Final results of the real-life observational VICTOR-6 study on metronomic chemotherapy in elderly metastatic breast cancer (MBC) patients.

Nowadays, treatment of metastatic breast cancer (MBC) has been enriched with novel therapeutical strategies. Metronomic chemotherapy (mCHT) is a continuous and frequent administration of chemotherapy at a lower dose and so whit less toxicity. Thus, this strategy could be attractive for elderly MBC patients. Aim of this analysis is to provide insights into mCHT's activity in a real-life setting of elderly MBC patients. Data of patients ≥ 75 years old included in VICTOR-6 study were analyzed. VICTOR-6 is a multicentre, Italian, retrospective study, which collected data on mCHT in MBC patients treated between 2011 and 2016. A total of 112 patients were included. At the beginning of mCHT, median age was 81 years (75-98) and in 33% of the patients mCHT was the first line choice. Overall Response Rate (ORR) and Disease Control Rate (DCR) were 27.9% and 79.3%, respectively. Median PFS ranged between 7.6 and 9.1 months, OS between 14.1 and 18.5 months. The most relevant toxicity was the hematological one (24.1%); severe toxicity (grade 3-4) ranged from 0.9% for skin toxicity up to 8% for hematologic one. This is a large study about mCHT in elderly MBC patients, providing insights to be further investigated in this subgroup of frail patients.

Metronomic chemotherapy (mCHT) in metastatic triple-negative breast cancer (TNBC) patients: results of the VICTOR-6 study.

PURPOSE: Triple-negative breast cancer (TNBC) represents a subtype of breast cancer which lacks the expression of oestrogen receptor (ER), progesterone receptor (PR) and human epidermal growth factor receptor-2 (HER2): TNBC accounts for approximately 20% of newly diagnosed breast cancers and is associated with younger age at diagnosis, greater recurrence risk and shorter survival time. Therapeutic options are very scarce. Aim of the present analysis is to provide further insights into the clinical activity of metronomic chemotherapy (mCHT), in a real-life setting. METHODS: We used data included in the VICTOR-6 study for the present analysis. VICTOR-6 is an Italian multicentre retrospective cohort study, which collected data of metastatic breast cancer (MBC) patients who have received mCHT between 2011 and 2016. Amongst the 584 patients included in the study, 97 were triple negative. In 40.2% of the TNBC patients, mCHT was the first chemotherapy treatment, whereas 32.9% had received 2 or more lines of treatment for the metastatic disease. 45.4% out of 97 TNBC patients received a vinorelbine (VRL)-based regimen, which resulted in the most used type of mCHT, followed by cyclophosphamide (CTX)-based regimens (30.9%) and capecitabine (CAPE)-based combinations (22.7%). RESULTS: Overall response rate (ORR) and disease control rate (DCR) were 17.5% and 64.9%, respectively. Median progression free survival (PFS) and overall survival (OS) were 6.0 months (95% CI: 4.9-7.2) and 12.1 months (95% CI: 9.6-16.7). Median PFS was 6.9 months for CAPE-based regimens (95% CI: 5.0-18.4), 6.1 months (95% CI: 4.0-8.9) for CTX-based and 5.3 months (95% CI: 4.1-9.5) for VRL-based ones. Median OS was 18.2 months (95% CI: 9.1-NE) for CAPE-based regimens and 11.8 months for VRL- (95% CI: 9.3-16.7 and CTX-based ones (95%CI: 8.7-52.8). Tumour response, PFS and OS decreased proportionally in later lines. CONCLUSION: This analysis represents the largest series of TNBC patients treated with mCHT in a real-life setting and provides further insights into the advantages of using this strategy even in this poor prognosis subpopulation.

Metronomic chemotherapy for advanced breast cancer patients in the real world practice: Final results of the VICTOR-6 study.

