Peri-operative management of an adult with POLG-related mitochondrial disease.

POLG-related mitochondrial disease is a rare mitochondrial disorder that is potentially associated with anaesthetic complications such as propofol-related infusion syndrome. A 19-year-old man with mitochondrial DNA deletions and POLG-related disorders presented for an elective robotic Heller-Dor myotomy for the treatment of oesophageal pseudo-achalasia associated with severe gastro-oesophageal reflux. The fasting period was minimised to reduce the risk of metabolic stress. The anaesthetic technique included a rapid sequence induction with propofol and rocuronium, a remifentanil and sevoflurane-based general anaesthesia with multimodal monitoring and peri-operative lactate-free intravenous fluids with added dextrose. The patient did not experience propofol-related infusion syndrome but did have delayed tracheal extubation due to residual neuromuscular blockade requiring a second dose of sugammadex. This report demonstrates the safety of single-use, low-dose propofol in this patient group. Patients with POLG-related mitochondrial disease may be at risk of prolonged neuromuscular blockade, and appropriate dosing of neuromuscular blocking agents with monitoring of neuromuscular blockade is strongly encouraged.

Modulation of Rb phosphorylation and antiproliferative response to palbociclib: the preoperative-palbociclib (POP) randomized clinical trial.

Background: The cyclin-dependent kinase 4 (CDK4)/6 inhibitor Palbociclib is a new standard treatment in hormone-receptor positive breast cancer patients. No predictive biomarkers have been identified and no pharmacodynamics has properly been described so far. Patients and methods: Patients with early-breast cancer were randomized 3 : 1 to oral palbociclib 125 mg daily for 14 days until the day before the surgery versus no treatment. Primary objective was antiproliferative response defined as a natural logarithm of Ki67 expression at day 15 below 1. Secondary end points were subgroups analyses and safety. Exploratory analyses included search for predictive biomarkers. Immunostainings (Ki67, RB, pRB, p16, pAKT, pER, pCDK2, CyclinD1), FISH (CCND1) and gene expression (GE) arrays were carried out at baseline and at surgery. In addition, activating PIK3CA and AKT1 mutations were assessed at baseline. Results: 74 patients were allocated to palbociclib and 26 to control. Most patients (93%) were hormone-receptor (HR)-positive, whereas 8% were HER2-positive. Palbociclib led to significantly more antiproliferative responses when compared with control (58% versus 12%, P < 0.001), and to a significantly higher Ki67 decrease (P < 0.001). In the HR-positive/HER2-negative subgroup, this antiproliferative effect was even more marked in the palbociclib arm when compared with control (70% versus 9%, P < 0.001). Palbociclib treatment led also to a significantly higher decrease from baseline in phospho-Rb when compared with control (P < 0.001). Among treated patients, changes in Ki67 correlated with changes in phospho-Rb (Spearman rank r = 0.41, P < 0.0001). GE analyses confirmed a major effect on proliferation and cell cycle genes. Among treated patients, CCNE2 expression was significantly more decreased in antiproliferative responders versus nonresponders (P = 0.006). Conclusion: Short-term preoperative palbociclib decreases Ki67 in early-breast cancer patients. Early decrease of Rb phosphorylation correlates with drug's effect on cell proliferation and could potentially identify patients with primary resistance. Clinical trial registration: NCT02008734.

[Pharmacoeconomics - Challenges for Health Professionals].

Over the last 30 years, medical expenditure has increased throughout the world. The main reasons estimated to lay behind it include aging, ever more chronic diseases and new emerging diseases, new drugs, expanded indications of current drugs, and development of pharmaceutical industry. A challenge for healthcare professionals is to sustain current quality of care and enable medical innovations while attempting to contain costs. The overall goal is to demonstrate the pharmacoeconomic value, i.e. a balance of economic, humanistic and clinical outcomes.

Influence of segmental chromosome abnormalities on survival in children over the age of 12 months with unresectable localised peripheral neuroblastic tumours without MYCN amplification.

