The chemotherapy-induced peripheral neuropathy outcome measures standardization study: from consensus to the first validity and reliability findings.

BACKGROUND: Chemotherapy-induced peripheral neuropathy (CIPN) is a debilitating and dose-limiting complication of cancer treatment. Thus far, the impact of CIPN has not been studied in a systematic clinimetric manner. The objective of the study was to select outcome measures for CIPN evaluation and to establish their validity and reproducibility in a cross-sectional multicenter study. PATIENTS AND METHODS: After literature review and a consensus meeting among experts, face/content validity were obtained for the following selected scales: the National Cancer Institute-Common Toxicity Criteria (NCI-CTC), the Total Neuropathy Score clinical version (TNSc), the modified Inflammatory Neuropathy Cause and Treatment (INCAT) group sensory sumscore (mISS), the European Organization for Research and Treatment of Cancer (EORTC) QLQ-C30, and CIPN20 quality-of-life measures. A total of 281 patients with stable CIPN were examined. Validity (correlation) and reliability studies were carried out. RESULTS: Good inter-/intra-observer scores were obtained for the TNSc, mISS, and NCI-CTC sensory/motor subscales. Test-retest values were also good for the EORTC QLQ-C30 and CIPN20. Acceptable validity scores were obtained through the correlation among the measures. CONCLUSION: Good validity and reliability scores were demonstrated for the set of selected impairment and quality-of-life outcome measures in CIPN. Future studies are planned to investigate the responsiveness aspects of these measures.

Two doses of NGR-hTNF in combination with capecitabine plus oxaliplatin in colorectal cancer patients failing standard therapies.

BACKGROUND: asparagine-glycine-arginine-human tumour necrosis factor (NGR-hTNF), an agent selectively damaging the tumour vasculature, showed a biphasic dose-response curve in preclinical models. Previous phase I trials of NGR-hTNF indicated 0.8 and 45 µg/m(2) as optimal biological and maximum-tolerated dose, respectively. PATIENTS AND METHODS: Two sequential cohorts of 12 colorectal cancer (CRC) patients who had failed standard therapies received NGR-hTNF 0.8 or 45 µg/m(2) in combination with capecitabine-oxaliplatin (XELOX). RESULTS: Median number of prior treatment lines was 3 in the low-dose and 2 in the high-dose cohort. Overall, 21 patients had been pretreated with oxaliplatin-based regimens. No grade 3-4 NGR-hTNF-related toxicities were observed. Grade 1-2 chills were reported in 43% and 40% of cycles in the low-dose and high-dose cohorts, respectively. In the low-dose cohort, one patient achieved a partial response and five had stable disease for a median of 4.6 months. In the high-dose cohort, six patients had stable disease for a median of 3.6 months. Three-month progression-free survival (PFS) rates were 50% and 33% in the low-dose and high-dose cohort, respectively. Three patients in low-dose cohort experienced PFS longer than PFS on last prior therapy. CONCLUSIONS: Both NGR-hTNF doses were safely combined with XELOX in pretreated CRC patients. Hint of activity was apparent only with low-dose NGR-hTNF.

NKp44, a triggering receptor involved in tumor cell lysis by activated human natural killer cells, is a novel member of the immunoglobulin superfamily.

Surface receptors involved in natural killer (NK) cell triggering during the process of tumor cell lysis have recently been identified. Of these receptors, NKp44 is selectively expressed by IL-2- activated NK cells and may contribute to the increased efficiency of activated NK cells to mediate tumor cell lysis. Here we describe the molecular cloning of NKp44. Analysis of the cloned cDNA indicated that NKp44 is a novel transmembrane glycoprotein belonging to the Immunoglobulin superfamily characterized by a single extracellular V-type domain. The charged amino acid lysine in the transmembrane region may be involved in the association of NKp44 with the signal transducing molecule killer activating receptor-associated polypeptide (KARAP)/DAP12. These molecules were found to be crucial for the surface expression of NKp44. In agreement with data of NKp44 surface expression, the NKp44 transcripts were strictly confined to activated NK cells and to a minor subset of TCR-gamma/delta+ T lymphocytes. Unlike genes coding for other receptors involved in NK cell triggering or inhibition, the NKp44 gene is on human chromosome 6.

