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The article presents a study regarding the concomitant wild-type glioblastoma and isocitrate dehydrogenase (IDH)-mutant anaplastic astrocytoma in a patient with constitutional mismatch repair deficiency syndrome (CMMRD). It cites the case of a 12-year-old girl who was presented with a history of progressive headache and balance disturbances. It also mentions the different laboratory examinations undergone by the patient that lead to her diagnosis.

The pharmacokinetics of flavone acetic acid (FAA) after a dose of 4.8 mg/m2 given i.v. over 1 h was investigated in 13 patients with different solid tumors. The mean volume of distribution and clearance were 52 +/- 4 l/m2 and 2.6 +/- 0.2 l/h x m2, respectively. A tumor or metastasis biopsy was obtained from six patients 2 h after the end of infusion. Tumor FAA levels ranged from 39.6 to 148.8 micrograms/g and were similar to those obtained after a therapeutic i.v. dose of 200 mg/kg FAA in animals bearing Pan/03 tumor, which is very sensitive to the drug. Although FAA tumor concentration could be detected only during one interval and we therefore cannot draw a definitive conclusion, differences in the agent's antitumor activity in mice and patients (i.e. very active in the former and inactive in the latter) are apparently not due to discrepancies in drug distribution and pharmacokinetics. [ABSTRACT FROM AUTHOR]

Backgrounds: MUTYH-associated polyposis (MAP) is an autosomal recessive disease caused by biallelic pathogenic variants (PV) of the MUTYH gene. The aim of this study was to investigate the genetic causes of unexplained polyposis patients with monoallelic MUTYH PV. The analysis focused on 26 patients with suspected MAP, belonging to 23 families. Ten probands carried also one or more additional MUTYH variants of unknown significance., Methods: Based on variant type and on the collected clinical and molecular data, these variants were reinterpreted by applying the ACMG/AMP rules. Moreover, supplementary analyses were carried out to investigate the presence of other variants and copy number variations in the coding and promoter regions of MUTYH, as well as other polyposis genes (APC, NTHL1, POLE, POLD1, MSH3, RNF43, and MCM9)., Results: We reclassified 4 out of 10 MUTYH variants as pathogenic or likely pathogenic, thus supporting the diagnosis of MAP in only four cases. Two other patients belonging to the same family showed a previously undetected deletion of the APC gene promoter. No PVs were found in the other investigated genes. However, 6 out of the 18 remaining families are still interesting MAP candidates, due to the co-presence of a class 3 MUTYH variant that could be reinterpreted in the next future., Conclusion: Several efforts are necessary to fully elucidate the genetic etiology of suspected MAP patients, especially those with the most severe polyposis/tumor phenotype. Clinical data, tumor molecular profile, family history, and polyposis inheritance mode may guide variant interpretation and address supplementary studies., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Background: Inherited epimutations of Mismatch Repair (MMR) genes are responsible for Lynch Syndrome (LS) in a small, but well defined, subset of patients. Methylation of the MSH2 promoter consequent to the deletion of the upstream EPCAM gene is found in about 1%-3% of the LS patients and represents a classical secondary, constitutional and tissue-specific epimutation. Several different EPCAM deletions have been reported worldwide, for the most part representing private variants caused by an Alu-mediated recombination., Methods: 712 patients with suspected LS were tested for MMR mutation in our Institute. EPCAM deletions were detected by multiplex ligation-dependent probe amplification (MLPA) and then defined by Long-Range polymerase chain reaction (PCR)/Sanger sequencing. A comprehensive molecular characterization of colorectal cancer (CRC) tissues was carried out by immunohistochemistry of MMR proteins, Microsatellite Instability (MSI) assay, methylation specific MLPA and transcript analyses. In addition, somatic deletions and/or variants were investigated by MLPA and next generation sequencing (NGS)., Results: An EPCAM deletion was found in five unrelated probands in Italy: variants c.556-490_*8438del and c.858+1193_*5826del are novel; c.859-1430_*2033del and c.859-670_*530del were previously reported. All probands were affected by CRC at young age; tumors showed MSI and abnormal MSH2/MSH6 proteins expression. MSH2 promoter methylation, as well as aberrant in-frame or out-of-frame EPCAM/MSH2 fusion transcripts, were detected in CRCs and normal mucosae., Conclusion: An EPCAM deletion was the causative variant in about 2% of our institutional series of 224 LS patients, consistent with previously estimated frequencies. Early age and multiple CRCs was the main clinical feature of this subset of patients., (© 2019 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals, Inc.)