Metronomic chemotherapy (mCHT) refers to the minimum biologically effective dose of a chemotherapy agent given as a continuous dosing regimen, with no prolonged drug-free breaks, that leads to antitumor activity. Aim of the present study is to describe the use of mCHT in a retrospective cohort of metastatic breast cancer (MBC) patients in order to collect data regarding the different types and regimens of drugs employed, their efficacy and safety. Between January 2011 and December 2016, data of 584 metastatic breast cancer patients treated with mCHT were collected. The use of VRL-based regimens increased during the time of observation (2011: 16.8% - 2016: 29.8%), as well as CTX-based ones (2011: 17.1% - 2016: 25.6%), whereas CAPE-based and MTX-based regimens remained stable. In the 1st-line setting, the highest ORR and DCR were observed for VRL-based regimens (single agent: 44% and 88%; combination: 36.7% and 82.4%, respectively). Assuming VRL-single agent as the referee treatment (median PFS: 7.2 months, 95% CI: 5.3-10.3), the longest median PFS were observed in VRL-combination regimens (9.5, 95%CI 88.8-11.3, HR = 0.72) and in CAPE-single agent (10.7, 95%CI 8.3-15.8, HR = 0.70). The VICTOR-6 study provides new data coming from the real-life setting, by adding new information regarding the use of mCHT as an option of treatment for MBC patients.

High-resolution array-CGH analysis on 46,XX patients affected by early onset primary ovarian insufficiency discloses new genes involved in ovarian function.

STUDY QUESTION: Can high resolution array-CGH analysis on a cohort of women showing a primary ovarian insufficiency (POI) phenotype in young age identify copy number variants (CNVs) with a deleterious effect on ovarian function? SUMMARY ANSWER: This approach has proved effective to clarify the role of CNVs in POI pathogenesis and to better unveil both novel candidate genes and pathogenic mechanisms. WHAT IS KNOWN ALREADY: POI describes the progression toward the cessation of ovarian function before the age of 40 years. Genetic causes are highly heterogeneous and despite several genes being associated with ovarian failure, most of genetic basis of POI still needs to be elucidated. STUDY DESIGN, SIZE, DURATION: The current study included 67 46,XX patients with early onset POI (<19 years) and 134 control females recruited between 2012 and 2016 at the Medical Cytogenetics and Molecular Genetics Lab, IRCCS Istituto Auxologico Italiano. PARTICIPANTS/MATERIALS, SETTING, METHODS: High resolution array-CGH analysis was carried out on POI patients' DNA. Results of patients and female controls were analyzed to search for rare CNVs. All variants were validated and subjected to a gene content analysis and disease gene prioritization based on the present literature to find out new ovary candidate genes. Case-control study with statistical analysis was carried out to validate our approach and evaluate any ovary CNVs/gene enrichment. Characterization of particular CNVs with molecular and functional studies was performed to assess their pathogenic involvement in POI. MAIN RESULTS AND THE ROLE OF CHANCE: We identified 37 ovary-related CNVs involving 44 genes with a role in ovary in 32 patients. All except one of the selected CNVs were not observed in the control group. Possible involvement of the CNVs in POI pathogenesis was further corroborated by a case-control analysis that showed a significant enrichment of ovary-related CNVs/genes in patients (P = 0.0132; P = 0.0126). Disease gene prioritization identified both previously reported POI genes (e.g. BMP15, DIAPH2, CPEB1, BNC1) and new candidates supported by transcript and functional studies, such as TP63 with a role in oocyte genomic integrity and VLDLR which is involved in steroidogenesis. LARGE SCALE DATA: ClinVar database (http://www.ncbi.nlm.nih.gov/clinvar/); accession numbers SCV000787656 to SCV000787743. LIMITATIONS, REASONS FOR CAUTION: This is a descriptive analysis for almost all of the CNVs identified. Inheritance studies of CNVs in some non-familial sporadic cases was not performed as the parents' DNA samples were not available. Addionally, RT-qPCR analyses were carried out in few cases as RNA samples were not always available and the genes were not expressed in blood. WIDER IMPLICATIONS OF THE FINDINGS: Our array-CGH screening turned out to be efficient in identifying different CNVs possibly implicated in disease onset, thus supporting the extremely wide genetic heterogeneity of POI. Since almost 50% of cases are negative rare ovary-related CNVs, array-CGH together with next generation sequencing might represent the most suitable approach to obtain a comprehensive genetic characterization of POI patients. STUDY FUNDING/COMPETING INTEREST(S): Supported by Italian Ministry of Health grants 'Ricerca Corrente' (08C203_2012) and 'Ricerca Finalizzata' (GR-2011-02351636, BIOEFFECT) to IRCCS Istituto Auxologico Italiano.

