Knowledge transfer as a tool towards improvement of cancer care in low- and middle-income countries. 6th European Roundtable Meeting (ERTM), June 14th, 2019, Berlin, Germany.

PURPOSE: To identify key factors for the best practice of knowledge transfer from high-income settings to low- and middle-income settings. RESULTS: Interactive sessions led to the identification of European learnings that can and should be shared beyond Europe. Furthermore, methods were characterised which may lead to successful knowledge transfer with subsequent quality improvement. CONCLUSION: To ensure successful implementation of knowledge and new methods, political support is extremely important. A strong focus should be an improvement of collaboration and network development. Rehabilitation, early and late pallative care, cost effectiveness and long-term follow-up are priorities. Limitations are budget constraints which limit the execution of NCCPs.

Nucleolar-derived ribonucleic acid in chromosomes, nuclear sap, and cytoplasm of Chironomus tentans salivary gland cells.

The distribution of monodisperse high molecular weight RNA (38, 30, 28, 23, and 18S RNA) was studied in the salivary gland cells of Chironomus tentans. RNA labeled in vitro and in vivo with tritiated cytidine and uridine was isolated from microdissected nucleoli, chromosomes, nuclear sap, and cytoplasm and analyzed by electrophoresis on agarose-acrylamide composite gels. As shown earlier, the nucleoli contain labeled 38, 30, and 23S RNA. In the chromosomes, labeled 18S RNA was found in addition to the 30 and 23S RNA previously reported. The nuclear sap contains labeled 30 and 18S RNA, and the cytoplasm labeled 28 and 18S RNA. On the basis of the present and earlier analyses, it was concluded that the chromosomal monodisperse high molecular weight RNA fractions (a) show a genuine chromosomal localization and are not due to unspecific contamination, (b) are not artefacts caused by in vitro conditions, but are present also in vivo, and (c) are very likely related to nucleolar and cytoplasmic (pre)ribosomal RNA. The 30 and 23S RNA components are likely to be precursors to 28 and 18S ribosomal RNA. The order of appearance of the monodisperse high molecular weight RNA fractions in the nucleus is in turn and order: (a) nucleolus, (b) chromosomes, and (c) nuclear sap. Since both 23 and 18S RNA are present in the chromosomes, the conversion to 18S RNA may take place there. On the other hand, 30S RNA is only found in the nucleus while 28S RNA can only be detected in the cytoplasm, suggesting that this conversion takes place in connection with the exit of the molecule from the nucleus.

In situ demonstration of DNA hybridizing with chromosomal and nuclear sap RNA in Chironomus tentans.

Cytological hybridization combined with microdissection of Chironomus tentans salivary gland cells was used to locate DNA complementary to newly synthesized RNA from chromosomes and nuclear sap and from a single chromosomal puff, the Balbiani ring 2 (BR 2). Salivary glands were incubated with tritiated nucleosides. The labeled RNA was extracted from microdissected nuclei and hybridized to denatured squash preparations of salivary gland cells under conditions which primarily allow repeated sequences to interact. The bound RNA, resistant to ribonuclease treatment, was detected radioautographically. It was found that BR 2 RNA hybridizes specifically with the BR 2 region of chromosome IV. Nuclear sap RNA was fractionated into high and low molecular-weight RNA; the former hybridizes with the BR 2 region of chromosome IV, the latter in a diffuse distribution over the whole chromosome set. RNA from chromosome I hybridizes diffusely with all chromosomes. Nucleolar RNA hybridizes specifically with the nucleolar organizers, contained in chromosomes II and III. It is concluded that the BR 2 region of chromosome IV contains repeated DNA sequences and that nuclear sap contains BR 2 RNA.

Intracellular distribution of low molecular weight RNA in Chironomus tentans.