Molecular cloning of NKp46: a novel member of the immunoglobulin superfamily involved in triggering of natural cytotoxicity.

NKp46 has been shown to represent a novel, natural killer (NK) cell-specific surface molecule, involved in human NK cell activation. In this study, we further analyzed the role of NKp46 in natural cytotoxicity against different tumor target cells. We provide direct evidence that NKp46 represents a major activating receptor involved in the recognition and lysis of both human and murine tumor cells. Although NKp46 may cooperate with other activating receptors (including the recently identified NKp44 molecule) in the induction of NK-mediated lysis of human tumor cells, it may represent the only human NK receptor involved in recognition of murine target cells. Molecular cloning of the cDNA encoding the NKp46 molecule revealed a novel member of the immunoglobulin (Ig) superfamily, characterized by two C2-type Ig-like domains in the extracellular portion. The transmembrane region contains the positively charged amino acid Arg, which is possibly involved in stabilizing the association with CD3zeta chain. The cytoplasmic portion, spanning 30 amino acids, does not contain immunoreceptor tyrosine-based activating motifs. Analysis of a panel of human/hamster somatic cell hybrids revealed segregation of the NKp46 gene on human chromosome 19. Assessment of the NKp46 mRNA expression in different tissues and cell types unambiguously confirmed the strict NK cell specificity of the NKp46 molecule. Remarkably, in line with the ability of NKp46 to recognize ligand(s) on murine target cells, the cDNA encoding NKp46 was found to be homologous to a cDNA expressed in murine spleen. In conclusion, this study reports the first characterization of the molecular structure of a NK-specific receptor involved in the mechanism of NK cell activation during natural cytotoxicity.

Identification and molecular characterization of NKp30, a novel triggering receptor involved in natural cytotoxicity mediated by human natural killer cells.

Two major receptors involved in human natural cytotoxicity, NKp46 and NKp44, have recently been identified. However, experimental evidence suggested the existence of additional such receptor(s). In this study, by the generation of monoclonal antibodies (mAbs), we identified NKp30, a novel 30-kD triggering receptor selectively expressed by all resting and activated human natural killer (NK) cells. Although mAb-mediated cross-linking of NKp30 induces strong NK cell activation, mAb-mediated masking inhibits the NK cytotoxicity against normal or tumor target cells. NKp30 cooperates with NKp46 and/or NKp44 in the induction of NK-mediated cytotoxicity against the majority of target cells, whereas it represents the major triggering receptor in the killing of certain tumors. This novel receptor is associated with CD3zeta chains that become tyrosine phosphorylated upon sodium pervanadate treatment of NK cells. Molecular cloning of NKp30 cDNA revealed a member of the immunoglobulin superfamily, characterized by a single V-type domain and a charged residue in the transmembrane portion. Moreover, we show that NKp30 is encoded by the previously identified 1C7 gene, for which the function and the cellular distribution of the putative product were not identified in previous studies.

Complex regulation of simple sugar transport in insulin-responsive cells.

Facilitated sugar entry into mammalian cells is catalysed by multiple isoforms of the glucose transporter and regulated by hormonal stimuli, nutritional status and oncogenesis. A large reserve of latent glucose transport capacity must be maintained by muscle and adipose cells that are sensitive to insulin, the primary activator of sugar uptake after feeding. Intracellular sequestration of sugar transporters accounts for a large part of this latent capacity, but new findings suggest that there is also reversible suppression of intrinsic catalytic activity of those glucose transporters residing at the cell surface. The mechanism of this suppression appears to be occlusion or disruption of the exofacial sugar-binding sites on the glucose-transporter proteins.

First-line single-agent cetuximab in patients with advanced colorectal cancer.