Hypoxic liver perfusion with mitomycin-C for treating multifocal metastases and unresectable primary tumours: a single-centre series of 42 patients.

PURPOSE: The purpose of our study was to retrospectively evaluate the feasibility, toxicity and impact on overall (OS) and disease-free (DFS) survival of intra-arterial liver perfusion with mitomycin-C (MMC) [hypoxic liver perfusion with MMC (HLPM)] in patients with multifocal liver metastases or with unresectable primary liver tumours. MATERIALS AND METHODS: Forty-two patients underwent 56 intra-arterial liver infusions with MMC between June 2001 and May 2009. The patients presented specific characteristics, i.e. they were all refractory to locoregional (LR) and/or systemic treatments. HLPM consists of selective catheterisation of the common hepatic artery, permanent occlusion of the gastroduodenal artery at its origin using metal coils, an inflated balloon catheter placement at the origin of the proper hepatic artery to block blood flow and induce hypoxia for around 10 min, MMC infusion and vascular-bed occlusion through injection of an absorbable haemostatic agent. During the procedure, the patients received anaesthesiological monitoring. Biochemical and morphological responses were evaluated, as were haematological, hepatic and systemic toxicity. RESULTS: Patients were hospitalised for 10 days on average (range 7-15). Side effects were liver toxicity in all cases, acute pancreatitis in one case and liver failure in one case. Computed tomography performed at 30 days documented a partial response (PR) in 29%, stable disease (SD) in 45% and progressive disease (PD) in 26% of patients. The response lasted 4 months on average (range 3-6). Mean overall survival (OS) was 20 months for all patients, reaching 30 months in those with colorectal carcinoma. CONCLUSIONS: The procedure is feasible, and treatmentrelated toxicity and mortality rates are acceptable. It may be considered a palliative treatment option in patients with advanced liver disease in centres with adequately experienced medical teams.

Dominant LMNA mutations can cause combined muscular dystrophy and peripheral neuropathy.

The coexistence of neurogenic and myogenic features in scapuloperoneal syndrome is rarely ascribed to a single gene. Defects in the nuclear envelope protein lamin A/C, encoded by the LMNA gene, have been shown to be associated with a variety of disorders affecting mainly the muscular and adipose tissues and, more recently, with autosomal recessive Charcot-Marie-Tooth type 2 neuropathy. This report is about a patient presenting features of myopathy and neuropathy due to a dominant LMNA mutation, suggesting that the peripheral nerve might be affected in primary LMNA myopathy. Our observations further support the marked intrafamilial and interfamilial phenotypic heterogeneity associated with lamin A/C defects.

Symptoms of depression and anxiety, and screening for mental disorders in migrainous patients.

The purpose of this clinic-based study was the assessment of symptoms of depression, anxiety, and non-specific psychiatric disorders amongst patients with migraine, compared with healthy subjects and with individuals with a non-neurological chronic disease. A cross-sectional study was carried out in which 178 individuals (migraine 51; psoriasis 35; healthy 92) were submitted to three scales: MADRS (depression), STAI-T (anxiety) and SRQ (screening for mental disorders). The subjects with migraine and psoriasis were from the Out-patient Clinics of Headache and of Dermatology, and the healthy volunteers were persons who were accompanying out-patients in the same hospital. Scores were analysed by manova and by association analysis and logistic regression. Scores of all instruments were higher in the migrainous group, but the univariate analysis of association (using cut-offs) showed significance only for suspicion of mental disorders (SRQ). By logistic regression, variables with strongest association to migraine were gender, education, and SRQ in decreasing order.

Familial periventricular heterotopia: missense and distal truncating mutations of the FLN1 gene.

