SNP of Aromatase Predict Long-term Survival and Aromatase Inhibitor Toxicity in Patients with Early Breast Cancer: A Biomarker Analysis of the GIM4 and GIM5 Trials.

PURPOSE: In estrogen receptor-positive (ER+) breast cancer, single-nucleotide polymorphisms (SNP) in the aromatase gene might affect aromatase inhibitors (AI) metabolism and efficacy. Here, we assessed the impact of SNP on prognosis and toxicity of patients receiving adjuvant letrozole. EXPERIMENTAL DESIGN: We enrolled 886 postmenopausal patients in the study. They were treated with letrozole for 2 to 5 years after taking tamoxifen for 2 to 6 years, continuing until they completed 5 to 10 years of therapy. Germline DNA was genotyped for SNP rs4646, rs10046, rs749292, and rs727479. Log-rank test and Cox model were used for disease-free survival (DFS) and overall survival (OS). Cumulative incidence (CI) of breast cancer metastasis was assessed through competing risk analysis, with contralateral breast cancer, second malignancies and non-breast cancer death as competing events. CI of skeletal and cardiovascular events were assessed using DFS events as competing events. Subdistribution HR (sHR) with 95% confidence intervals were calculated through Fine-Gray method. RESULTS: No SNP was associated with DFS. Variants rs10046 [sHR 2.03, (1.04-2.94)], rs749292 [sHR 2.11, (1.12-3.94)], and rs727479 [sHR 2.62, (1.17-5.83)] were associated with breast cancer metastasis. Three groups were identified on the basis of the number of these variants (0, 1, >1). Variant-based groups were associated with breast cancer metastasis (10-year CI 2.5%, 7.6%, 10.7%, P = 0.035) and OS (10-year estimates 96.5%, 93.0%, 89.6%, P = 0.030). Co-occurrence of rs10046 and rs749292 was negatively associated with 10-year CI of skeletal events (3.2% vs. 10%, P = 0.033). A similar association emerged between rs727479 and cardiovascular events (0.3% vs. 2.1%, P = 0.026). CONCLUSIONS: SNP of aromatase gene predict risk of metastasis and AI-related toxicity in ER+ early breast cancer, opening an opportunity for better treatment individualization.

Identification and characterization of novel ETV4 splice variants in prostate cancer.

ETV4, one of ETS proteins overexpressed in prostate cancer, promotes migration, invasion, and proliferation in prostate cells. This study identifies a series of previously unknown ETV4 alternatively spliced transcripts in human prostate cell lines. Their expression has been validated using several unbiased techniques, including Nanopore sequencing. Most of these transcripts originate from an in-frame exon skipping and, thus, are expected to be translated into ETV4 protein isoforms. Functional analysis of the most abundant among these isoforms shows that they still bear an activity, namely a reduced ability to promote proliferation and a residual ability to regulate the transcription of ETV4 target genes. Alternatively spliced genes are common in cancer cells: an analysis of the TCGA dataset confirms the abundance of these novel ETV4 transcripts in prostate tumors, in contrast to peritumoral tissues. Since none of their translated isoforms have acquired a higher oncogenic potential, such abundance is likely to reflect the tumor deranged splicing machinery. However, it is also possible that their interaction with the canonical variants may contribute to the biology and the clinics of prostate cancer. Further investigations are needed to elucidate the biological role of these ETV4 transcripts and of their putative isoforms.

ETV4 promotes late development of prostatic intraepithelial neoplasia and cell proliferation through direct and p53-mediated downregulation of p21.

BACKGROUND: ETV4 is one of the ETS proteins overexpressed in prostate cancer (PC) as a result of recurrent chromosomal translocations. In human prostate cell lines, ETV4 promotes migration, invasion, and proliferation; however, its role in PC has been unclear. In this study, we have explored the effects of ETV4 expression in the prostate in a novel transgenic mouse model. METHODS: We have created a mouse model with prostate-specific expression of ETV4 (ETV4 mice). By histochemical and molecular analysis, we have investigated in these engineered mice the expression of p21, p27, and p53. The implications of our in vivo findings have been further investigated in human cells lines by chromatin-immunoprecipitation (ChIP) and luciferase assays. RESULTS: ETV4 mice, from two independent transgenic lines, have increased cell proliferation in their prostate and two-thirds of them, by the age of 10 months, developed mouse prostatic intraepithelial neoplasia (mPIN). In these mice, cdkn1a and its p21 protein product were reduced compared to controls; p27 protein was also reduced. By ChIP assay in human prostate cell lines, we show that ETV4 binds to a specific site (-704/-696 bp upstream of the transcription start) in the CDKN1A promoter that was proven, by luciferase assay, to be functionally competent. ETV4 further controls CDKN1A expression by downregulating p53 protein: this reduction of p53 was confirmed in vivo in ETV4 mice. CONCLUSIONS: ETV4 overexpression results in the development of mPIN but not in progression to cancer. ETV4 increases prostate cell proliferation through multiple mechanisms, including downregulation of CDKN1A and its p21 protein product: this in turn is mediated through direct binding of ETV4 to the CDKN1A promoter and through the ETV4-mediated decrease of p53. This multi-faceted role of ETV4 in prostate cancer makes it a potential target for novel therapeutic approaches that could be explored in this ETV4 transgenic model.