BACKGROUND: The anti-epidermal growth factor receptor (EGFR) antibody cetuximab is active in heavily pretreated patients with metastatic colorectal cancer (mCRC) both in monotherapy and in combination with chemotherapy (CT). This study assesses the antitumor activity of single-agent cetuximab in CT-naive patients. PATIENTS AND METHODS: Phase II clinical trial was used. Patients were EGFR positive by immunohistochemistry and were not candidate for radical surgery, even in the case of substantial tumor shrinkage. Cetuximab was administered weekly. RESULTS: Thirty-nine patients were treated and evaluated. The most common adverse event was skin toxicity (89% any grade; 48% grade 1; 31% grade 2; 10% grade 3). One patient had a complete response and three obtained partial responses (10% overall response rate). Thirteen patients had stable disease (34%). Twenty-two patients experienced progressive disease (56%). Overall median time to progression (TTP) was 2 months, and the responders individual TTP was 12, 9, 9, and 6 months. CONCLUSIONS: Even in chemo-naive patients, cetuximab as single agent is active only in a small fraction of mCRC, similarly to what has been reported for heavily pretreated patients. The extent of benefit when response occurs is, however, such that it is mandatory to intensify the search for the predictive markers of response to cetuximab therapy.

Differentiation of HL60 promyelocytic cells is promoted by a 'differentiation enhancing factor' produced by erythroleukemia cells.

A differentiation enhancing factor isolated from murine erythroleukemia cells is also a potent enhancer of the differentiation of HL60 human promyelocytic leukemia cells, induced by retinoic acid and by phorbol ester. This stimulating effect is the result of a large increase in the sensitivity of HL60 cells for retinoic acid and for phorbol 12-myristate 13-acetate (20-fold and 40-fold, respectively). Accelerated differentiation induced by the protein factor, and monitored by the appearance of marker enzymes, is accompanied by a large increase in the fluctuation of the levels of protein kinase C (PKC) isozymes in HL60 cells. These results provide further support for the role of this new protein factor in cell differentiation and indicate that other cell types are susceptible to its biological effect.

Protein kinase C isoforms in murine erythroleukemia cells and their involvement in the differentiation process.

In addition to alpha, delta and epsilon-protein kinase C, murine erythroleukemia cells contain zeta-PKC and also a c-PKC isoform, named alpha 1, which shows cross-reactivity with an anti-alpha-PKC antipeptide antibody. In a C44 MEL cell clone, characterized by a high rate of differentiation, both c-PKC forms are expressed at a level higher than that of the N23 MEL cell clone which differentiates at a low rate and contains higher levels of epsilon-PKC and particularly of the delta-PKC isozyme. In the course of MEL cell differentiation, delta-PKC in N23 cells and alpha 1-PKC in C44 cells are rapidly down-regulated and the overall process is almost completed before cell commitment. Of the other three PKC isozymes present in both clones, only alpha-PKC is down-regulated to a significant extent. It is proposed that modulation of the signal delivered by each PKC isozyme is one of the biochemical mechanisms involved in MEL cell differentiation.

Extracellular release of the 'differentiation enhancing factor', a HMG1 protein type, is an early step in murine erythroleukemia cell differentiation.

Differentiation enhancing factor (DEF) is a 29 kDa protein expressed in murine erythroleukemia (MEL) cells and active in promoting a significant increase in the rate of hexamethylenebisacetamide induced differentiation of these cells. The factor was recently shown to possess an amino acid sequence identical to that reported for one of the HMG1 proteins, designated as 'amphoterin' on the basis of its highly dipolar sequence. In the present study, we have expressed DEF cDNA in an E. coli strain and found that the recombinant protein has functional properties identical to those observed with native DEF. Furthermore, we demonstrate that, following MEL cell stimulation with the chemical inducer, DEF is secreted in large amounts in the extracellular medium. In fact, the N-terminal sequence and the partial amino acid sequence of tryptic peptides from the secreted protein correspond to those of DEF isolated from the soluble fraction of resting MEL cells. These results are indicative for an extracellular localization as the site of action of DEF and suggest a novel function for proteins belonging to the HMG1 family. Finally, the early decay of DEF mRNA, in chemical induced MEL cells, support the hypothesis that the involvement of the enhancing factor occurs and is completed in the early phases of cell differentiation.

P49, a putative HLA-G1 specific inhibitory NK receptor belonging to the immunoglobulin Superfamily.