8-Oxoguanine, a common mutagenic DNA lesion, generates G:C>T:A transversions via mispairing with adenine during DNA replication. When operating normally, the MUTYH DNA glycosylase prevents 8-oxoguanine-related mutagenesis by excising the incorporated adenine. Biallelic MUTYH mutations impair this enzymatic function and are associated with colorectal cancer (CRC) in MUTYH-Associated Polyposis (MAP) syndrome. Here, we perform whole-exome sequencing that reveals a modest mutator phenotype in MAP CRCs compared to sporadic CRC stem cell lines or bulk tumours. The excess G:C>T:A transversion mutations in MAP CRCs exhibits a novel mutational signature, termed Signature 36, with a strong sequence dependence. The MUTYH mutational signature reflecting persistent 8-oxoG:A mismatches occurs frequently in the APC, KRAS, PIK3CA, FAT4, TP53, FAT1, AMER1, KDM6A, SMAD4 and SMAD2 genes that are associated with CRC. The occurrence of Signature 36 in other types of human cancer indicates that DNA 8-oxoguanine-related mutations might contribute to the development of cancer in other organs., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

To determine prevalence, spectrum and genotype-phenotype correlations of MUTYH variants in Italian patients with suspected MAP (MUTYH-associated polyposis), a retrospective analysis was conducted to identify patients who had undergone MUTYH genetic testing from September 2002 to February 2014. Results of genetic testing and patient clinical characteristics were collected (gender, number of polyps, age at polyp diagnosis, presence of colorectal cancer (CRC) and/or other cancers, family data). The presence of large rearrangements of the MUTYH gene was evaluated by Multiplex Ligation-dependent Probe Amplification analysis. In all, 299 patients with colorectal neoplasia were evaluated: 61.2% were males, the median age at polyps or cancer diagnosis was 50 years (16-80 years), 65.2% had <100 polyps and 51.8% had CRC. A total of 36 different MUTYH variants were identified: 13 (36.1%) were classified as pathogenetic, whereas 23 (63.9%) were variants of unknown significance (VUS). Two pathogenetic variants were observed in 78 patients (26.1%). A large homozygous deletion of exon 15 was found in one patient (<1.0%). MAP patients were younger than those with negative MUTYH testing at polyps diagnosis (P<0.0001) and at first cancer diagnosis (P=0.007). MAP patients carrying the p.Glu480del variant presented with a younger age at polyp diagnosis as compared to patients carrying p.Gly396Asp and p.Tyr179Cys variants. A high heterogeneity of MUTYH variants and a high rate of VUS were identified in a cohort of Italian patients with suspected MAP. Genotype-phenotype analysis suggests that the p.Glu480del variant is associated with a severe phenotype.

Lynch syndrome (LS) is an inherited predisposition cancer syndrome, typically caused by germline mutations in the mismatch repair genes MLH1, MSH2, MSH6 and PMS2. In the last years, a role for epimutations of the same genes has also been reported. MLH1 promoter methylation is a well known mechanism of somatic inactivation in tumors, and more recently, several cases of constitutional methylation have been identified. In four subjects affected by multiple tumors and belonging to a suspected LS family, we detected a novel secondary MLH1 gene epimutation. The methylation of MLH1 promoter was always linked in cis with a 997 bp-deletion (c.-168&#95;c.116+713del), that removed exon 1 and partially involved the promoter of the same gene. Differently from cases with constitutional primary MLH1 inactivation, this secondary methylation was allele-specific and CpGs of the residual promoter region were totally methylated, leading to complete allele silencing. In the colon tumor of the proband, MLH1 and PMS2 expression was completely lost as a consequence of a pathogenic somatic point mutation (MLH1 c.199G>A, p.Gly67Arg) that also abrogated local methylation by destroying a CpG site. The evidences obtained highlight how MLH1 mutations and epimutations can reciprocally influence each other and suggest that an altered structure of the MLH1 locus results in epigenetic alteration., (© The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

