Monitoring serum levels ELR+ CXC chemokines and the relationship between microvessel density and angiogenic growth factors in multiple myeloma.

BACKGROUND: The ELR+ CXC chemokines are important mediators of tumorigenesis, related to their angiogenic properties. Angiogenesis appears to be a prominent feature in the progression of multiple myeloma (MM). CXC chemokines have four highly conserved cysteine amino acid residues, with the first two cysteine molecules separated by a single amino acid. The angiogenic potential of this group is determined by the presence of three amino acid residues (Glu-Leu-Arg: the ELR motif) preceding the first cysteine amino acid, in the NH2 terminus. AIMS: The purpose of this study was to determine serum concentrations of angiogenesis-related chemokines ELR+ motif, such as interleukin-8 (IL-8), epithelial neutrophil activating protein-78 (ENA-78) and growth-related gene alpha (GRO-α), as well the bone marrow microvascular density (MVD) in patients with MM at diagnosis and after treatment, in plateau phase. We also evaluated the relationship among them with other known growth factors involved in angiogenesis. METHODS: Serum levels of the ELR+ CXC chemokines: IL-8, ENA-78 and GRO-α as well as of the angiogenic factors: hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF) and tumor necrosis factor-α (TNF-α) were determined in 63 newly diagnosed MM patients, in 30 in plateau phase and in 20 healthy controls. Serum measurements of them were performed with commercially available kits for ELISA. Bone marrow biopsies were performed before and after treatment, in plateau phase, in order to determine MVD by staining vessels with anti-CD31. RESULTS: Serum concentrations of IL-8, ENA-78, GRO-α and TNF-α were significantly higher in the group of MM patients (44.5±25.3, 765±572.1, 186.5±129.1 and 4.2±2.8 pg/ml, respectively) in comparison to control group (27.3±6.4, 335.1±268.6, 112.5±76.1 and 1.3±0.8 pg/ml) (p<0.02 for GRO-α, p<0.001 for other cases). We also found that untreated patients had higher levels of IL-8, ENA-78, GRO-α than post treatment patients, but statistical significant difference was found only for IL-8 (48.36±30.93 pg/ml vs. 35.05±19.77 pg/ml, p<0.001). Furthermore IL-8, GRO-α, TNF-α, HGF and VEGF were significantly higher with increasing disease stage (p<0.001 in all cases). ENA-78 serum levels were higher in stage III than in stage I and II, but without statistical significance. Additionally we correlated each proinflammatory cytokine with well known angiogenic factors such as HGF, VEGF and TNF-α. A positive correlation was found between serum HGF and IL-8 and GRO-α (r=0.316 p<0.01, r=0.297 p<0.02, respectively). Similarly serum VEGF correlated with ENA-78 and GRO-α (r=0.323 p<0.01, r=0.469 p<0.001, respectively). In the pretreatment group of patients a positive correlation between bone marrow MVD and serum levels of GRO-α was found (r=0.304 p<0.01). There was a difference in survival times between patients with higher than median versus low IL-8, ENA-78 and GRO-α levels, but the differences could not reach statistical significance in either case. CONCLUSIONS: These findings support the hypothesis that ELR+ motif CXC chemokines, such as IL-8, ENA-78 and GRO-α correlate with angiogenic growth factors and may play a role in the progression of MM. Further studies are needed to determine their prognostic and predictive significance.

Angiogenesis-related cytokines, RANKL, and osteoprotegerin in multiple myeloma patients in relation to clinical features and response to treatment.

An essential cytokine system for the osteoclast biology in multiple myeloma (MM) consists of the receptor of activator of NF-κB ligand (RANKL), its receptor (RANK), and the soluble decoy receptor, osteoprotegerin (OPG). Myeloma cells cause imbalance in OPG/RANKL interactions. We measured serum levels of OPG, soluble (s) RANKL, sRANKL/OPG ratio, markers of disease activity [LDH, CRP, interleukin-6 (IL-6), ß2-microglobulin (B2M)], and angiogenic factors [hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF)], in 54 newly diagnosed MM patients and in 25 of them in plateau phase. All the above values were higher in MM patients compared to controls and decreased in plateau phase. sRANKL and RANKL/OPG were higher with advancing disease stage and skeletal grade. Significant correlations were found among RANKL and RANKL/OPG with HGF, LDH, VEGF, IL-6, and B2M. In conclusion, RANKL and OPG play significant roles in MM pathophysiology, as regulators of bone turnover and mediators of angiogenesis.

