no induction of LAK activity. We conclude that for optimal induction of LAK activity by IL-2 in rat splenocyte cultures 2-mercaptoethanol is essential, while indomethacin can only marginally further improve this induction.

Notes: Abstract. In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanol-sensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.

Notes: Abstract In vivo targeting of lymphokine-activated killer (LAK) cells to tumour deposits by bispecific monoclonal antibodies (bimAb) may be a way to improve adoptive immunotherapy. We developed a bimAb against adherent LAK (ALAK) cells and colon tumour CC531 in Wag rats. The bimAb was produced by somatic hybridization of two mouse hybridomas, one producing monoclonal antibodies (mAb) against CD8 (IgG2b, OX8), and the other producing mAb against a CC531-associated antigen (IgG1, CC52). A bimAb-producing clone was selected by an enzyme-linked immunosorbent assay with CC531 tumour cells. BimAb were purified from ascitic fluid by protein A affinity chromatography. Each of five pooled peak fractions was analysed by flow cytometry for the presence of bimAb. Most bimAb were found in a fraction that was eluted at pH 4.5 from protein A. FPLC analysis of this fraction revealed that no parental antibodies were present. The OX8 × CC52 bimAb greatly increased conjugate formation in vitro between ALAK cells and CC531. Results of51Cr-release assays with CC531 as target cells and ALAK cells as effector cells were not significantly different in the presence or in the absence of the bimAb. The methods we used here, a cell enzyme-linked immunosorbent assay and flow cytometry, are simple methods for development and purification of a bimAb when a functional selection method is not a priori available. The OX8 × CC52 bimAb we developed this way may increase in vivo tumour targeting of ALAK cells and thus augment antitumour effect in vivo.

Notes: Abstract In the present study we investigated the inhibition of interleukin-2(IL-2)-induced lymphokine-activated killer (LAK) activity in rat splenocyte cultures in relation to the presence of 2-mercaptoethanol and macrophages/monocytes. The presence of 2-mercaptoethanol is necessary for induction of LAK activity in rat splenocyte cultures. Removal of macrophages/monocytes from rat splenocytes by plastic or nylon-wool adherence, or iron ingestion resulted in LAK induction by IL-2 in the absence of 2-mercaptoethanol. The effect of macrophages/monocytes on LAK activity was also studied in transwell co-cultures. In the absence of 2-mercaptoethanol, the induction of LAK activity was very low in macrophage/monocyte-depleted splenocytes with macrophages/monocytes in the upper compartment of a transwell culture. In contrast, in the presence of 2-mercaptoethanol a high level of LAK activity was induced in these transwell cultures, showing that 2-mercaptoethanol abolished the LAK-inhibiting capacity of macrophages/monocytes. In addition, established LAK activity was strongly inhibited when, after LAK induction, splenocytes were cultured with supernatant of unfractionated splenocytes, which were cultured with IL-2 but in the absence of 2-mercaptoethanol. Addition of 2-mercaptoethanol abrogated the inhibiting effect of the supernatant completely. These experiments demonstrate that rat macrophages/monocytes produce 2-mercaptoethanolsensitive soluble LAK-inhibiting factors. Ultrafiltration of conditioned culture medium of macrophages/monocytes revealed the presence of LAK-inhibiting factors larger than 10 kDa. We concluded that 2-mercaptoethanol-sensitive soluble factors produced by macrophages/monocytes determine the level of LAK induction in rat splenocyte cultures.

Notes: Abstract The relative importance of prognostic factors in papillary and follicular thyroid cancer was studied in 113 patients using Cox's proportional hazards model. Prognostic factors studied were: histology, tumor grade, extrathyroidal growth, nodal involvement, distant métastases at diagnosis, nuclear DNA content, age at diagnosis, and sex. Nuclear DNA content was measured in primary tumors by flow cytometry. Total thyroidectomy and postoperative131I ablation was the standard treatment. The results showed that nuclear DNA content correlated significantly with histologic type and, in papillary cancer, also with tumor grade. The presence of distant metastases at diagnosis was, by far, the most important prognostic factor. In the patient group without distant metastases (n=91), multiploidy (i.e., presence of 2 or more aberrant stemlines) was the only significant prognostic factor for overall survival. With respect to diseasefree survival, multiploidy was second only to the age factor. In the patient group with distant métastases (n=22), all 6 patients with multiploid tumors died compared to 8 (50%) of 16 of those with other ploidy tumors. However, the small number in this group precluded significant results. The present study demonstrates that nuclear DNA content is a prognostic factor in those patients with papillary and follicular thyroid cancer without distant metastases at diagnosis.