Low molecular weight RNA species are described in isolated nuclear components and cytoplasm of salivary gland cells of Chironomus tentans. In addition to 4S and 5S RNA and RNA in the 4-5S range previously described, at least three other components in the range below 16S are present. RNA, the molecular weight of which was estimated to 2.3 x 10(5) and designed 10S RNA, can be observed only in nucleoli; other RNA, the molecular weight of which was estimated to 1.3 x 10(5) and designed 8S RNA, was detected in the chromosomes, the nuclear sap, and the cytoplasm but not in the nucleoli; and a third type of RNA, the molecular weight of which was estimated to 8.5 x 10(4) and designed 7S RNA, was present in nucleoli, chromosomes, nuclear sap, and cytoplasm. The substituted benzimidazole, 5,6-dichloro-1 (beta-D-ribofuranosyl)benzimidazole (DRB), which gives a differential inhibition of the labeling of heterodisperse, mainly high molecular weight RNA in the chromosomes, does not inhibit the labeling of 8S RNA. The relative amounts of label in 8S RNA and 4-5S RNA (including 4S RNA and 5S RNA) in different isolated chromosomes, are distributed in proportion to the chromosomal DNA contents. The 8S RNA as well as the 7S RNA show a relative accumulation in chromosomes and nuclear sap with prolonged incubation time and are in this respect similar to intranuclear low molecular weight RNA species described by previous workers. Our data suggest, however, that these two types of RNA may differ in an important aspect from the previously described types since they are also present in the cytoplasm.

Long-term survival in malignant melanoma with special reference to age and sex as prognostic factors.

A total of 12,353 (97.5%) of all patients with a first malignant melanoma newly diagnosed in Sweden during the period 1960-82 were subjected to a complete computerized follow-up with respect to survival through December 31, 1982. Calculation of relative survival rates (RSs) revealed a consistently more favorable course in women than in men, the 5-year RSs being 80.8 and 68.0% and the 10-year RSs being 75.0 and 61.8%, respectively. Prolonged follow-up and analyses of annual excess mortality showed, in addition, that men surviving about 10 years constituted an apparently cured fraction, whereas among women there was an excess mortality throughout the observation period. The prognosis was increasingly more favorable at younger ages in males, whereas no regular age trend emerged in the female group of patients. A multivariate analysis indicated that the findings were not confounded by temporal trends in RSs or by differences in tumor location between the groups compared and also that the relative hazard was significantly higher for men than for women only during the first 8 years after diagnosis.

Trends in survival from malignant melanoma: remarkable improvement in 23 years.

Trends in incidence of and mortality and survival from malignant melanoma in Sweden for 1960 through 1982 were analyzed. Incidence rates increased annually by 5.4% for females and by 5.8% for males, whereas mortality rates increased annually by 2.7% for females and 3.3% for males. For females, the 5-year relative survival (RS) rates increased by approximately 15 percentage points before 1970. In contrast, males before 1970 had a successive improvement in RS rates of 4.6-8.2 percentage points for each 5-year period of diagnosis. Multivariate analyses revealed that during the study period the malignant melanoma-specific hazard decreased by 71% [95% confidence interval (CI) = 59%-79%] for females and by 64% (95% CI = 54%-73%) for males during the first 5 years of follow-up.

Screening of germline mutations in the CDKN2A and CDKN2B genes in Swedish families with hereditary cutaneous melanoma.