OBJECTIVE: To examine the clinical and MRI associations in bilateral periventricular nodular heterotopia (BPNH) (MIM # 300049) in two families segregating a missense mutation and a C-terminal deletion of the filamin 1(FLN1) gene. BACKGROUND: Classical familial BPNH, an X-linked dominant disorder, has been associated with protein truncations or splicing mutations, which tend to cluster at the N-terminal of the FLN1 protein, causing severe predicted loss of the protein function. The clinical syndrome includes symmetrical contiguous nodular heterotopia lining the lateral ventricles, epilepsy, mild retardation to normal cognitive level in affected females, and prenatal lethality in hemizygous boys. METHODS: Clinical examination, cognitive testing, MRI, mutation analysis (direct sequencing, single-strand conformation polymorphism) in seven patients from two families with BPNH. RESULTS: In Family 1, harboring an A > T change in exon 2 (E82V), heterotopic nodules were few, asymmetric, and noncontiguous. Five boys born from affected females had died unexpectedly early in life. In Family 2, harboring an 8 base pair deletion in exon 47 (7627_7634del TGTGCCCC), heterotopic nodules were thick and contiguous. Affected females in both families showed normal to borderline IQ and epilepsy. CONCLUSION: Missense mutations and distal truncations consistent with partial loss of FLN1 function cause familial BPNH with the classical clinical phenotype including epilepsy and mild mental retardation, if any. However, missense mutations have milder anatomic consequences in affected females and are possibly compatible with live birth but short survival of boys.

Clinical and molecular genetic spectrum of autosomal dominant Emery-Dreifuss muscular dystrophy due to mutations of the lamin A/C gene.

Emery-Dreifuss muscular dystrophy (EDMD) is characterized by early contractures of the elbows and Achilles tendons, slowly progressive muscle wasting and weakness, and life-threatening cardiomyopathy with conduction blocks. We recently identified LMNA encoding two nuclear envelope proteins, lamins A and C, to be implicated in the autosomal dominant form of EDMD. Here, we report on the variability of the phenotype and spectrum of LMNA mutations in 53 autosomal dominant EDMD patients (36 members of 6 families and 17 sporadic cases). Twelve of the 53 patients showed cardiac involvement exclusively, although the remaining 41 all showed muscle weakness and contractures. We were able to identify a common phenotype among the patients with skeletal muscle involvement, consisting of humeroperoneal wasting and weakness, scapular winging, rigidity of the spine, and elbow and Achilles tendon contractures. The disease course was generally slow, but we observed either a milder phenotype characterized by late onset and a mild degree of weakness and contractures or a more severe phenotype with early presentation and a rapidly progressive course in a few cases. Mutation analysis identified 18 mutations in LMNA (i.e., 1 nonsense mutation, 2 deletions of a codon, and 15 missense mutations). All the mutations were distributed between exons 1 and 9 in the region of LMNA that is common to lamins A and C. LMNA mutations arose de novo in 76% of the cases; 2 of these de novo mutations were typical hot spots, and 2 others were identified in 2 unrelated cases. There was no clear correlation between the phenotype and type or localization of the mutations within the gene. Moreover, a marked inter- and intra-familial variability in the clinical expression of LMNA mutations exists, ranging from patients expressing the full clinical picture of EDMD to those characterized only by cardiac involvement, which points toward a significant role of possible modifier genes in the course of this disease. In conclusion, the high proportion of de novo mutations together with the large spectrum of both LMNA mutations and the expression of the disease should now prompt screening for LMNA in familial and sporadic cases of both EDMD and dilated cardiomyopathy associated with conduction system disease.

In search of the MRX genes.

Mental retardation (MR) is one of the most common human disorders. MR may be just one of the clinical signs of a complex syndrome or it may be associated with metabolic disorders or with disorders of brain development, but in many patients [nonspecific MR (NSMR)], it is the only consistent clinical manifestation. It is expected that NSMR is caused by alterations in molecular pathways important for cognitive functions. Insights into NSMR have recently come from the study of X-linked MR as eight genes were identified during the last few years. This development has represented a fundamental breakthrough in our understanding of NSMR and of cognitive functions and has opened new perspectives in the study of MR. The new genes identified are a heterogeneous group, but it is very intriguing that they are all directly or indirectly involved in signaling pathways and that the majority are proteins that regulate members of the Ras superfamily of small GTP binding proteins.

Neurite extension occurs in the absence of regulated exocytosis in PC12 subclones.