Notes: Summary To enable the treatment of hepatic metastasis with higher, theoretically more effective, doses of systemically toxic anticancer drugs, an isolated liver perfusion (ILP) technique was developed in WAG/Ola rats. First, in a toxicity study the maximally tolerated dose (MTD) of mitomycin C (MMC) was determined for a 25-min ILP and for hepatic artery infusion (HAI) after the administration of a bolus dose. The MTD in the ILP setting (4.8 mg/kg) was 4 times that using HAI (1.2 mg/kg). Subsequently, in a rat colorectal hepatic-metastasis model, concentrations of MMC in tumour, liver, plasma and perfusate were measured during a 25-min ILP to investigate the expected pharmacokinetic advantage of ILP. The mean plasma level determined after ILP (1.2 as well as 4.8 mg/kg MMC) was significantly lower (P〈0.001) than that obtained following HAI. This may explain both the absence of severe systemic toxicity and the higher MTD in ILP-treated groups. No significant difference in mean tumour and liver tissue concentrations of MMC were found when the groups treated with 1.2 mg/kg drug via HAI vs ILP were compared. The mean MMC concentration in tumour tissue was significantly higher (almost 5 times;P〈0.05) in rats treated by ILP with the MTD (4.8 mg/kg) than in those treated via HAI with the MTD (1.2 mg/kg). ILP of MMC can be safely performed using a dose 4 times higher than the MTD in the HAI setting, leading to an almost 5-fold concentration of MMC in hepatic metastasis. ILP of MMC may therefore represent a promising therapy for metastasis confined to the liver.

Notes: Abstract Purpose: Glutathione (GSH) plays an important role in the resistance of tumors to cytostatics. Therefore, depletion of GSH by the GSH synthesis inhibitor buthionine sulfoximine (BSO) has been proposed to enhance the efficacy of certain anticancer agents. We studied the effect of BSO in rats bearing intrahepatically implanted tumors of the CC531 colorectal cancer cell line on the antitumor activity of melphalan (L-PAM). Since these liver tumors tend to derive most of their blood supply from the hepatic artery, we evaluated whether delivery of BSO into the hepatic artery would more selectively decrease GSH levels in the implanted tumor tissue as compared with normal liver and extrahepatic tissues. Methods: Tumor-bearing rats were treated with a 24-h continuous infusion of 0.375 mmol/kg BSO via the jugular vein, immediately followed by a bolus L-PAM (15 μmol/kg; 4.5 mg/kg) infusion via the hepatic artery. Laparotomy was performed on day 14 and 28 after treatment for measurement of the liver tumors. For the evaluation of locoregional administration of BSO, a 24-h continuous infusion of 0.375 mmol/kg BSO was delivered into either the hepatic artery, the portal vein, or the jugular vein in freely moving rats and GSH levels in the tumor, liver, kidney, lung, heart, bone marrow, and blood were measured. Results: BSO infusion via the jugular vein increased the antitumor efficacy of L-PAM injected into the hepatic artery 2-fold as determined at 14 days after treatment. Although infusion of BSO via the hepatic artery depleted GSH more severely in the tumor as compared with jugular vein or portal vein administration, the additional effect was only slight (10%). No difference was observed in any other tissue. Conclusion: GSH depletion increased the cytostatic efficacy of L-PAM 2-fold in vivo as determined at 14 days after treatment. Hepatic artery infusion of BSO translated into a statistically significant, but probably not therapeutically relevant, increase in tumor GSH depletion as compared with the other routes of BSO administration.