MUTYH variants are differently distributed in geographical areas of the world. In MUTYH-associated polyposis (MAP) patients from North-Eastern Italy, c.933+3A>C (IVS10+3A>C), a transversion causing an aberrant splicing process, accounts for nearly 1/5 of all mutations. The aim of this study was to verify whether its high frequency in North-Eastern Italy is due to a founder effect and to clarify its impact on MUTYH transcripts and protein. Haplotype analysis and age estimate performed on members of eleven Italian MAP families and cancer-free controls provided evidence that c.933+3A>C is a founder mutation originated about 83 generations ago. In addition, the Italian haplotype associated with the c.933+3A>C was also found in German families segregating the same mutation, indicating it had a common origin in Western Europe. Altogether c.933+3A>C and the two common Caucasian mutations p.Tyr179Cys and p.Gly396Asp represent about 60% of MUTYH alterations in MAP patients from North-Eastern Italy, suggesting the opportunity to perform targeted molecular screening for these variants in the diagnostic setting. Expression analyses performed on lymphoblastoid cell lines supported the notion that MUTYH c.933+3A>C alters splicing causing the synthesis of a non functional protein. However, some primary transcripts escape aberrant splicing, producing traces of full-length transcript and wild-type protein in a homozygote; this is in agreement with clinical findings that suggest a relatively mild phenotypic effect for this mutation. Overall, these data, that demonstrate a founder effect and further elucidate the splicing alterations caused by the MUTYH c.933+3A>C mutation, have important implications for genetic counseling and molecular diagnosis of MAP., (Copyright © 2012 UICC.)

Background: Duodenal cancer and ampullary cancer are major causes of death after a prophylactic colectomy in patients with familial adenomatous polyposis (FAP). Forward-viewing endoscopy and side-viewing endoscopy are recommended in patients with FAP for surveillance of periampullary and duodenal polyposis. The study of polyps distal to the duodenum in FAP is limited. A capsule endoscopy (CE) allows visualization of the mucosa of the entire small bowel., Objective: The objective was to detect whether CE has clinical value or any utility for the surveillance of small-bowel polyps in patients with FAP and to evaluate whether there are genotypic factors that predict which patients are at a lower risk of small-bowel polyps., Setting: Two Italian tertiary-referral centers., Patients: Twenty-three patients with FAP who presented for a CE., Main Outcome Measurements: Patients with FAP were examined by CE to assess the location, size, and number of small-bowel polyps. Patient age at CE, sex, years of observation after surgery, type of surgery, duodenal adenomas, and colorectal cancer at surgery were analyzed. All patients were selected for mutation analysis, and the germline adenomatous polyposis coli (APC) gene mutation was detected., Results: Eleven of 23 patients with FAP had duodenal polyps. During CE, jejunal-ileal polyps were detected in 7 of 23 FAPs, with a total number of 15 polyps in the ileum. The presence of duodenal adenomas was the only clinical feature predictive of small-bowel polyps. Identification of the ampulla of Vater was not achieved with CE; duodenal polyps were only seen in 4 of 11 patients identified endoscopically, with an underestimation of polyp numbers. APC mutations between codons 499 and 805 were associated with the absence of small-bowel polyps., Conclusions: CE is useful and safe for the surveillance of jejunal-ileal polyps in selected patients with FAP. CE is not useful in the surveillance of the duodenum where the majority of small-bowel cancers occur.

In an investigation devoted to the search for plasma markers for colorectal cancer (CRC), carried out by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry, a series of overexpressed peptides were identified in the plasma of patients. Among them the peptide with molecular weight 903 Da was the most abundant one, with a mean +/- (SD) relative abundance of 37 +/- 17% and a frequency over 60%. Interestingly, also in plasma samples of ten subjects affected by familial adenomatous polyposis (FAP), the peptide with molecular weight 903 was overexpressed. In this investigation, MALDI/MS/MS experiments were carried out on the ion at m/z 904 detected in the MALDI mass spectra of CRC and FAP patients. The data analysis by SwissProt.2007.01.09 indicates that this peptide is due to the sequence RPPGFSPF, found in the kininogen-1 precursor, which is an alpha-2-thiol proteinase inhibitor. In the case of subjects affected by a particular FAP syndrome, the MALDI/MS/MS spectra were quite different from those obtained from CRC and FAP patients. In fact, two sequences have been evidenced: RPPGFSPF belonging to kininogen-1 precursor, and PRKSSSSR belonging to Forkhead box protein 01A.