Viral DNA detection and RAS mutations in actinic keratosis and nonmelanoma skin cancers.

BACKGROUND: Actinic keratosis (AK) is a well-established precancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes. A number of viruses have been proposed to play a role in the development of nonmelanoma skin cancers (NMSC), but the most plausible evidence to date suggests that cutaneous human papillomavirus (HPV) is the key instigating factor. OBJECTIVES: To evaluate the prevalence of HPV, cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) and investigate their relationship with the presence of RAS gene mutations in cutaneous lesions obtained from nonimmunosuppressed patients. METHODS: HPV, CMV, HSV and EBV detection was performed using polymerase chain reaction (PCR) in skin biopsies (26 AK, 12 SCC and 15 BCC samples) that were collected from immunocompetent patients. The RAS mutation incidence was also investigated in all cutaneous lesions by use of PCR/restriction fragment length polymorphism and direct DNA sequencing. RESULTS: Seventeen out of 53 (32%) skin lesions were found to be positive for HPV DNA. The highest incidences of HPV infection were five of 15 (33%) in BCC and four of 12 (33%) in SCC specimens. The HPV incidence was eight of 26 (31%) in AK and eight of 53 (15%) in normal skin tissue. Twelve out of 53 (23%) skin lesions were CMV-positive. The highest incidence of CMV infection was six of 15 (40%), observed in BCC specimens. The CMV incidence was two of 26 (8%) in AK and four of 12 (33%) in SCC. No normal skin biopsy was found to be positive for CMV. All cutaneous samples were negative for HSV and EBV DNA, as assessed by our PCR-based assays. Only three samples, one AK (4%), one BCC (6%) and one SCC (8%), were found to carry a G>T transversion at the second position of HRAS codon 12. Both HRAS mutant SCC and BCC biopsies were HPV- and CMV-positive, as well. CONCLUSIONS: HPV DNA is detected in NMSC, AK and normal skin biopsies. Our results also indicate that CMV is involved in NMSC at higher levels than in premalignant lesions, whereas the virus was not detected in normal skin biopsies. HSV and EBV do not appear to be involved in the pathogenesis of cutaneous lesions. Moreover, we suggest that the HRAS codon 12 mutation is not a very common event in AK or NMSC. Finally, both viral infection and HRAS activation appear to represent independent factors in the aetiology of NMSC, samples of which were obtained from immunocompetent patients.

Deregulation of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in basal cell carcinoma.

BACKGROUND: Basal cell carcinoma (BCC) is a locally aggressive slowly growing tumour that rarely metastasizes and is mostly seen in older members of the population. OBJECTIVES: To determine the involvement of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in BCC. METHODS: We investigated the integrity of the CDKN2A locus in 15 BCC samples by analysing the presence of allelic imbalance/loss of heterozygosity (LOH). Moreover, we studied the mRNA expression levels of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in the BCC samples and compared them with mRNA levels in the corresponding normal tissue. The presence of mutations was examined by sequencing for exons 1a and 2 of p16(INK4a). RESULTS: We found LOH in one BCC sample for the marker D9S1748. A polymorphism (G442A) of exon 2 was detected in three cases. p14(ARF), p15(INK4b) and p53 presented high expression levels, whereas p16(INK4a) exhibited low mRNA levels compared with the corresponding normal tissue. Significant correlations were detected among the genes studied. CONCLUSIONS: Our results demonstrate a different expression profile between p16(INK4a) and p14(ARF), p15(INK4b) and p53 in BCC. Moreover, we found a low percentage of LOH and of a polymorphic sequence variant (Ala148Thr) for the CDKN2A locus.

Genomic instability, mutations and expression analysis of the tumour suppressor genes p14(ARF), p15(INK4b), p16(INK4a) and p53 in actinic keratosis.