BACKGROUND: Approximately 10% of human cutaneous melanomas occur in families in which several members are affected. The familial predisposition to this disease is often associated with dysplastic nevus syndrome, a condition in which afflicted family members have multiple dysplastic nevi (atypical moles). The chromosome region 9p21 and markers on chromosomes 1p and 6p have been linked to melanoma susceptibility. The tumor suppressor genes CDKN2A and CDKN2B have been mapped to the 9p21 region, and genetic analyses have revealed the presence of germline CDKN2A alterations in melanoma families. The reported frequencies of such alterations, however, vary among these families. PURPOSE: The present investigation was carried out to determine the frequencies of CDKN2A and CDKN2B germline gene mutations among members in a population-based cohort of Swedish melanoma families (i.e., melanoma kindreds). METHODS: DNA was prepared from blood samples obtained from 181 individuals belonging to 100 melanoma kindreds. The polymerase chain reaction (PCR) technique, followed by single-strand conformation polymorphism (SSCP) and nucleotide sequence analyses, were used to identify the types and frequencies of mutations in exons 1, 1beta, 2, and 3 of the CDKN2A gene and in exons 1 and 2 of the CDKN2B gene. RESULTS: CDKN2A gene aberrations were independently identified by both SSCP and nucleotide-sequence analyses. Nucleotide-sequence analysis identified a single point mutation leading to a substitution of leucine for proline in codon 48 of exon 1 in a family with a history of melanoma and several other cancers. A second abnormality, leading to an insertion of an extra arginine residue at codon number 113 of exon 2, was seen in four separate families. The CDKN2A exon-3 coding region had the wild-type sequence in all samples. No germline mutations were found in the alternative exon 1beta of the CDKN2A gene or in exons 1 and 2 of the CDKN2B gene. CONCLUSIONS: The present investigation demonstrates that CDKN2A germline gene mutations were observed in 7.8% of the 64 Swedish melanoma kindreds that each included at least two first-degree relatives with melanoma and dysplastic nevus syndrome. No CDKN2A exon 1beta or CDKN2B mutations were identified. The critical genes responsible for the inheritance of a susceptibility to develop melanoma among family members in this population have yet to be identified.

Age-related decrease of ultraviolet light-induced DNA repair synthesis in human peripheral leukocytes.

The capacity for ultraviolet light-induced DNA repair synthesis, studied in peripheral leukocytes from 58 healthy subjects 13 to 94 years old, was found to vary greatly between individuals. A negative, statistically significant correlation was obtained between age and this synthesis, indicating a decrease in repair capacity with age. An age-related decrease in DNA repair may increase the susceptibility of cells to agents causing DNA damage, i.e. carcinogens and certain cytostatic drugs.

Different activities of unscheduled DNA synthesis in human melanoma and bone marrow cells.

Unscheduled DNA synthesis (UDS) indicated by melphalan was studied in freshly collected tumor cells from human melanoma metastases. Comparative studies were done on human bone marrow blast cells. Significant levels of UDS comparable with those in myeloblasts were found in only two of eight melanoma cell populations. This difference between melanoma and blast cells was not related to different cellular uptake of melphalan. When UDS was induced by ultraviolet irradiation, significant levels of UDS were found in all melanoma and blast cell populations studied. Also, in a human melanoma cell line, high levels of UDS were found after exposure to ultraviolet irradiation, while treatment with melphalan did not result in detectable levels of UDS. Possible explanations for the divergent results of UDS in melphalan-exposed melanoma cells are discussed.

Formation and removal of DNA cross-links induced by melphalan and nitrogen mustard in relation to drug-induced cytotoxicity in human melanoma cells.

The formation and removal of nitrogen mustard (HN2)- and melphalan-induced DNA cross-links (DNA interstrand and DNA-protein cross-links) in a human melanoma cell line (RPMI 8322), as determined by alkaline elution of DNA, was compared and related to the cytotoxic effect of each drug. HN2 was considerably more cytotoxic than melphalan as determined by inhibition of colony formation. Immediately following exposure to HN2 maximum levels of DNA cross-links were found. Melphalan, in contrast, caused a protracted induction of DNA cross-links with maximum levels obtained 6-12 h following drug exposure. HN2 induced approximately 13 times higher peak levels of DNA cross-links compared to equal concentrations of melphalan. Removal of DNA cross-links following exposure to both drugs followed an exponential time course. The rate of removal of HN2-induced DNA cross-links was, however, 1.5-2.4 times more rapid than that of melphalan-induced cross-links. A strong correlation was obtained between the cytotoxicity of both drugs and the total area under the curve for DNA interstrand cross-links, indicating that both the initial induction of as well as the rate of removal of DNA interstrand cross-links are important for the cytotoxic effects of bifunctional alkylating agents.

CDKN2A germ-line mutations in individuals with multiple cutaneous melanomas.