Purpose: We report the case of two siblings, clinically andendoscopically followed for 12 years, who displayed anattenuated adenomatous polyposis coli phenotype., Methods: On workup for rectal bleeding with colonoscopy, we found multiple adenomas mainly right-sided in a 21-year-old female and the same colonic phenotype was observed in her 27-year-old brother. We made a clinical diagnosis of attenuated adenomatous polyposis coli and performed APC gene testing. Because they had refused the proposed ileorectal anastomosis surgical option, we planned a periodic, endoscopic follow-up., Results: Gene testing did not confirm the clinical suspicion of attenuated adenomatous polyposis coli. Actually, we did not find anypathogenic mutation in APC gene and we recently identified a biallelic Y125C MYH defect. During the endoscopic follow-up, a progressive reduction of adenomas was seen., Conclusions: New insight colorectal cancer genetics have allowed definition of a new class of polyposis that applies to some patients with attenuated adenomatous polyposis coli phenotype as in the siblings we have described. To prevent colorectal cancer without recurring to surgery, colonoscopic polypectomy may be a suitable tool in controlling MYH polyposis.

IL-6-mediated B-cell growth promotion is involved in the pathogenesis of EBV+ lymphoproliferative disorders of immunosuppressed patients. Since retinoic acid (RA) inhibits the proliferation of EBV-immortalized lymphoblastoid B-cell lines (LCLs), we have investigated the effects of RA on IL-6 signaling in these cells. RA down-regulated IL-6-receptor components with IL-6 agonist activity (membrane and soluble gp80) and increased the levels of soluble gp130, an IL-6 antagonist. These changes, however, were not related to the enhanced production of endogenous IL-6 induced by RA in LCLs. RA-induced modulation of IL-6 receptor components did not abolish IL-6-mediated phosphorylation of gp130, whereas JAK1 and STAT3 phosphorylation and activation induced by IL-6 were markedly inhibited. Overall, the effects of RA resulted in the induction of a complete resistance of LCLs to IL-6-mediated growth promotion. Conversely, RA did not inhibit the constitutive activation of JAK1, TYK2, STAT3 and ERK1/2, ruling out that the JAK/STAT and MAPK pathways may mediate the antiproliferative activity of RA. The finding that RA severely impairs IL-6-dependent signalings in LCLs and inhibits their growth despite the presence of constitutively active JAK/STAT and MAPK cascades provide additional support for a role of RA in the prevention and treatment of EBV-related lymphoproliferative disorders of immunosuppressed patients.

Human Epstein-Barr virus-immortalized lymphoblastoid B-cell lines tested positive by PCR for simian virus 40 (SV40) DNA (22 of 42 cell lines, 52.3%). B lymphocytes or tissues from which B-cell lines derived were also SV40 positive. In situ hybridization showed that SV40 DNA was present in the nucleus of a small fraction (1/250) of cells. SV40 T-antigen mRNA was detected by reverse transcription-PCR. Lymphoblastoid B-cell lines (n = 4) infected with SV40 remained SV40 positive for 4 to 6 months. SV40-positive B-cell lines were more tumorigenic in SCID mice than were SV40-negative cell lines (4 of 5 [80%] SV40-positive cell lines versus 2 of 4 [50%] SV40-negative cell lines). These results suggest that SV40 may play a role in the early phases of human lymphomagenesis.

In this study we examined a series of 52 patients belonging to hereditary nonpolyposis colorectal cancer (HNPCC) or HNPCC-related families, all who had previously tested negative for mismatch repair (MMR) gene point mutations. Southern blot mutational screening of MLH1 and MSH2 genes was carried out with the aim of detecting large genomic rearrangements and of identifying the molecular mechanisms underlying the inactivation of the MMR genes. Three patients had abnormal restriction patterns and were found to carry distinct MLH1 internal deletions. Long-range PCRs identified the loss of DNA tracts spanning exon 6 (about 2.4 kb in proband A-AV20 and 0.8 kb in proband A-PD5) and exon 3 (about 2.5 kb in proband R-RM2). In A-AV20 the breakpoints occurred into identical 33-bp regions in introns 5 and 6 and a mechanism of classical Alu-mediated homologous recombination was evident. Also, in patient A-PD5 the breakpoints were located in these introns, but without direct involvement of repetitive sequences. In patient R-RM2 the breakpoints were located within repetitive L1 elements with poor homology in intron 2 and 3 and the rearranged allele was characterized by a complex insertion deletion (delCCinsACATAGTA), giving rise to a palindromic CTTAACATAGTATGTTAAG sequence in proximity of the fusion site. This study confirms that genomic rearrangements are an important component of the spectrum of MMR mutations. Although Alu repeats are likely to be implicated in the majority of cases, different molecular mechanisms may also be responsible for the observed MLH1 intragenic deletions. In particular, HNPCC resulting from L1-mediated recombination has been identified as a novel mechanism for MMR inactivating mutation., (Copyright 2002 Wiley-Liss, Inc.)