NK cells display several killer inhibitory receptors (KIRs) specific for different alleles of major histocompatibility complex (MHC) class I molecules. A family of KIRs are represented by type I transmembrane proteins belonging to the Immunoglobulin Superfamily (Ig-SF). In the present study we describe a cDNA, termed cl.15.212, that encodes for a type I transmembrane protein displaying approximately 50% sequence homology with other Ig-SF members. The protein encoded by cl.15.212 (termed p49 according to its apparent molecular weight of 49 kDa) is characterized by two extracellular Ig-like domains, a 115-amino acid cytoplasmic tail containing a single immuno-receptor tyrosine-based inhibitory motif (ITIM) typical of KIR. Different from the other KIRs, the cl.15.212 transcript is expressed by all NK cells and by a fraction of T-cell clones expressing KIR. To determine the specificity of the cl.15.212-encoded receptor, we generated a chimeric protein, formed by the ectodomain of p49 and the Fc portion of human IgG1 (p49-Fc). Soluble molecules bound efficiently to LCL721.221 (221) cells transfected with HLA-G1, -A3, -B46 alleles and weakly to the -B7 allele. On the other hand, they did not bind to 221 cells either untransfected or transfected with HLA-A2, -B51, -Cw3, or-Cw4.

Rasch-built Overall Disability Scale for patients with chemotherapy-induced peripheral neuropathy (CIPN-R-ODS).

Chemotherapy-induced peripheral neuropathy (CIPN) is a common neurological side-effect of cancer treatment and may lead to declines in patients' daily functioning and quality of life. To date, there are no modern clinimetrically well-evaluated outcome measures available to assess disability in CIPN patients. The objective of the study was to develop an interval-weighted scale to capture activity limitations and participation restrictions in CIPN patients using the Rasch methodology and to determine its validity and reliability properties. A preliminary Rasch-built Overall Disability Scale (pre-R-ODS) comprising 146 items was assessed twice (interval: 2-3 weeks; test-retest reliability) in 281 CIPN patients with a stable clinical condition. The obtained data were subjected to Rasch analyses to determine whether model expectations would be met, and if necessarily, adaptations were made to obtain proper model fit (internal validity). External validity was obtained by correlating the CIPN-R-ODS with the National Cancer Institute-Common Toxicity Criteria (NCI-CTC) neuropathy scales and the Pain-Intensity Numeric-Rating-Scale (PI-NRS). The preliminary R-ODS did not meet Rasch model's expectations. Items displaying misfit statistics, disordered thresholds, item bias or local dependency were systematically removed. The final CIPN-R-ODS consisting of 28 items fulfilled all the model's expectations with proper validity and reliability, and was unidimensional. The final CIPN-R-ODS is a Rasch-built disease-specific, interval measure suitable to detect disability in CIPN patients and bypasses the shortcomings of classical test theory ordinal-based measures. Its use is recommended in future clinical trials in CIPN.

Characterization of the biological role of murine erythroleukemia cells "differentiation enhancing factor" using antisense oligodeoxynucleotides.

On the basis of the amino acid sequence of isolated tryptic peptides, it has been established that the differentiation enhancing factor, produced and active on murine erythroleukemia (MEL) cells, possesses a unique sequence, with no similarity to that of known proteins. Accordingly, this factor can be defined as a novel biologically active peptide. An antisense oligodeoxynucleotide, deduced from the sequence of a non-decapeptide (produced by tryptic digestion of the factor), decreases the rate and the extent of MEL cell differentiation, induced by hexamethylenebisacetamide. In these cells the amount of the factor is reduced to one third of that constitutively present in untreated cells. Exogenous addition of the factor restores cell inducibility to normal values. Taken together, these results demonstrate the presence in MEL cells of a new factor, structurally and functionally unrelated to any of the known biologically active peptides, and suggest its crucial role in the promotion of an initial signal, in chemically induced erythroid differentiation.

Modulation of the intracellular Ca(2+)-dependent proteolytic system is critically correlated with the kinetics of differentiation of murine erythroleukemia cells.

Calpain has been identified as the intracellular proteinase that catalyzes the selective down-regulation of protein kinase C (PKC) isoforms, occurring in the early stages of commitment to terminal erythroid differentiation of murine erythroleukemia (MEL) cells induced by hexamethylenebisacetamide. This conclusion has been reached through direct experiments performed with two MEL cell clones, one characterized by a high and the other by a low rate of differentiation. In both cell types, introduction of an anti-calpain antibody resulted in a significant delay in the onset of down-regulation of PKC isoforms, and in an increase in the latent period that precedes differentiation. Both cell lines also displayed reduced rates of PKC decay and accumulation of mature erythroid cells. Furthermore, in the fast-responding clone, calpastatin, the natural calpain-inhibitor protein, was found to be almost completely absent, resulting in activation and expression of proteolytic activity of calpain even at micromolar concentrations of Ca2+, a condition not sufficient to trigger calpain activation in the slowly responding clone which contains high levels of calpastatin. The fast-responding MEL cell clone, enriched with calpastatin, displayed a lower rate of cell differentiation, with a kinetics almost identical to that observed following introduction of the anti-calpain antibody. It is proposed that Ca(2+)-dependent proteolysis plays a crucial role for the progress of MEL cell differentiation through the specific degradation of PKC isozymes.