BACKGROUND: The prognostic impact of segmental chromosome alterations (SCAs) in children older than 1 year, diagnosed with localised unresectable neuroblastoma (NB) without MYCN amplification enrolled in the European Unresectable Neuroblastoma (EUNB) protocol is still to be clarified, while, for other group of patients, the presence of SCAs is associated with poor prognosis. METHODS: To understand the role of SCAs we performed multilocus/pangenomic analysis of 98 tumour samples from patients enrolled in the EUNB protocol. RESULTS: Age at diagnosis was categorised into two groups using 18 months as the age cutoff. Significant difference in the presence of SCAs was seen in tumours of patients between 12 and 18 months and over 18 months of age at diagnosis, respectively (P=0.04). A significant correlation (P=0.03) was observed between number of SCAs per tumour and age. Event-free (EFS) and overall survival (OS) were calculated in both age groups, according to both the presence and number of SCAs. In older patients, a poorer survival was associated with the presence of SCAs (EFS=46% vs 75%, P=0.023; OS=66.8% vs 100%, P=0.003). Moreover, OS of older patients inversely correlated with number of SCAs (P=0.002). Finally, SCAs provided additional prognostic information beyond histoprognosis, as their presence was associated with poorer OS in patients over 18 months with unfavourable International Neuroblastoma Pathology Classification (INPC) histopathology (P=0.018). CONCLUSIONS: The presence of SCAs is a negative prognostic marker that impairs outcome of patients over the age of 18 months with localised unresectable NB without MYCN amplification, especially when more than one SCA is present. Moreover, in older patients with unfavourable INPC tumour histoprognosis, the presence of SCAs significantly affects OS.

Immunohistochemistry to identify EGFR mutations or ALK rearrangements in patients with lung adenocarcinoma.

BACKGROUND: Immunohistochemistry has been proposed as a specific and sensitive method to identify EGFR mutations or ALK rearrangements in lung tumours. PATIENTS AND METHODS: We assessed EGFR and KRAS by direct sequencing in 154 patients with lung adenocarcinoma. ALK rearrangements were assayed by FISH and RT-PCR. Immunohistochemistry was carried out and evaluated closely following published methods using recommended monoclonal rabbit or mouse antibodies. RESULTS: Thirteen of 36 exon 19 EGFR-mutated tumours (36%)-including 12 of 22 with p.Glu746_Ala750del (55%)-were positive with the 6B6 antibody that was raised against p.Glu746_Ala750del. One hundred eleven of 114 EGFR exon 19 wild-type tumours (97%) were negative with 6B6. Four of 21 exon 21 EGFR-mutated tumours (19%)-including 4 of 17 with p.Leu858Arg (24%)-were positive with the 43B2 antibody that was raised against p.Leu858Arg. One hundred twenty-two of 124 (98%) EGFR exon 21 wild-type tumours were negative with 43B2. Two of four ALK rearrangements-including two of three with ELM4-ALK fusion transcripts-were identified with the 5A4 antibody. Eleven of 13 tumours without ALK rearrangement (85%) were negative with 5A4. CONCLUSIONS: Immunohistochemistry is a specific means for identification of EGFR mutations and ALK rearrangements. It suffers, however, from poor sensitivity.

Interaction of a copper(II)-Schiff base complexes with calf thymus DNA and their antimicrobial activity.

The interaction of a copper complexes containing Schiff bases with calf thymus (CT) DNA was investigated by spectroscopic methods. UV-vis, fluorescence and CD spectroscopies were conducted to assess their binding ability with CT DNA. The binding constants K have been estimated from 0.8 to 9.1×10(4) M(-1). The percentage of hypochromism is found to be over 70% (from spectral titrations). The results showed that the copper(II) complexes could bind to DNA with an intercalative mode. Synergic action of Cu(II) complexes with ascorbic acid against Candida albicans induced the generation of free radicals and increased (more than 60 times) antimicrobial effect of these complexes.

Partial mosaic trisomy 5: a new case report with ocular involvement.

We describe a new case of a male patient with a small marker chromosome present as 80% mosaic, derived from chromosome 5 with presence of posterior iridolenticular synechia, high hyperopia, epicanthic folds, hypertelorism, moderate developmental delay with lack of speech, macrocephaly, and subtle dysmorphic features including micrognathia, slightly rotated ears, and polydactyly. The karyotype of our patient was as follows: 46, XY/47, XY +mar, characterized by FISH (fluorescence in situ hybridization) using the chromosome five painting probe. Ocular involvement in trisomic 5 subjects is a very rare event. To our knowledge, only two cases have been described to date. The present case contributes to the description of the ocular presentation and the distinct clinical phenotype of de novo partial trisomy 5 syndrome.

Status of HER1 and HER2 in peritoneal, ovarian and colorectal endometriosis and ovarian endometrioid adenocarcinoma.