Glucocorticoids are able to release Epstein-Barr virus-immortalized (EBV-immortalized) lymphoblastoid B cell lines (LCLs) from the persistent growth arrest induced in these cells by retinoic acid (RA). Moreover, physiologic concentrations of glucocorticoids efficiently antagonized LCL growth inhibition induced by 13-cis-RA; 9-cis-RA; all-trans-RA; and Ro 40-6055, an RA alpha receptor (RAR alpha) selective agonist. RAR alpha expression levels, however, were not affected by glucocorticoids. Glucocorticoids, but not other steroid hormones, directly promote LCL proliferation, a phenomenon that was mainly mediated by down-regulation of the cyclin-dependent kinase (CDK) inhibitor p27(Kip-1). Moreover, glucocorticoids contrasted the up-regulation of p27(Kip-1), which was underlying the RA-induced LCL growth arrest, thereby indicating that glucocorticoids and RA signalings probably converge on p27(Kip-1). Both antagonism of RA-mediated growth inhibition and promotion of LCL proliferation were efficiently reversed by the glucocorticoid receptor (GR) antagonist RU486, indicating that all of these effects were mediated by GR. Of note, RU486 also proved to be effective in vivo and, in mice, was able to significantly inhibit the growth of untreated LCLs as well as LCLs growth-arrested by RA in vitro. These findings provide a rational background to further evaluate the possible role of glucocorticoids in the pathogenesis of EBV-related lymphoproliferations of immunosuppressed patients. Moreover, GR antagonists deserve further consideration for their possible efficacy in the management of these disorders, and the use of schedules, including both RA and a GR antagonist, may allow a more thorough evaluation of the therapeutic potential of RA in this setting. (Blood. 2000;96:711-718)

We have previously demonstrated that 13-cis-retinoic acid (RA), 9-cis-RA and all-trans-RA (ATRA) powerfully inhibit the proliferation of Epstein-Barr virus-immortalized B-lymphoblastoid cell lines (LCLs). The aim of the present study was to assess whether these compounds are effective at inhibiting the growth of B cells at more advanced stages of lymphomagenesis, including fully transformed B lymphocytes. To this end, c-myc-transfected LCLs (myc-LCLs) and Burkitt's lymphoma (BL) cell lines were used. We report that 13-cis-RA, 9-cis-RA and ATRA also markedly inhibit the proliferation of myc-LCLs by inducing G(0)/G(1) growth arrest as well as enhancing rates of apoptosis. Conversely, all but 1 (DG75) of the 8 BL cell lines investigated were poorly RA-responsive. Moreover, unlike LCLs and myc-LCLs, RA-treated DG75 cells rapidly resumed proliferation upon drug removal. Analysis of cell cycle-regulatory proteins showed that, as in LCLs, strong up-regulation of p27(Kip-1) and increased levels of under-phosphorylated pRb and p130 were detected in RA-treated DG75 cells. While the catalytic activity of all 3 G(1)-associated CDKs (CDK2, CDK4 and CDK6) was strongly inhibited in RA-treated LCLs, only CDK2-associated kinase activity was reduced in DG75 cells arrested in G(0)/G(1) by RA. Moreover, RA-treated DG75 cells failed to show the down-regulation of cyclin D3 observed in LCLs. Use of receptor-selective agonists and antagonists showed that in LCLs and RA-responsive BL cells, RA-induced growth arrest is mainly mediated by RARalpha. The RARalpha-selective agonist Ro 40-6055 was also effective at very low concentrations (10(-10) M). Nevertheless, comparable levels of RARalpha mRNA were found in RA-responsive and -resistant BL cell lines, indicating that mechanisms different from transcriptional deregulation of RARalpha probably underlie the differential responsiveness of BL cells., (Copyright 2000 Wiley-Liss, Inc.)