Differential expression of protein kinase C isoform genes in three murine erythroleukemia cell variants: implication for chemical induced differentiation.

The presence of alpha, delta, epsilon, theta, and zeta protein kinase C isoforms in DS19 murine erythroleukemia cells has been established in this study. In addition, the mRNA levels of these isozymes have been measured by quantitative reverse transcriptase-polymerase chain reaction. Isoform delta has been found to be the most abundant isotype, whereas isoform zeta resulted to be present in only few copies. Furthermore, the expression levels of all five protein kinase C isozymes have been studied in three cell clones, derived from parental DS19 cells and characterized by different susceptibilities to differentiation. This comparative analysis indicated that the calcium-independent isozymes (delta, epsilon, zeta, and theta) display significantly higher expression levels in cells less prone to differentiation. On the other hand, the mRNA levels of the only calcium-dependent isoform present (alpha) fluctuate poorly from one cell clone to the other, but are the highest in the cell clone characterized by the fastest rate of differentiation. This study represents the first complete characterization of the basal levels of specific protein kinase C isotypes in different murine erythroleukemia cell clones and provides further evidence for the role of individual isozymes in the early events that trigger chemical induced murine erithroleukemia cell differentiation.

Antipeptide antibodies toward the extracellular domain of insulin receptor beta-subunit.

In order to investigate structure and function of beta-subunit extracellular portion, four polyclonal antibodies (AP1, AP2, AP3 and AP4) toward peptides comprised in this region were generated. None of them recognizes native human and rat insulin receptor both in vitro and in whole cells. Two antibodies, AP1 and AP2, immunoprecipitate isolated (DTT-reduced) human beta-subunits and bind to human IM-9 cell after alpha-subunit tryptic cleavage. Only AP1 recognizes rat beta-subunit both in vitro and in trypsin treated rat FAD cells. These findings suggest that: (i) the extracellular portion of the insulin receptor beta-subunit is partially covered by the alpha-subunit in human and rat native insulin receptors; (ii) human and rat beta-subunit extracellular domains are different, at least in the amino acid sequence corresponding to residues 785-796 of the human insulin receptor.

Activation of cell surface glucose transporters measured by photoaffinity labeling of insulin-sensitive 3T3-L1 adipocytes.

Several studies have demonstrated that the intrinsic catalytic activity of cell surface glucose transporters is highly regulated in 3T3-L1 adipocytes expressing GLUT1 (erythrocyte/brain) and GLUT4 (adipocyte/skeletal muscle) glucose transporter isoforms. For example, inhibition of protein synthesis in these cells by anisomycin or cycloheximide leads to marked increases in hexose transport without a change in the levels of cell surface glucose transporter proteins (Clancy, B. M., Harrison, S. A., Buxton, J. M., and Czech, M. P. (1991) J. Biol. Chem. 266, 10122-10130). In the present work the exofacial hexose binding sites on GLUT1 and GLUT4 in anisomycin-treated 3T3-L1 adipocytes were labeled with the cell-impermeant photoaffinity reagent [2-3H]2-N-[4-(1-azitrifluoroethyl)benzoyl]-1,3-bis- (D-mannos-4-yloxy)-2-propylamine [( 2-3H] ATB-BMPA) to determine which isoform is activated by protein synthetic blockade. As expected, a 15-fold increase in 2-deoxyglucose uptake in response to insulin was associated with 1.7- and 2.6-fold elevations in plasma membrane GLUT1 and GLUT4 protein levels, respectively. Anisomycin treatment of cultured adipocytes for 5 h produced an 8-fold stimulation of hexose transport but no increase in the content of glucose transporters in the plasma membrane fraction as measured by protein immunoblot analysis. Cell surface GLUT1 levels were also shown to be unaffected on 3T3-L1 adipocytes in response to anisomycin using an independent method, the binding of an antiexofacial GLUT1 antibody to intact cells. In contrast, anisomycin fully mimicked the action of insulin to stimulate (about 4-fold) the radiolabeling of GLUT1 transporters specifically immunoprecipitated from intact 3T3-L1 adipocytes irradiated after incubation with [2-3H] ATB-BMPA. Photolabeling of GLUT4 under these conditions was also significantly enhanced (1.8-fold) by anisomycin treatment, but this effect was only 15% of that caused by insulin. These results suggest that: 1) the photoaffinity reagent [2-3H]ATB-BMPA labels those cell surface glucose transporters present in a catalytically active state rather than total cell surface transporters as assumed previously and 2) inhibition of protein synthesis in 3T3-L1 adipocytes stimulates sugar transport primarily by enhancing the intrinsic catalytic activity of cell surface GLUT1, and to a lesser extent, GLUT4 proteins.