New NO- and H2S-releasing doxorubicins as targeted therapy against chemoresistance in castration-resistant prostate cancer: in vitro and in vivo evaluations.

Chemotherapy for castration-resistant prostate cancer (CRPC) is only temporarily effective due to the onset of chemoresistance. We investigated the efficacy of NO- and H2S-releasing doxorubicins (NitDox and H2SDox) in overcoming drug resistance and evaluated their safety. New and innovative NO- and H2S-releasing doxorubicins (NitDox and H2SDox) showed a good intracellular accumulation and high cytotoxic activity in vitro in an androgen-independent and doxorubicin-resistant DU-145 prostate cancer cell line. Nude mice were subcutaneously injected with 4*106 DU-145 cells and treated once a week for 3 weeks with 5 mg/kg doxorubicin, NitDox, H2SDox or vehicle, i.p. Animal weight, tumor volume, intra-tumoral drug accumulation, apoptosis and the presence of nitrotyrosine and sulfhydryl (SH) groups within the tumor, were evaluated. Cardiotoxicity was assessed by measuring troponin plasma levels and the left ventricular wall thickness. In vivo, NitDox and H2SDox accumulated inside the tumors, significantly reduced tumor volumes by 60%, increased the percentage of apoptotic cells in both the inner and the outer parts of the tumors and the presence of nitrotyrosine and SH groups. Doxorubicin treatment was associated with reduced body weight and cardiotoxicity. On the contrary, NitDox and H2SDox were well tolerated and had a better safety profile. Combining efficacy with reduced cardiovascular side effects, NitDox and H2SDox are promising novel therapeutic agents for reversing chemoresistance in CRCP.

Eculizumab treatment: stochastic occurrence of C3 binding to individual PNH erythrocytes.

BACKGROUND: C5 blockade by eculizumab prevents complement-mediated intravascular hemolysis in paroxysmal nocturnal hemoglobinuria (PNH). However, C3-bound PNH red blood cells (RBCs), arising in almost all treated patients, may undergo extravascular hemolysis reducing clinical benefits. Despite the uniform deficiency of CD55 and of CD59, there are always two distinct populations of PNH RBCs, with (C3+) and without (C3-) C3 binding. METHODS: To investigate this paradox, the phenomenon has been modeled in vitro by incubating RBCs from eculizumab untreated PNH patients with compatible sera containing eculizumab, and by assessing the C3 binding after activation of complement alternative pathway. RESULTS: When RBCs from untreated patients were exposed in vitro to activated complement in the context of C5-blockade, there was the prompt appearance of a distinct C3+ PNH RBC population whose size increased with time and also with the rate of complement activation. Eventually, all PNH RBCs become C3+ to the same extent, without differences between old and young (reticulocytes) PNH RBCs. CONCLUSIONS: This study indicates that the distinct (C3+ and C3-) PNH RBC populations are not intrinsically different; rather, they result from a stochastic all-or-nothing phenomenon linked to the time-dependent cumulative probability of each individual PNH red cell to be exposed to levels of complement activation able to trigger C3 binding. These findings may envision novel approaches to reduce C3 opsonization and the subsequent extravascular hemolysis in PNH patients on eculizumab.

Assessment of the InSiGHT Interpretation Criteria for the Clinical Classification of 24 MLH1 and MSH2 Gene Variants.

Pathogenicity assessment of DNA variants in disease genes to explain their clinical consequences is an integral component of diagnostic molecular testing. The International Society for Gastrointestinal Hereditary Tumors (InSiGHT) has developed specific criteria for the interpretation of mismatch repair (MMR) gene variants. Here, we performed a systematic investigation of 24 MLH1 and MSH2 variants. The assessments were done by analyzing population frequency, segregation, tumor molecular characteristics, RNA effects, protein expression levels, and in vitro MMR activity. Classifications were confirmed for 15 variants and changed for three, and for the first time determined for six novel variants. Overall, based on our results, we propose the introduction of some refinements to the InSiGHT classification rules. The proposed changes have the advantage of homogenizing the InSIGHT interpretation criteria with those set out by the Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium for the BRCA1/BRCA2 genes. We also observed that the addition of only few clinical data was sufficient to obtain a more stable classification for variants considered as "likely pathogenic" or "likely nonpathogenic." This shows the importance of obtaining as many as possible points of evidence for variant interpretation, especially from the clinical setting.