Epstein-Barr virus (EBV) isolates show a wide genomic heterogeneity, and a key issue is whether distinct strain variations may contribute to the development and/or malignancy of EBV-related disorders. Herein, we report on the virologic and biologic characterization of an EBV strain derived from a cyto-histologically aggressive EBV-related Hodgkin's disease (HD) (case HD-3) showing a high number of "anaplastic" Reed-Sternberg cells expressing markedly high levels of CD30, CD40 and LMP-1. The HD-3-derived EBV showed strong in vitro immortalizing properties, as suggested by the unusually high number of spontaneous lymphoblastoid cell lines (LCLs) obtained from the patient. Immunofluorescence and immuno-cytochemical analyses showed that HD-3 LCLs expressed significantly higher levels of CD23, CD30, CD38, CD39, CD40 and CD71 antigens and CD54 and CD58 adhesion molecules than B95.8 LCLs. In contrast, the expression of CD11a, CD24, CD95, bcl-2, LMP-1 and EBNA-2 was similar in both groups of LCLs. These phenotypic changes are consistent with the induction of a pronounced activation status and are not dependent on the cellular background, having been closely reproduced by the same virus in LCLs from an unrelated donor (DEN-HD-3 LCLs). HD-3 LCLs were able to grow in vitro at low serum concentrations (up to 0.1%) and were significantly more clonogenic in soft agarose than B95.8 LCLs. Moreover, although no evidence of tumor formation was observed in nude mice injected with B95.8 LCLs, all 5 spontaneous LCLs of patient HD-3 and the 2 DEN-HD-3 LCLs grew in transplanted animals as lymphoproliferations composed of EBER+, LMP-1+ cells. Our findings indicate that the biologic properties of the HD-3 EBV strain are significantly different from those of the B95.8 virus and may have contributed to the cytologic and histo-pathologic malignancy of this HD case. Moreover, molecular characterization of the HD-3 EBV genome identified a 63-bp deletion within the 3' end of the LMP-1 gene as a likely significant change that may be responsible, at least in part, for the biologically relevant phenotypic modifications and enhanced in vitro and in vivo growth potential induced in B lymphocytes by this virus strain.

Serum interleukin-6 (IL-6) and soluble intercellular adhesion molecule-1 (sICAM-1) levels were measured by enzyme-linked immunosorbent assay in 62 renal cancer patients: 30 were tumor-free after radical nephrectomy and 32 presented with metastatic disease. Serum IL-6 was undetectable in all but one of the tumor-free patients, whereas 41% (13 of 32) of the metastatic patients presented serum IL-6 levels. Furthermore, there was a significant correlation between serum IL-6 levels and a shorter overall survival (p = 0.009). Moreover, serum sICAM-1 levels were significantly higher (p = 0.05) in the metastatic patients with detectable serum IL-6 than in those without IL-6, suggesting a possible link between IL-6 and sICAM-1. The probability of a shorter overall survival was greater in the metastatic patients with both serum IL-6 and elevated sICAM-1 levels (>635 ng/ml), than in those with elevated sICAM-1 but without IL-6 (p = 0.01). The production of IL-6 by 16 freshly dissociated renal cancer cells cultured in vitro was also observed. It appeared that IL-6 levels did not correlate with the expression and release of ICAM-1 by cultured cells, although the highest values of ICAM-1 release were found in cultures synthesizing the highest values of IL-6. In conclusion, in vivo presence of serum IL-6 and elevated sICAM-1 levels is related to an unfavorable prognosis; it can be speculated that the cells capable of releasing high levels of sICAM-1 and IL-6 may negatively influence the antitumor response.