Germ-line CDKN2A mutations are present in some kindreds with hereditary cutaneous melanoma, and in Sweden a founder mutation with an extra arginine in codon 113 (113insR) has been identified. We screened 80 individuals with at least two primary cutaneous melanomas, who were identified mainly by a search of a regional cancer registry, for germ-line CDKN2A mutations. In nine patients, CDKN2A alterations that may contribute to melanoma predisposition were detected. In six individuals with a family history of melanoma, the 113insR founder mutation was present. One patient, who also had a family history of melanoma, had a 24-bp deletion that included codons 62-69. An in vitro binding assay established that the resulting mutant p16 protein was unable to bind cyclin-dependent kinase 4 and cyclin-dependent kinase 6. Two patients without a family history of melanoma had CDKN2A alterations: (a) one had a mutation in the 5' noncoding sequence (-14C/T); and (b) the other had an insertion of an extra T in codon 28, which results in a stop signal in codon 43. The median age at diagnosis of the first melanoma was significantly lower, the number of primary melanomas was significantly higher, and the presence of a family history of melanoma was significantly more common in patients with CDKN2A mutations than in those without germ-line mutations. The proportion of CDKN2A mutation carriers was significantly higher among patients treated for three or more primary melanomas compared with those with two tumors only. We conclude that mutation screening of individuals with multiple primary melanomas is a useful strategy to identify new melanoma kindreds with CDKN2A germ-line mutations.

Effect of D,L-buthionine-S,R-sulfoximine on cytotoxicity and DNA cross-linking induced by bifunctional DNA-reactive cytostatic drugs in human melanoma cells.

The effects of D,L-buthionine-S,R-sulfoximine (BSO) on cytotoxicity and DNA cross-linking induced by bifunctional DNA-reactive cytostatic agents in a human melanoma cell line (RPMI 8322) were investigated. RPMI 8322 cells were exposed to 0.01 mM BSO for 24 h, which resulted in a decrease in cellular glutathione to 14% without any reduction of cell proliferation or plating efficiency. BSO pretreatment significantly enhanced cytotoxicity of melphalan with a dose modification factor (DMF) of 3.4 and nitrogen mustard (HN2) (DMF 3.3). The increased cytotoxicity was paralleled by similar increases in DNA cross-linking (melphalan: DMF 2.2, HN2: DNF 2.5). A small but significant potentiation by BSO of cis-diamminedichloroplatinum(II) toxicity was seen (DMF 1.5), with a corresponding minor but significant increase in DNA cross-linking (DMF 1.1). Similarly, the potentiation of bis-chloroethylnitrosurea toxicity was small but significant (DMF 1.1), with no significant increase in DNA cross-linking (DMF 1.0). No effect of BSO pretreatment on the rate of removal of HN2-induced DNA cross-links was observed. Thus, the observed sensitization of RPMI 8322 cells to melphalan, HN2, cis-diamminedichloroplatinum(II), and bis-chloroethylnitrosourea was correlated to similar changes in drug-induced DNA cross-linking. Despite the increased cytotoxicity and DNA cross-linking BSO did not significantly increase the intracellular concentration of intact melphalan. These findings support the hypothesis that the potentiation of the cytotoxicity of bifunctional alkylating agents by BSO is due to an increased DNA cross-linking caused by a reduced intracellular conjugation of drug with glutathione, which results in an increased binding of drug to DNA targets.

Sensitization of human melanoma cells to the cytotoxic effect of melphalan by the glutathione transferase inhibitor ethacrynic acid.