Polymorphism of the complement receptor 1 gene correlates with the hematologic response to eculizumab in patients with paroxysmal nocturnal hemoglobinuria.

Complement blockade by eculizumab is clinically effective in hemolytic paroxysmal nocturnal hemoglobinuria. However, the response is variable and some patients remain dependent on red blood cell transfusions. In 72 patients with hemolytic paroxysmal nocturnal hemoglobinuria on eculizumab we tested the hypothesis that response may depend on genetic polymorphisms of complement-related genes. We found no correlation between the complement component C3 genotypes and the need for blood transfusions. On the other hand, we found a significant correlation with the HindIII polymorphism of a complement regulatory gene, the complement receptor 1 (CR1) gene. At this locus two co-dominant alleles are known, of which H (common) is associated with high expression, whereas L (rare) is associated with low expression of CR1 on red blood cells. Patients who still needed blood transfusion on eculizumab accounted for 18% of the H/H homozygotes, 33% of the H/L heterozygotes and 68% of the L/L homozygotes (P=0.016). Thus, patients with paroxysmal nocturnal hemoglobinuria who have the L/L genotype are seven times more likely to be sub-optimal responders to eculizumab. Both in vitro and in vivo we found that the CR1 HindIII genotype correlates with the abundance of paroxysmal nocturnal hemoglobinuria red cells that have bound C3, and with the kinetics of C3 binding. These results are consistent with the notion that by affecting C3 binding the CR1 genotype influences the response to eculizumab treatment, and this emerges as a novel example of pharmacogenetics.

Identification and molecular characterization of a novel 55-kb deletion recurrent in southern Italy: the Italian (G) γ((A) γδß)°-thalassemia.

OBJECTIVES: To characterize the molecular basis of a ß-thalassemia defect in subjects with mild microcytosis associated with normal Hb A2 and increased levels of Hb F. METHODS: Six subjects from three apparently unrelated families from Campania (southern Italy) have been investigated using DNA restriction analysis, inverse PCR, cloning, sequencing, multiplex ligation-dependent probe amplification (MLPA), quantitative real-time PCR, and gap-PCR. RESULTS: We have identified a novel 55-kb ß-globin gene cluster deletion in three unrelated families: the Italian (G) γ((A) γδß)°-thalassemia. This deletion removes most of the ß-globin cluster. The 5' breakpoint was within the (A) γ-globin exon 2, and the 3' breakpoint was within a 160-bp palindrome: the breakpoint-flanking regions present a microhomology (5'-TGGG-3') that, together with the palindromic structure, may have contributed to the recombination. CONCLUSIONS: Large deletions of ß-globin gene cluster are usually found in single families. Here, we report about the novel Italian (G) γ((A) γδß)°-thalassemia we have found in three families. Twenty years ago, the characterization of the first family was challenging, whereas that of the other families has taken advantage of nowadays techniques. The relatively high frequency of this novel deletion in southern Italy suggests that it should be tested, together with the Sicilian (δß)°-thalassemia, in Italian and Mediterranean families with microcytosis, normal Hb A2, and increased Hb F levels.

Easy genotyping of complement C3 'slow' and 'fast' allotypes by tetra-primer amplification refractory mutation system PCR.

Complement C3 'slow' and 'fast' allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 'slow' and 'fast' alleles by a single tube reaction.

Multiplex tetra-primer amplification refractory mutation system PCR to detect 6 common germline mutations of the MUTYH gene associated with polyposis and colorectal cancer.

BACKGROUND: We describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) for detecting MUTYH mutations, which are associated with colorectal adenomas and colorectal cancer. METHODS: We designed specific T-ARMS-PCR assays for 6 mutations (Y165C, G382D, 1395_7delGGA, Y90X, 1103delC, and R231H) selected on the basis of the frequency of their occurrence. We also designed a set of 3 multiplex T-ARMS PCR assays, each for detection of 2 mutations. We tested DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations. RESULTS: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing. CONCLUSIONS: T-ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 common MUTYH mutations with use of as few as 3 single tube PCR reactions. It could be useful to carry out large population-based epidemiologic studies.