A role for the EGF system, in particular HER1 and 2, in growth of the endometrium has been suggested but HER1 and 2 have not been studied in all locations of endometriosis and in ovarian endometrioid adenocarcinoma (OEC) which is a rare form of malignant transformation of endometriosis. Immunohistochemistry (IHC) was used for studying HER1 and HER2 in ovarian (n = 10), peritoneal (n = 10), colorectal endometriosis (n = 20) and OEC (n = 10). Fluorescent in situ hybridisation (FISH) was used for analysing the status of HER2 gene in colorectal endometriosis and OEC. All samples were negative for HER2 in both glandular and stromal cells and in glandular cells for HER1 by IHC. In 15 out of 20 colorectal endometriosis, there was a weak expression in stromal cells. Following FISH, two colorectal samples had a partial 17 aneusomy and three OEC, a 17 polysomy. The other samples were 17 disomic without HER2 amplification; HER1 and 2 do not seem to have a role in endometriosis physiopathology.

Cytotoxicity of copper(II) complexes of N-salicylidene-L-glutamate: modulation by ascorbic acid.

Cytotoxic/cytostatic activity of N-salicylidene-L-glutamato diaqua copper(II) complex (CuC) against mice leukemia cells L1210 has been estimated and their bioactivity was enhanced by addition of ascorbic acid. The Cu-complex with isoquinoline ligand (IQ-CuC) had stronger cytostatic effect (IC50 =15.6 microM) than parental complex (CuC) and its cytotoxicity several times increased in the presence of 0.1 mM ascorbic acid (IC50 =1.0 microM). The cytotoxicity has been caused by oxidative stress, enhanced creation of TBARS has been confirmed, and formation of 2',7'-dichlorofluorescein from 2',7'- dichlorodihydrofluorescein has been observed, also. Some hallmarks of apoptotic/necrotic death of L1210 cells have been observed by fluorescent microscopy after dyeing of cell with propidium iodide and Hoechst 33342. In addition, it was confirmed that both complexes in the presence of ascorbic acid cleavaged of pDNA. Although these copper complexes were initially prepared as substances with antioxidant properties we have showed that combined treatment of L1210 cells with IQCuC and ascorbic acid induced strong oxidative stress and death of cells. Our results confirmed that physiological concentration of ascorbic acid increases the cytostatic/cytotoxic efficiency of N-salicylidene-L-glutamato diaqua copper(II) complexes.

[Diagnosis of HER2 gene amplification in breast carcinoma]. / Diagnostic de l'amplification du gène HER2 dans les cancers du sein Le point de vue génétique.

Amplification of the HER2 gene, mapping to 17q21.1, is present in about 20 % of breast carcinomas. Amplification leads to an overexpression of the protein that made it possible to develop a targeted therapy by the monoclonal antibody trastuzumab (Herceptin). A good response to the treatment requires a stringent assessment of the gene status in tumours; only patients whose tumour shows a high expression of the protein or an amplification of the gene are eligible. Cases with intermediate level expression are checked by in situ hybridization, mainly by FISH, to identify amplifications in this subset of tumours. Results are sometimes difficult to interpret due to the frequent aneuploidy of the tumours. Moreover, copy number cut-offs of the gene for defining an amplification are variable according to the studies. A tumour is considered now as amplified when showing more than six HER2 copies per nucleus, or a ratio HER2 to centromere 17 greater than 2.2. The phenomenon of HER2 amplification in breast cancers is discussed in this paper, and distinguished from gene overrepresentation. It is recommended that tumours showing six to seven copies of HER2 are assessed with a kit including the centromere 17. Clusters of signals are characteristic of amplifications. The process designed for the assessment of HER2 is a model of strategies that will be used for the evaluation of markers involved in future targeted therapies.

The potential contribution of fluorescent in situ hybridization analysis to the cytopathological diagnosis of Merkel cell carcinoma.

We report the cases of two patients with head and neck Merkel cell carcinoma (MCC) who developed local recurrences confirmed by cytopathology. Interphase fluorescent in situ hybridization (FISH) analysis was performed for research purposes using centromeric probes of chromosomes 6 and 8, on cytological slides. Trisomy of chromosome 6 was found in 85% of tumour cells in the first case of MCC and case 2 exhibited trisomy 8 in 77% of tumour cells. In the absence of specific molecular markers, detection of trisomy 6 and/or trisomy 8 could help in identifying MCC. FISH analysis is easily and quickly performed on interphase nuclei obtained through fine needle aspiration and may be extended to the study of other relevant genetic abnormalities.

Molecular techniques in cytopathology practice.