Glutathione transferases are enzymes implied in the resistance of tumor cells to bifunctional alkylating cytostatic drugs. We have investigated the effect of the glutathione transferase inhibitor by ethacrynic acid on the cytotoxicity of melphalan to a human melanoma cell line (RPMI 8322) with a high level of glutathione transferase activity. Using 1-chloro-2,4-dinitrobenzene as substrate, ethacrynic acid was shown to inhibit the activity of purified human glutathione transferases, with 50% inhibition values of 1, 10, and 15 microM for transferase mu (class mu), transferase epsilon (class alpha) and transferase pi (class pi), respectively, all of which occur in RPMI 8322 cells. Ethacrynic acid at a concentration of 20 microM, which by itself was noncytotoxic, increased the cytotoxicity of melphalan to RPMI 8322 human melanoma cells approximately 2-fold. The induction of DNA interstrand cross-links by 40 microM melphalan was increased 1.4-fold by 30 microM ethacrynic acid. These results indicate that a potentiation of the cytotoxic effect of bifunctional alkylating agents can be achieved by inhibition of glutathione transferase and that the enhanced cytotoxicity may be caused at least in part by increased formation of drug-DNA adducts.

Contribution of glutathione transferase M3-3 to 1,3-bis(2-chloroethyl)-1-nitrosourea resistance in a human non-small cell lung cancer cell line.

The glutathione transferase (GST) isoenzyme profile was determined in two human tumor cell lines, U1690 derived from a small cell lung cancer and U1810 derived from a non-small cell lung cancer. U1810 cells are 3.2-fold more resistant to 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) than are U1690 cells, a finding ascribable in part to the expression of O6-alkylguanine-DNA alkyltransferase activity in the U1810 cells. GST P1-1 and GST A1-1 were determined quantitatively by enzyme-linked immunoassay and were found to be 1.3- and 15-fold higher in the cytosol fraction of U1690 cells as compared to U1810 cells, respectively. The higher BCNU resistance in U1810 cells can, therefore, not be correlated with the expression of these isoenzymes. However, sodium dodecyl sulfate/polyacrylamide gel electrophoresis in combination with immunoblot analysis demonstrated a class Mu GST, which was identified as GST M3-3 on the basis of electrophoretic mobility and cross-reaction with anti-rat GST 3-3 antibodies. This isoenzyme was detectable in U1810 cells but not in U1690 cells. Studies with purified human GST A1-1, GST M1-1, GST M3-3, and GST P1-1 demonstrated that GST M3-3, but not the other isoenzymes, catalyzed the denitrosation of BCNU. Such inactivation of BCNU has previously been demonstrated with rat class Mu GSTs (M. T. Smith et al., Cancer Res., 49: 2621-2625, 1989) but not with any human GST. These findings suggest that GST M3-3 contributes to BCNU resistance in the U1810 cells.

Excellent translational research in oncology: A journey towards novel and more effective anti-cancer therapies.

Comprehensive Cancer Centres (CCCs) serve as critical drivers for improving cancer survival. In Europe, we have developed an Excellence Designation System (EDS) consisting of criteria to assess "excellence" of CCCs in translational research (bench to bedside and back), with the expectation that many European CCCs will aspire to this status.

Serum S-100b protein as a prognostic marker in malignant cutaneous melanoma.

PURPOSE: To evaluate whether S-100B protein in serum is an independent prognostic marker in malignant melanoma. MATERIALS AND METHODS: S-100B protein in serum was analyzed in 1,007 consecutive patients with histologically verified cutaneous malignant melanoma. At the time of blood sampling, 876 patients were in clinical stage I, 35 were in stage II, and 96 were in stage III. The serum concentrations of S-100B protein were measured by a luminescence immunoassay (LIA). RESULTS: The mean serum concentration of S-100B protein was significantly related to clinical stage, with the lowest level in stage I and the highest in stage III. In a multivariate analysis, S-100B protein levels in serum showed the strongest prognostic impact of the factors analyzed with respect to disease-specific survival in clinical stages II to III, followed by clinical stage. Serum S-100B protein was not a significant independent prognostic factor in clinical stage I, where tumor thickness showed the strongest relation to melanoma-specific survival, followed by ulceration and satellites. CONCLUSION: This investigation contains the largest material of patients so far analyzed with the new LIA assay of S-100B protein in serum and confirms that S-100B protein in serum is correlated with clinical stage and is an independent prognostic marker in clinical stages II and III.

Regional hyperthermic perfusion with melphalan after surgery for recurrent malignant melanoma of the extremities. Swedish Melanoma Study Group.