Beta-thalassaemia-87 C-->G: relationship of the Hb F modulation and polymorphisms in compound heterozygous patients.

A clinical, haematological, biochemical and molecular study was carried out in 17 patients affected with thalassaemia intermedia, who were compound heterozygotes for the beta-thalassaemia mutation beta-87 C-->G to determine the genetic basis of their clinical heterogeneity. The beta-87 was found associated with haplotype VIII (beta-87/VIII) or V (beta-87/V). The 10 patients with the beta-87/VIII showed milder clinical conditions, with significantly higher levels of haemoglobin (Hb) (9.8 +/- 1.1 g/dl vs. 8.5 +/- 1.3 g/dl) and fetal haemoglobin (Hb F) (6.2 +/- 1.5 g/dl vs. 2.6 +/- 1.5 g/dl; P = 0.0034) and higher synthesis of (G)gamma ((G)gamma/(Total)gamma 69.4 +/- 2.6% vs. 42.8 +/- 16.2%; P = 0.0042) than the seven patients with the beta-87/V. The beta-87/VIII showed a configuration of rare polymorphisms in the 5' sub-haplotype, which have been reported to exert an increasing effect on Hb F. They were "T"-158 (G)gamma-globin gene, T-A-G in pre-(G)gamma framework, (TG)(11)(CG)(3) in the (G)gamma-IVS2, (AT)(9)N(12)(AT)(10) in LCR-HS2; in contrast, the haplotype V had, respectively, "C", T-G-A (TG)(19)(CG)(2)CACG in the (G)gamma-IVS2, and (AT)(10)N(12)(AT)(11). In all patients the beta-87 was associated with the (AT)(9)T(5) motif 5' beta-globin gene with increased affinity for the BP-1 protein, and with the (TG)(13) in the (A)gamma-IVS2. The high increase of the Hb F, mostly of the (G)gamma-type, strongly suggests the hypothesis that the 'T'-158 (G)gamma plays a principal role and that the other polymorphisms could exert a cooperative role in the modulation of Hb F in patients with erythropoietic stress.

Hb Gun Hill: a further de novo observation.

Here we report the third observation (the second de novo) of unstable Hb Gun Hill or [b91(F7)-95(FG2)Leu-His-Cys-Asp-Lys-->0]. The two-year old male carrier showed low mean corpuscular hemoglobin (MCH) and mean hemoglobim concentration (MCHC), 8.5% fetal hemoglobin and trade mark 30% variant hemoglobin. Mild hemolytic symptoms were detected seven years later. DNA sequencing and functional studies of mRNA and globin chains were performed.

In vivo gene marking of rhesus macaque long-term repopulating hematopoietic cells using a VSV-G pseudotyped versus amphotropic oncoretroviral vector.

BACKGROUND: Gene transfer efficiency into primitive hematopoietic cells may be limited by their expression of surface receptors allowing vector entry. Vectors pseudotyped with the vesicular stomatitis virus (VSV-G) envelope do not need receptors to enter cells, and therefore may provide superior transduction efficiency. METHODS: Using a competitive repopulation model in the rhesus macaque, we examined in vivo gene marking levels of blood cells transduced with two vectors: (i) a VSV-G pseudotyped retrovirus and (ii) a conventional amphotropic retrovirus. The VSV-G vector, containing the human glucose-6-phosphate dehydrogenase (G6PD) gene, was constructed for treatment of severe hemolytic anemia caused by G6PD deficiency. Three myeloablated animals were transplanted with peripheral blood CD34+ cells, half of which were transduced with the VSV-G vector and the other half with the amphotropic vector. RESULTS: In all animals post-transplantation, levels of in vivo marking in circulating granulocytes and mononuclear cells were similar: 1% or less with both vectors. In one animal, the human G6PD enzyme transferred by the VSV-G vector was expressed in erythrocytes, early after transplantation, at a level of 45% of the endogenous rhesus G6PD protein. CONCLUSIONS: In a clinically relevant animal model, we found similar in vivo marking with a VSV-G pseudotyped and a standard amphotropic oncoretroviral vector. Amphotropic receptor expression may not be a limiting factor in transduction efficiency, but VSV-G pseudotypes possess other practical advantages that may make them advantageous for clinical use.

Beta+45 G --> C: a novel silent beta-thalassaemia mutation, the first in the Kozak sequence.