We have investigated the process leading to differentiation of PC12 cells. This process is known to include extension of neurites and changes in the expression of subsets of proteins involved in cytoskeletal rearrangements or in neurosecretion. To this aim, we have studied a PC12 clone (trk-PC12) stably transfected with the nerve growth factor receptor TrkA. These cells are able to undergo both spontaneous and neurotrophin-induced morphological differentiation. However, both undifferentiated and nerve growth factor-differentiated trk-PC12 cells appear to be completely defective in the expression of proteins of the secretory apparatus, including proteins of synaptic vesicles and large dense-core granules, neurotransmitter transporters, and neurotransmitter-synthesizing enzymes. These results indicate that neurite extension can occur independently of the presence of the neurosecretory machinery, including the proteins that constitute the fusion machine, suggesting the existence of differential activation pathways for the two processes during neuronal differentiation. These findings have been confirmed in independent clones obtained from PC12-27, a previously characterized PC12 variant clone globally incompetent for regulated secretion. In contrast, the integrity of the Rab cycle appears to be necessary for neurite extension, because antisense oligonucleotides against the neurospecific isoform of Rab-guanosine diphosphate-dissociation inhibitor significantly interfere with process formation.

Mutations in GDI1 are responsible for X-linked non-specific mental retardation.

Rab GDP-dissociation inhibitors (GDI) are evolutionarily conserved proteins that play an essential role in the recycling of Rab GTPases required for vesicular transport through the secretory pathway. We have found mutations in the GDI1 gene (which encodes uGDI) in two families affected with X-linked non-specific mental retardation. One of the mutations caused a non-conservative substitution (L92P) which reduced binding and recycling of RAB3A, the second was a null mutation. Our results show that both functional and developmental alterations in the neuron may account for the severe impairment of learning abilities as a consequence of mutations in GDI1, emphasizing its critical role in development of human intellectual and learning abilities.

X-linked Emery-Dreifuss muscular dystrophy can be diagnosed from skin biopsy or blood sample.

We have raised an anti-emerin polyclonal antibody against a fusion protein encompassing most of the hydrophilic portion of emerin. Using this antibody, we have analyzed emerin expression in Emery-Dreifuss muscular dystrophy (EDMD) patients and controls, by immunocytochemistry, in skeletal muscle and skin, and by immunoblot, in peripheral blood mononuclear cells and lymphoblasts. Emerin was localized on the surfaces of nuclei in control skeletal muscle and skin but was absent or reduced in patient skeletal muscle, was absent from the skin of patients, and was expressed only in a few nuclei in a patient's mother. Immunoblot of peripheral blood cells from EDMD patients showed absence of the emerin band, altered-size emerin, or a protein of normal molecular mass but slightly reduced quantity. The diagnosis of X-linked EDMD is normally confirmed by genetic analysis of the STA gene coding for emerin. We propose immunocytochemical evaluation of emerin expression in skin biopsies as a sensitive and more convenient tool for diagnosing X-linked EDMD and, in particular, for distinguishing it from the autosomal dominant form. This technique may be applied to suspected EDMD patients, especially sporadic cases or those with incomplete clinical phenotype, and also suspected carriers. Immunoblot of peripheral blood cells is also useful, but it may not unequivocally identify carriers and some patients.

A family of transmembrane proteins with homology to the MET-hepatocyte growth factor receptor.

In hunting for unknown genes on the human X chromosome, we identified a cDNA in Xq28 encoding a transmembrane protein (SEX) of 1871 amino acids. SEX shares significant homology with the extracellular domain of the receptors encoded by the oncogenes MET, RON, and SEA [hepatocyte growth factor (HGF) receptor family]. Further screenings of cDNA libraries identified three additional sequences closely related to SEX: these were named SEP, OCT, and NOV and were located on human chromosomes 3p, 1, and 3q, respectively. The proteins encoded by these genes contain large cytoplasmic domains characterized by a distinctive highly conserved sequence (SEX domain). Northern blot analysis revealed different expression of the SEX family of genes in fetal tissues, with SEX, OCT, and NOV predominantly expressed in brain, and SEP expressed at highest levels in kidney. In situ hybridization analysis revealed that SEX has a distinctive pattern of expression in the developing nervous system of the mouse, where it is found in postmitotic neurons from the first stages of neuronal differentiation (9.5 day postcoitus). The SEX protein (220 kDa) is glycosylated and exposed at the cell surface. Unlike the receptors of the HGF family, p220SEX, a MET-SEX chimera or a constitutively dimerized TPR-SEX does not show tyrosine kinase activity. These data define a gene family (SEX family) involved in the development of neural and epithelial tissues, which encodes putative receptors with unexpected enzymatic or binding properties.