We examined ICAM-1 expression in 37 freshly dissociated renal-cancer cell populations. Immunoperoxidase analysis revealed that 31 of the 37 renal tumors expressed ICAM-1 to various degrees; ICAM-1 expression was significantly lower in tumor cells obtained from patients remaining tumor-free after a median follow-up of 60 months (mean value 24.4% +/- 21) than in tumor cells obtained from the relapsed patients (mean value 40.8% +/- 22), and the low expression of this molecule on the cell surface seemed to correlate with favorable clinical behavior. In 41 patients, the mean level of sICAM-I was 551 +/- 260 ng/ml, significantly higher than normal. However, sICAM-1 levels were significantly lower in the 20 tumor-free (mean 467 +/- 158 ng/ml) than in the 21 metastatic patients (mean 631 +/- 318 ng/ml). Eleven renal-cancer cell populations were cultured in order to examine the expression and release of ICAM-1. All of these cells were positive for ICAM-1 expression, which was elevated in 6 cases (> 50%) and low in the remaining 5 cases (18-35%). However, only the 5 cell populations expressing low levels of ICAM-1 released this molecule, showing an inverse correlation with cellular expression. Five of the cell populations were treated for 48 hr with rIFN-gamma, in these cells, both ICAM-1 expression and sICAM-1 levels increased, although sICAM-1 levels in the supernatants of the cell populations with constitutive high ICAM-1 expression remained very low.

Monocyte-mediated cytotoxicity (determined in a 72-h111In release assay) and the circulating levels of tumor necrosis factor alpha (TNF-alpha), interleukin (IL) 1beta, IL-6, IFN-gamma, C-reactive protein, and beta2-microglobulin were determined in 14 melanoma patients treated with multilamellar vesicle liposomes containing muramyl tripeptide phosphatidylethanolamine, 4 mg twice a week for 12 weeks. Monocyte-mediated cytotoxicity increased 24 h after the first infusion in 9 of 14 patients and had reached maximum levels (mean, 44% +/- 8) in all patients by the sixth week; similar values were observed at the 12th week. Once increased in vivo, peripheral blood monocyte cytotoxicity was not susceptible to any further increase after a subsequent in vitro incubation of the monocytes with liposomes. However, the peripheral blood monocytes which were not cytotoxic in vivo were activated by in vitro incubation with liposomes and not by medium. TNF-alpha and IL-6 peaked 2 h after the first infusion and returned to baseline values at 24 h; they were not significantly increased by subsequent treatments. The induction of fever in patients, observed 2 h after the first infusion, correlated with TNF-alpha and IL-6 levels. Similarly, C-reactive protein levels also increased at 24 h, but only after the first dose. No increase in beta2-microglobulin and IL-1beta levels was observed, and IFN-gamma was never detected in serum. Two patients experienced stable disease lasting 7 and 12 months, and 12 patients progressed. These results show that multilamellar vesicle muramyl tripeptide phosphatidylethanolamine administration activates monocyte cytotoxicity and cytokine production (TNF-alpha, IL-6). Chronic treatment with multilamellar vesicle muramyl tripeptide phosphatidylethanolamine results in tachyphylaxis in terms of cytokine secretion but not cytotoxicity. There was no difference between the maximum cytotoxicity levels obtained in vivo and those obtained in vitro using the same agent. A better understanding of immunoregulation is required for a rational application of this and related immunotherapies.

Aims and Background: A soluble form of intercellular adhesion molecule-1 (sICAM-1) has been recently identified in patients with malignant melanoma. It has been demonstrated that inflammatory cytokines can modulate the cellular expression of ICAM-1 and the shedding of this molecule by cells. To our knowledge, few data exist on serum sICAM-1 levels in cancer patients treated with immunomodulators. Liposomes containing muramyl tripeptide (MLV MTP-PE) can activate monocytes from cancer patients in vitro and in vivo, making them cytotoxic such as tumor necrosis factor-alpha (TNF-alpha) and Interleukin-6 (IL-6). The purpose of the present study was to evaluate the levels of sICAM-1 and their possible correlation with serum inflammatory cytokine levels in melanoma patients treated with MLV MTP-PE., Methods: The sera from 9 patients with metastatic melanoma treated with MLV MTP-PE, 4 mg i.v. twice a week for 12 weeks, were tested in ELISA system to detect sICAM-1, TNF-alpha, IL-6, Interleukin-1 beta (IL-1 beta) and Interferon-gamma (IFN-gamma) before, and 2 and 24 h after the 1st, 12th and 24th infusion of MLV MTP-PE., Results: Baseline levels of sICAM-1 were elevated in all patients (median 540 ng/ml: range 400-1030 ng/ml). Twenty-four h after the 1st infusion of MLV MTP-PE, we observed 6 increases in sICAM-1 levels, 1 decrease and 2 stable values (median 720 ng/ml: range 410-1820; P = 0.060). Twenty-four h after the 12th infusion, sICAM-1 increased in 3 patients and did not change in 4 (median 790 ng/ml: range 495-1650 ng/ml; P = 0.069). At the 24th infusion, sICAM-1 increased in 4 of 6 evaluable patients and remained stable in 2 (median 802 ng/ml: range 510-1450 ng/ml; P = 0.045). To better analyze the variations in sICAM-1, the patients were arbitrarily divided into two groups according to their clinical behavior: 4 presented stabilization (all lesions, n = 2; some lesions, n = 2) (Group A); 5 presented progressive disease (Group B). In Group A, sICAM-1 levels remained stable or showed a modest increase during treatment (except in 1 patient, who exhibited a substantial variation after the 12th infusion). In contrast, in Group B very high levels of sICAM-1 were observed at the beginning of the study therapy in 1 patient and after the 1st infusion in 3 patients; these values remained high until the 24th infusion. In most of the patients, TNF-alpha and IL-6 increased after the 1st infusion, but not thereafter. IFN-gamma was never detected; IL-1 beta was detectable in a few cases, but only before the infusions., Conclusions: baseline levels of sICAM-1 were elevated in all patients and further increased during treatment only in patients with more aggressive disease. No correlation was found between sICAM-1 and inflammatory cytokines. It would therefore seem that in patients with advanced disease, higher levels and a progressive increase in sICAM-1 may be unfavorable prognostic factors.