A prospective randomized trial testing regional hyperthermic perfusion with melphalan has been conducted. Sixty-nine patients with recurrent malignant melanoma of the extremities were randomly allocated to surgery (36 patients) or surgery plus regional perfusion (33 patients). Prognostic variables concerning primary tumor as well as the recurrent disease were evenly distributed in the groups, excluding any bias in the randomization. Median tumor-free survival after randomization was 17 months in the perfusion group and 10 months in the control group. There were 15 locoregional recurrences in the perfusion group and 24 in the control group. The tumor-free survival curve was significantly (P = .044) better for the perfusion group than for the control group. Median survival time after randomization was 57 months in the perfusion group and 35 months in the control group. This difference was not significant. One patient died within 1 month after perfusion of pulmonary embolism. Regional hyperthermic perfusion after surgery of recurrent malignant melanoma should only be recommended in prospective and controlled trials, until its value has been proven in several randomized studies.

Adjuvant chemotherapy with doxorubicin in high-grade soft tissue sarcoma: a randomized trial of the Scandinavian Sarcoma Group.

From January 1981 to February 1986, a total of 240 patients with primary, malignancy-grade III or IV soft tissue sarcoma were entered into an adjuvant chemotherapy multicenter trial conducted by the Scandinavian Sarcoma Group (SSG). Of these patients, 181 were evaluable. The tumor was located in the extremities in 155 patients. After radical surgery (wide and compartmental) the patients were randomized to treatment with single-agent doxorubicin 60 mg/m2 administered as an intravenous (IV) bolus once a month for 9 months (group 1, n = 77) or to control (group 2, n = 77). If the surgical procedure was marginal, the patients initially received postoperative radiotherapy, followed by doxorubicin (group 3, n = 16) or control (group 4, n = 11). The control groups did not receive any adjuvant chemotherapy. Adjuvant therapy was initiated within 6 weeks of surgery (group 1) or within 10 weeks of surgery for patients receiving postoperative radiotherapy (group 3). With a median follow-up of 40 months, there was no significant difference between the four treatment groups in overall survival (group 1, 75%; group 2, 70%; group 3, 69%; group 4, 73%), disease-free survival (group 1, 62%; group 2, 56%; group 3, 62%; group 4, 64%), or local tumor control (group 1, 92%; group 2, 92%; group 3, 87%; group 4, 90%). The conclusions were the same whether the total group or evaluable patients only were included in the analysis. The local recurrence rate for patients undergoing radical surgery was 8% and for patients undergoing marginal surgery followed by radiotherapy was 12%. This study indicates that the use of single-agent doxorubicin as postoperative adjuvant chemotherapy has no significant clinical benefit in patients with high-grade soft tissue sarcoma.

Novel O6-methylguanine-DNA methyltransferase SNPs: a frequency comparison of patients with familial melanoma and healthy individuals in Sweden.

The DNA repair protein O6-methylguanine-DNA methyltransferase (MGMT) is involved in the cellular defense against alkylating agents. Genetic alterations in the MGMT gene may impair the protein's ability to remove alkyl groups from the O6-position of guanine, thereby raising the mutation rate and increasing the risk of cancer. We assessed polymorphisms in the promoter region and the 5 exons of the MGMT gene by PCR/SSCP and nucleotide sequence analysis of DNA extracted from blood samples. The population studied consisted of 89 melanoma patients, each belonging to a different family with a hereditary predisposition for melanoma, and 76 healthy individuals (blood donors). A total of 11 single nucleotide polymorphisms (SNPs) were detected, five in the promoter region, one in exon 1, two in exon 3 and three in exon 5. Six of the alterations were novel polymorphisms, of which five were located in the promoter region and one in exon 5. When the distribution of specific SNPs in cases and controls with only one variant was calculated; C575A was present only in melanoma patients (p=0.072). Moreover, while 20% of the healthy individuals had no SNPs this was the case in only 12.4% of the melanoma patients. However, no statistically significant differences were seen between cases and controls for any of the 11 SNPs.