In the last decade, new molecular techniques were introduced into pathology laboratories. Cytology also benefited from the innovations emerging from this new era. Molecular cytopathology (MCP) can be defined as molecular studies applied on all types of cytological specimens, namely gynaecology cytology, exfoliative non- gynaecology cytology and fine needle aspirates. The development of many new ancillary techniques has paralleled the emergence of clinical cytology as a major diagnostic specialty. Clinical applications of these techniques have been growing in the last decade. The widespread acceptance of liquid-based systems in gynaecological cytology emphasises the relation between cells and molecules. The increased use of morphology and molecular biology in human papillomavirus-induced lesions for example, showed the potential to optimise, in one single brushed sample, diagnosis and research. Cytology samples from serous effusions, the pulmonary tree, urine, and aspirations, among others, are now likely to be studied by different molecular techniques for diagnosis, prognosis, or even assessment of therapeutic targets. In this review, the main published results concerning the application of molecular techniques in different fields of cytopathology are highlighted, and their applications discussed.

Conventional and array-based comparative genomic hybridization analysis of nasopharyngeal carcinomas from the Mediterranean area.

Nasopharyngeal carcinoma (NPC) occurs with a high incidence in Southeast Asia and to a lesser extent in the Mediterranean area, especially in Tunisia, Algeria, and Morocco. Cellular gene alterations combined with latent Epstein-Barr virus infection are thought to be essential for NPC oncogenesis. To date, chromosome analysis with comparative genomic hybridization (CGH) has been reported exclusively for NPCs from Southeast Asia. Although NPCs from the Mediterranean area have several distinct clinical and epidemiological features, CGH investigations have been lacking. Chromosome analysis was therefore undertaken on a series of NPC xenografts and biopsies derived from patients of Mediterranean origin. Four xenografts were investigated with a combination of conventional CGH, array-based CGH, and comparative expressed sequence hybridization. In addition, 23 fresh NPC biopsies were analyzed with conventional CGH. Data obtained from xenografts and fresh biopsies were consistent, except that amplification of genes at 18p was observed only in xenografts derived from metastatic tissues. Frequent gains associated with gene overexpression were detected at 1q25 approximately qter (64%) and 12p13 (50%). Losses were noticed mainly at 11q14 approximately q23 (50%), 13q12 approximately q31 (50%), 14q24 approximately q31 (43%), and 3p13 approximately p23 (43%). Comparison with previous reports suggests that Mediterranean NPCs have higher frequencies of gains at 1q and losses at 13q than their Asian counterparts.

Complex translocation (8;12;21): a new variant of t(8;21) in acute myeloid leukemia.

Variants of the t(8;21)(q22;q22) involving chromosomes 8, 21, and other chromosomes account for approximately 3% of all t(8;21)(q22;q22) in acute myeloid leukemia (AML) patients. In this paper, we report a case of AML-M2 with a t(8;12;21)(q22;p12 approximately p13;q22) associated with chromosomal abnormalities, including loss of the Y chromosome and trisomy 8q22 approximately qter. Using a dual-color fluorescence in situ hybridization (FISH) analysis with ETO and AML1 probes, we demonstrated an ETO/AML1 fusion signal on the derivative chromosome 8. Using whole painting probes for chromosomes 8 and 12, we demonstrated a three-way translocation, t(8;12;21)(q22;p12 approximately p13;q22). Reverse transcription polymerase chain reaction (RT-PCR) analysis showed the presence of AML1/ETO fusion transcript. The present case highlights the importance of the combination of approaches, i.e., standard karyotyping, FISH, and RT-PCR, for the detection of variants of t(8;21)(q22;q22), shedding light on region 8q22 approximately qter which could harbor potential genes responsible for leukemogenesis.

No topoisomerase I alteration in a neuroblastoma model with in vivo acquired resistance to irinotecan.

CPT-11 (irinotecan) is a DNA-topoisomerase I inhibitor with preclinical activity against neuroblastoma (NB) xenografts. The aim was to establish in vivo an NB xenograft resistant to CPT-11 in order to study the resistance mechanisms acquired in a therapeutic setting. IGR-NB8 is an immature NB xenograft with MYCN amplification and 1p deletion, which is sensitive to CPT-11. Athymic mice bearing advanced-stage subcutaneous tumours were treated with CPT-11 (27 mg kg(-1) day(-1) x 5) every 21 days (1 cycle) for a maximum of four cycles. After tumour regrowth, a new in vivo passage was performed and the CPT-11 treatment was repeated. After the third passage, a resistant xenograft was obtained (IGRNB8-R). The tumour growth delay (TGD) was reduced from 115 at passage 1 to 40 at passage 4 and no complete or partial regression was observed. After further exposure to the drug, up to 28 passages, the resistant xenograft was definitively established with a TGD from 17 at passage 28. Resistant tumours reverted to sensitive tumours after 15 passages without treatment. IGR-NB8-R remained sensitive to cyclophosphamide and cisplatin and cross-resistance was observed with the topoisomerase I inhibitor topotecan. No quantitative or qualitative topoisomerase I modifications were observed. The level of expression of multidrug resistance 1 (MDR1), MDR-associated protein 1 (MRP1) and, breast cancer resistance protein, three members of the ATP-binding cassette transporter family was not modified over passages. Our results suggest a novel resistance mechanism, probably not involving the mechanisms usually observed in vitro.