Aims: The purpose of the present study was to investigate whether human recombinant interferon-gamma (hrIFN-gamma) can act synergically with various activators in increasing the cytotoxicity of cancer patient monocytes against fresh autologous and allogeneic tumor cells., Methods: Fresh target cells were obtained by means on the mechanical and enzymatic dissociation of human renal carcinomas. A 375 and SW 626 cell lines were used as positive controls. Monocytes from renal cancer patients and normal volunteers were activated in vitro with lipopolysaccharide, muramyl tripeptide (MTP-PE) or liposomes containing MTP-PE (MTP-PE liposomes), with or without a pre-incubation with hrIFN-gamma and were tested for cytotoxicity by means of a 72-hr 111indium-release assay. All of the patients were tumor free at the time of the study., Results: Cancer patient peripheral blood monocytes were activated in vitro by different immunomodulators and became cytotoxic to freshly dissociated autologous or allogeneic tumor cells. A synergic effect producing maximal cytotoxicity was obtained with an appropriately scheduled combination of hrIFN-gamma (10 U/ml) and MTP-PE liposomes (50 nm/ml), free lipopolysaccharide (10 micrograms/ml) or MTP-PE (100 micrograms/ml). The synergic cytotoxicity was observed against fresh allogeneic and autologous tumor cells, as well as against cultured cells., Conclusions: All of these data support the possibility of a combined treatment using hrIFN-gamma and MTP-PE liposomes in human studies, particularly when it is borne in mind that liposomes can prevent the direct toxicity of many immunomodulators and that the low levels of hrIFN-gamma required for the synergic activation are not toxic in vivo.

Our purpose was to evaluate the ability of blood monocytes of renal cancer patients to become cytotoxic against fresh, autologous tumor cells. Fresh target cells were obtained by mechanical enzymatic dissociation of tumor and normal renal tissue. The A375 cell line, derived from a human melanoma, and the SW626 cell line, derived from a human ovarian carcinoma, were used as positive target cell controls. Monocytes from renal cancer patients and normal volunteers were activated in vitro with lipopolysaccharide (LPS), or muramyl tripeptide (MTP-PE), or multilamellar vesicle liposomes containing MTP-PE (MLV-MTP-PE), with or without a pre-incubation with r-IFN-gamma, and tested for cytotoxicity in a 72-hr 111Indium-release assay. All patients were tumor-free at the time of the monocyte study. No difference in cytotoxic activity was observed between monocytes from healthy volunteers and those from cancer patients. Freshly dissociated tumor cells were as susceptible to tumoricidal monocytes as the 2 cell lines. Moreover, no cell population appeared to be resistant to activated monocytes, which were cytotoxic to both allogeneic and autologous fresh tumor cells. Activated monocytes maintained their ability to discriminate between normal and neoplastic cells and were not cytotoxic against autologous or allogeneic normal non-neoplastic cells. Our data indicate that MLV MTP-PE liposomes activate peripheral blood monocytes from cancer patients to a tumoricidal status against fresh, dissociated non-cultured autologous tumor cells.