An X chromosome-linked gene encoding a protein with characteristics of a rhoGAP predominantly expressed in hematopoietic cells.

An increasingly large number of proteins involved in signal transduction have been identified in recent years and shown to control different steps of cell survival, proliferation, and differentiation. Among the genes recently identified at the tip of the long arm of the human X chromosome, a novel gene, C1, encodes a protein that appears to represent a newly discovered member of the group of signaling proteins involved in regulation of the small GTP binding proteins of the ras superfamily. The protein encoded by C1, p115, is synthesized predominantly in cells of hematopoietic origin. It is characterized by two regions of similarity to motifs present in known proteins: GAP and SH3 homologous regions. Its localization in a narrow cytoplasmic region just below the plasma membrane and its inhibitory effect on stress fiber organization indicate that p115 may down regulate rho-like GTPases in hematopoietic cells.

Identification of novel RFLPs in the vicinity of CpG islands in Xq28: application to the analysis of the pattern of X chromosome inactivation.

Probes for CpG islands were cloned from the distal long arm of the human X chromosome; three of them were found to be polymorphic. A HindIII RFLP was identified by the probe 2-25 (DXS606), and it was mapped to the Xq27-Xq28 boundary. Probes 2-19 (DXS605) and 2-55 (DXS707), which identify EcoRI and MspI polymorphisms, respectively, have been mapped to the distal part of Xq28, in the G6PD-RCP/GCP gene region. Probe 2-19 has been further localized about 16 kb from the 3' end of the G6PD gene. The new RFLPs may be useful for the precise mapping of the many disease genes localized in this part of the human X chromosome. Probe 2-19 is highly informative, and it has been studied in greater detail. Using the methylation-sensitive rare-cutter enzyme EagI in conjunction with the polymorphic EcoRI site, we were able to demonstrate that the RFLP may be used both to study randomness of X chromosome inactivation and for carrier detection in X-linked syndromes where nonrandom X inactivation occurs. It is conceivable that the combined use of 2-19 and of the probes described so far (pSPT-PGK and M27 beta) will make analysis of X inactivation feasible in virtually every female.

The CpG island in the 5' region of the G6PD gene of man and mouse.

The nucleotide (nt) sequence of the entire CpG island in the 5' region of the human glucose-6-phosphate dehydrogenase-encoding gene (G6PD) and of the corresponding region in mouse was determined. In comparison to the human gene, the 5' region of the mouse G6PD gene has highly reduced G + C and CpG dinucleotide content, but maintains the functional features of a CpG island, as it is differentially methylated on the active vs. the inactive X chromosome. In addition to the expected conservation of exons, nt sequence comparison showed that several boxes are highly conserved between the two species in the 5' flanking DNA and in the first intron. Moreover, the conservation of the position of most CpG dinucleotides in the promoter region and in one of the upstream boxes, at about -900, gives support to the hypothesis that, in each island, specific CpGs play a major role in the regulation of gene expression.

Molecular cloning and analysis of the fragile X region in man.

The fragile X syndrome (FraX), the most common inherited form of mental retardation, has been located to Xq27.3. As a step in the molecular analysis of this mutation, we have cloned a contiguous 1.8 Mb region containing the entire fragile X region in YAC and cosmid clones. The cloned area defines a region of 50 kb containing a CpG island, found to be selectively methylated in patients expressing the fragile X phenotype. In this 50kb area we have localised the breakpoints of four somatic cell hybrids selected to break at the position of the fragile site. Fluorescence in-situ hybridisation of cosmids flanking this area shows that the breakpoints, the CpG island and the fragile site coincide.

Isolation of sequences that span the fragile X and identification of a fragile X-related CpG island.

Yeast artificial chromosomes (YACs) were obtained from a 550-kilobase region that contains three probes previously mapped as very close to the locus of the fragile X syndrome. These YACs spanned the fragile site in Xq27.3 as shown by fluorescent in situ hybridization. An internal 200-kilobase segment contained four chromosomal breakpoints generated by induction of fragile X expression. A single CpG island was identified in the cloned region between markers DXS463 and DXS465 that appears methylated in mentally retarded fragile X males, but not in nonexpressing male carriers of the mutation nor in normal males. This CpG island may indicate the presence of a gene involved in the clinical phenotype of the syndrome.