A family from the Southeast of Italy was found to have a novel beta-globin mutant, beta+45 G-->C, with the features of a silent beta-thalassaemia mutation. It was asymptomatic in two heterozygotes, but its interaction with the severe thalassaemia mutation beta-IVS-II-654 C-->T worsened the haematological and biosynthetic phenotype in two compound heterozygotes; moreover, another compound heterozygote, who was also heterozygote for the alphaalphaalpha(anti3.7), suffered from thalassaemia intermedia. The mutation was found associated in cis with the IVS-II-754 T-->C substitution, which did not lead to abnormally spliced mRNA. Furthermore, the amount of beta+45 mRNA was the same as the betaA mRNA in the reticulocytes of the carriers. In vitro transcription/translation experiments demonstrated that the beta+45 G-->C decreased the efficiency of translation of the beta-globin chain by about 30%: this slight impairment was consistent with the observed clinical phenotype. The beta+45 G-->C is the first mutation found in the Kozak sequence (GACACCATGG) of the beta-globin gene and the first one at the position -6 upstream the ATG. The Kozak consensus sequence plays a major role in the initiation of translation process. The present finding supports the hypothesis that the G in position -6 is important in this process.

Hb Bronte or alpha93(FG5)Val-->Gly: a new unstable variant of the alpha2-globin gene, associated with a mild alpha(+)-thalassemia phenotype.

We report a new unstable variant identified in three carriers of a family from East Sicily; it was named Hb Bronte after the place from which the family originated. DNA sequencing from nucleotides -181 to +894 (alpha1) and to +884 (alpha2) revealed a GTG-->GGG substitution at codon 93 of the alpha2-globin gene. The MCV and MCH values were at the lower end of the normal range in the carriers. On cation exchange high performance liquid chromatography (HPLC), the Hb A2 level was apparently increased to around 6%, and a small abnormal peak (0.3-0.4%) was detected after Hb A2. Two abnormal bands were detected by cellulose acetate electrophoresis: a major band (about 3-4%) migrated between Hb A and Hb F; a minor band (<1%) migrated between Hb A2 and carbonic anhydrase. Normal values of Hb A2 were detected by DEAE microchromatography. On reversed phase HPLC the variant chain was not detected, and most likely it was eluted with the alpha chain peak. The isopropanol stability test was very slightly positive in the carriers. Hemolytic symptoms were absent with the exception of indirect bilirubin, which was at high borderline in 2/3 carriers. In biosynthesis in vitro, the specific activity of the alpha chains was much higher than that of the beta-globin chains, and the alpha/beta biosynthetic ratio in the mother and proband was of the beta-thalassemia (thal) type (2.24 and 2.54, respectively). Time course experiments showed that the increase of the 3H-specific activity of the peak containing normal and variant alpha chains was not linear and was much higher than that of beta chains; moreover, the alpha/beta biosynthetic ratio varied during the 2 hours incubation.

Epidemiology of the delta globin alleles in southern Italy shows complex molecular, genetic, and phenotypic features.

We characterized mutations and haplotypes of the delta-globin gene (HBD, MIM# 142000) in two regions of southern Italy. Mutations were discovered by screening for individuals with Hb A2<2%. In Basilicata, about 10,000 students were screened and 53 carriers in 43 unrelated families were diagnosed; in Campania, cases were referred through a routine thalassemia counseling service. Twelve alleles were detected. Four were novel variants [Hb A2-Metaponto (g.238C>A), Hb A2-Campania (g.302C>A), Hb A2-Lucania (g.393C>G), and Hb A2-Capri (g.443G>T)]. Hb A2-Lucania was not inherited but had arisen in the propositus. Two were novel mutations in the noncoding regions: the substitutions IVS2+6T>A, presumably affecting the splicing, and g.-126A>T in the GATA motif presumably affecting transcription. All novel alleles were found associated with haplotypes common in the Mediterranean area. The remaining six were alleles already described. The Hb A2-Yialousa (g.82G>T) was the most prevalent (42/63 families). Recurrent homologous crossing-over events have, most likely, linked this allele to Haplotypes IX (24 families), IV (10 families), or III (seven families). The ratio of Haplotypes IX:IV:III was about the same in the two regions. The rare allele Hb A2-NYU (g.39T>A) was found in 11 families from Basilicata associated with Haplotype I. All the 11 families lived in a restricted area extending from the Ionian Coast for 15 km along the Angri and Sinni Rivers. A founder effect most probably gave origin to this isolated group. The remaining four alleles were rare: the 7.2-kb deletion Corfù type (HBD g.-5946_1262del), Hb A2-Mitsero (g.14C>T), Hb A2-Etolia (g.385T>C), Hb A2-Coburg (g.1376G>A). Correlation between genotype and phenotype was established in 103 carriers.