Identity in molecular structure between "differentiation enhancing factor" of murine erythroleukemia cells and the 30 kD heparin-binding protein of developing rat brain.

A 29 kD protein previously isolated from murine erythroleukemia (MEL) cells and shown to enhance the rate of differentiation of these cells has now been demonstrated to possess an amino acid sequence identical to that reported for the 30 kD heparin-binding protein from developing rat brain, named amphoterin after its highly dipolar structure. The identity between the two proteins has been established on the basis of a strong heparin binding affinity and a complete homology in the amino acid sequences of N-terminal region as well as of several tryptic peptides. Furthermore, the cDNA encoding this protein has been isolated from MEL cell mRNA, by means of reverse transcriptase-polymerase chain reaction, and its sequence was found to correspond to that of amphoterin. The MEL cell differentiation enhancing factor, previously abbreviated as DEF, is again confirmed to reduce the latent period preceding the appearance of hexamethylenebisacetamide induced cell commitment and to stimulate the catalytic activity of alpha-protein kinase C. Thus, here we demonstrate that a protein expressed in MEL cells, whose sequence is identical to that previously reported for amphoterin, plays an essential role in promoting cell differentiation, thereby indicating a new relevant function of amphoterin.

Correlation between levels of delta protein kinase C and resistance to differentiation in murine erythroleukemia cells.

It has been demonstrated that the level of delta protein kinase C is inversely correlated to the responsiveness of murine erythroleukemia cells to chemical induction to terminal erythroid differentiation. In these cells, deltaPKC is largely present in a membrane associated form, and thus in a constitutively active state, a condition which characterizes the undifferentiated phenotype. Accordingly, commitment to cell differentiation has been shown to be preceded by down regulation of deltaPKC, a process significantly accelerated and induced to almost completion by the differentiation enhancing factor (DEF) in a dose dependent manner. The present results provide a better understanding of the role of deltaPKC in characterizing the undifferentiated MEL cell phenotype and suggest a relationship between the acceleration in the rate of differentiation induced by DEF and the down regulation of this kinase form.

Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells.

The potential regulatory role of delta protein kinase C (delta PKC) in murine erythroleukaemia cell differentiation was studied by using antisense oligodeoxynucleotides targeting the translation initiation region of mouse delta PKC mRNA. Cell treatment with antisense oligonucleotides, at a concentration of 20 microM, followed by hexamethylenebisacetamide induction, produced a specific 2-fold increase in the differentiation rate of both slowly and rapidly differentiating murine erythroleukaemia cell clones. Cell permeabilization by a cationic lipid resulted in a decrease of one order of magnitude in the amounts of antisense oligonucleotides necessary to elicit the maximal response, and accelerated the kinetics of the stimulatory effect. These changes in murine erythroleukaemia cell differentiation rates, observed in both cell clones, were associated with 60% and 50% decreases, respectively, in delta PKC immunoreactive protein in slowly and rapidly differentiating cells. The present results indicate strongly that basal levels of delta PKC in murine erythroleukaemia cells are essential in regulating the initial differentiation rate of these cells in response to chemical induction, and provide further evidence that this PKC isoform plays a fundamental role in maintaining the undifferentiated phenotype of murine erythroleukaemia cells.