Blastoid mantle cell lymphoma: evidence for nonrandom cytogenetic abnormalities additional to t(11;14) and generation of a mouse model.

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32), which is associated with cyclin D1 hyperexpression and a poor prognosis. MCL cases have been shown to progress to a more aggressive disease but the molecular events responsible of this phenomenon have not been determined. We have established two cell lines from the pleural effusions of two patients with MCL that we have used for further cytogenetic characterization to better define the incidence and nature of secondary chromosome abnormalities using multicolor fluorescence in situ hybridization, whole chromosome paint, and specific probes. Both cell lines grew independently without growth factors. Using CCND1/IGH-specific probes, patient UPN1 was found to have a masked t(11;14). Numerous and complex chromosomal abnormalities were found in both cell lines affecting chromosomes 2, 8, 13, 18, 22, X, and Y. These abnormalities included 8p losses, suggesting the presence of an anti-oncogene in this region, rearrangements of 8q24, MYC gene, and translocations involving 8, X, and Y chromosomes, which might be significant in the pathogenesis of MCL progression. The use of the cell lines (UPN1) allowed us to generate a mouse model of human MCL, mimicking a disseminated lymphoma and leading to the death of the animals in 4 weeks. This blastoid MCL model could be of major interest to determine molecular events involved in MCL progression, allowing isolation of involved genes and their functional characterization, and to study the effects of new chemotherapy regimens in mouse models.

Quality assessment of genetic markers used for therapy stratification.

PURPOSE: Therapy stratification based on genetic markers is becoming increasingly important, which makes commitment to the highest possible reliability of the involved markers mandatory. In neuroblastic tumors, amplification of the MYCN gene is an unequivocal marker that indicates aggressive tumor behavior and is consequently used for therapy stratification. To guarantee reliable and standardized quality of genetic features, a quality-assessment study was initiated by the European Neuroblastoma Quality Assessment (ENQUA; connected to International Society of Pediatric Oncology) Group. MATERIALS AND METHODS: One hundred thirty-seven coded specimens from 17 tumors were analyzed in 11 European national/regional reference laboratories using molecular techniques, in situ hybridization, and flow and image cytometry. Tumor samples with divergent results were re-evaluated. RESULTS: Three hundred fifty-two investigations were performed, which resulted in 23 divergent findings, 17 of which were judged as errors after re-evaluation. MYCN analyses determined by Southern blot and in situ hybridization led to 3.7% and 4% of errors, respectively. Tumor cell content was not indicated in 32% of the samples, and 11% of seemingly correct MYCN results were based on the investigation of normal cells (eg, Schwann cells). Thirty-eight investigations were considered nonassessable. CONCLUSION: This study demonstrated the importance of revealing the difficulties and limitations for each technique and problems in interpreting results, which are crucial for therapeutic decisions. Moreover, it led to the formulation of guidelines that are applicable to all kinds of tumors and that contain the standardization of techniques, including the exact determination of the tumor cell content. Finally, the group has developed a common terminology for molecular-genetic results.

Lack of interstitial chromosome 1p deletions in clinically-detected neuroblastoma.

Loss of heterozygosity (LOH) of the distal part of the short arm of chromosome 1 in neuroblastoma is a well characterised phenomenon. In addition, previous reports have described interstitial deletions outside the common region of loss on chromosome 1p36, suggesting additional tumour suppressor loci. In this study, we have searched extensively for interstitial 1p deletions in a panel of 67 neuroblastoma samples from clinically-detected cases. We used three VNTR probes and 10 dinucleotide markers from the 1p32-36 regions reported to show interstitial deletions. Fifteen (22%) tumours showed telomeric LOH without evidence for more proximal interstitial deletions. Forty-five tumours showed no LOH or allelic imbalance. Seven (10%) tumours demonstrated allelic imbalance for one or more markers. These tumours were subsequently analysed by fluorescent in situ hybridisation (FISH) and flow cytometry. The patterns found in all seven tumours were consistent with copy number changes of the entire chromosome 1, without evidence for interstitial deletions. This study indicates that interstitial deletions of chromosome 1p are rare in clinically-detected neuroblastoma when analysed by a combination of molecular and cytogenetic techniques.