Actinic keratosis (AK) is a well-established pre-cancerous skin lesion that has the potential to progress to squamous cell carcinoma (SCC). We investigated the involvement of the CDKN2A, CDKN2B and p53 genes in AK and in the progression of AK to SCC. Mutational analysis on exons 1a, 1b and 2 of the CDKN2A locus and exon 1 of the CDKN2B locus as well as allelic imbalance was performed in 26 AK specimens. Expression levels of the genes p14(ARF), p15(INK4b), p16(INK4a) and p53 were examined in 16 AKs and 12 SCCs by real-time RT-PCR. A previously described polymorphism of p16(INK4a) (Ala148Thr) was detected at an allelic frequency of 12%. Six samples carried novel mutations at codon 71 of the CDKN2A locus and one sample presented an additional mutation at codon 65. Two AK samples carried a not-previously described non-UV type missense mutation at codon 184 (Val184Glu) of exon 1b in the p14(ARF) gene. Regarding the CDKN2B locus a new mutation at codon 50 (Ala50Thr) and another at codon 24 (Arg24Arg), were detected. Microsatellite instability (MSI) was found in 15% of AKs in at least one marker, indicating that genetic instability has some implication in the development of AK. Down-regulation of p16(INK4a) and p53 mRNA levels was noted in SCC compared to AK. TSGs expression levels in sun-exposed morphologically normal-appearing skin, suggests that abnormal growth stimuli might exist in these tissues as well. Furthermore, we suggest a possible role of p15(INK4b), independently from the intracellular pathway mediated by p16(INK4a), and of p14(ARF) in AK development, as well as in the progression of AK to SCC. The deregulation of the expression profiles of the CDKN2A, CDKN2B and p53 genes may, independently of mutations and LOH at 9p21, play a significant role in AK and progression of AK to SCC.

Circulating HER2 mRNA-positive cells in the peripheral blood of patients with stage I and II breast cancer after the administration of adjuvant chemotherapy: evaluation of their clinical relevance.

BACKGROUND: The purpose of this study was to evaluate the prognostic value of circulating tumor cells (CTCs) expressing HER2 messenger RNA (mRNA) after the administration of adjuvant chemotherapy in women with operable breast cancer. PATIENTS AND METHODS: HER2 mRNA-positive CTCs were detected by nested RT-PCR in the peripheral blood of 214 patients with stage I and II breast cancer after the completion of adjuvant chemotherapy. RESULTS: HER2 mRNA-positive CTCs were detected in 45 (21%) patients. Adjuvant chemotherapy could eliminate HER2 mRNA-positive CTCs in 16 (30.2%) prechemotherapy-positive patients. Moreover, HER2 mRNA-positive CTCs were detected in eight (5%) of 161 prechemotherapy-negative patients. The detection of HER2 mRNA-positive CTCs after chemotherapy was associated with reduced disease-free interval (DFI) (P = 0.006) but not with overall survival (P = 0.2); this effect was mainly observed in node-negative patients (P = 0.04) and to a lesser extent in node-positive (P = 0.06). Multivariate analysis revealed that the detection of HER2 mRNA-positive CTCs was an independent predictive factor for DFI (hazard ratio 3.238, P < 0.0005). CONCLUSIONS: The detection of HER2 mRNA-positive CTCs after the completion of adjuvant chemotherapy may provide clinically useful information concerning the efficacy of treatment and the prognosis of patients with operable breast cancer.

A phase I study of the optimized cryptic peptide TERT(572y) in patients with advanced malignancies.

OBJECTIVE: It was the aim of this study to evaluate the safety of the optimized cryptic peptide TERT(572Y) in pretreated patients with advanced cancer. METHODS: Nineteen patients with progressive and chemotherapy-refractory tumors received escalated doses (2-6 mg) of 2 subcutaneous injections of the optimized TERT(572Y) peptide followed by 4 subcutaneous injections of the native TERT(572) peptide every 3 weeks. Both TERT peptides were coinjected with adjuvant Montanide ISA51. Toxicity was evaluated every 3 weeks and peptide-specific CD8+ cells were detected by flow cytometry using TERT(572Y) tetramers. RESULTS: Fourteen out of 19 patients completed the vaccination program. No grade III/IV toxicity was observed. Grade I anemia was observed in 4 patients and local skin reaction at the injection site in 11 patients. Other nonhematologic toxicities were mild, and no late toxicity was observed after a median postvaccination follow-up period of 10.7 months. There was no dose-limiting toxicity. Peripheral blood TERT(572Y)-specific CD8+ lymphocytes were detected in 13 out of 14 evaluable patients after 2 injections with the optimized TERT(572Y) peptide. There was no complete or partial response, but 4 patients (21%) with persistent TERT(572Y)-specific CD8+ experienced stable disease for a median of 10.5 months. CONCLUSION: TERT(572Y) peptide vaccine is well tolerated and effective in eliciting specific TERT(572Y) CD8+ lymphocytes in pretreated cancer patients, demonstrating that cryptic peptides could be used in cancer immunotherapy.