Imprint cytology in tumor tissue bank quality control: an efficient method to evaluate tumor necrosis and to detect samples without tumor cells

Quality assessment of the tissue stored in a tumor biobank is crucial because it is estimated that approximately 10% of the frozen samples are unsuitable for a molecular analysis mainly because of sampling problems in the tissue. We studied the value of imprint cytology (IC) versus frozen section to quantify necrosis and tumor cells in the tissue. The amount of tumor cells and necrosis was assessed by one pathologist on the frozen sections and ICs independently on 100 consecutive tumor samples. It was expressed as a percentage on frozen sections and on a four-level semiquantitative scale for IC (0 to 3+). Overall agreement between the quantity of tumor cells on IC and on frozen section was fair (Κ = 0.23). Sensitivity and specificity of IC to detect the absence of tumor cell on the frozen section were 57% (4/7) and 98% (91/93), respectively. Overall agreement between necrosis quantification on IC and on frozen section was substantial (Κ = 0.66).Sensitivity and specificity of IC to detect significant necrosis (defined as more than 30% necrosis) were 100% (3/3) and 98% (95/97), respectively. We show that IC is efficient to semiquantify necrosis in a tumor sample and to detect significant necrosis. IC seems to be less efficient to quantify tumor cells.     

Quality assessment of intraoperative frozen sections in a teaching hospital: an analysis of 847 consecutive cases

We evaluated the accuracy of 847 consecutive frozen section diagnoses in order to develop a quality control. We also evaluated the time needed to perform them. Frozen sections and final diagnoses agreed in 92.6% and disagreed in 1.7% (14 cases). 5.8% of the cases were deferred. The only case of false-positive frozen sections (0.1%) was due to a pathologist's misinterpretation. False-negative frozen sections were due to sampling errors: in 5 cases, diagnostic tissue was present only in permanent sections of the frozen block and in 8 cases diagnostic tissue was present only in the portion of the specimen not sampled by the frozen section. One hundred and ninety two frozen sections concerned thyroid neoplasms. Thirteen cancers were diagnosed on frozen sections, 2 cancers were considered as benign and 9 cancers had a differed diagnosis. The mean duration to perform the frozen sections was 21 minutes. In conclusion, intra operative frozen section diagnosis is rapid and reliable. Discrepancies are more often false negatives due to sampling errors. Although a high rate of differed diagnosis was observed in thyroid neoplasms, frozen sections remain useful for these lesions. Imprint cytology of thyroid nodules is advisable.     

In Vitro Experiments and Numerical Simulations of Airflow in Realistic Nasal Airway Geometry

Pressure–flow relationships measured in human plastinated specimen of both nasal cavities and maxillary sinuses were compared to those obtained by numerical airflow simulations in a numerical three-dimensional reconstruction issued from CT scans of the plastinated specimen. For experiments, flow rates up to 1500 ml/s were tested using three different gases: HeO₂, Air, and SF₆. Numerical inspiratory airflow simulations were performed for flow rates up to 353 ml/s in both the nostrils using a finite-volume-based method under steady-state conditions with CFD software using a laminar model. The good agreement between measured and numerically computed total pressure drops observed up to a flow rate of 250 ml/s is an important step to validate the ability of CFD software to describe flow in a physiologically realistic binasal model. The major total pressure drop was localized in the nasal valve region. Airflow was found to be predominant in the inferior median part of nasal cavities. Two main vortices were observed downstream from the nasal valve and toward the olfactory region. In the future, CFD software will be a useful tool for the clinician by providing a better understanding of the complexity of three-dimensional breathing flow in the nasal cavities allowing more appropriate management of the patient's symptoms.     

Spectrum of molecular alterations in colorectal, upper urinary tract, endocervical, and renal carcinomas arising in a patient with hereditary non-polyposis colorectal cancer

Hereditary nonpolyposis colon cancer (HNPCC) syndrome is the most frequent hereditary cancer syndrome predisposing to cancers of various locations, especially colon, endometrium, stomach, and upper urinary tract. Carcinomas of the kidney parenchyma are not considered as an HNPCC-related tumor. HNPCC tumors are characterized by microsatellite instability (MSI) due to a defect in mismatch repair (MMR) and carry somatic frameshift mutations in mononucleotide repeats within the coding regions of key genes. We report the first case of a papillary carcinoma of the kidney in an HNPCC patient who developed carcinomas of the upper urinary tract, endocervix, and colon. Whereas the HNPCC-related tumors demonstrated MSI phenotype, loss of MSH2 protein expression, and frameshift mutations in several of the 13 target genes analyzed, the kidney cancer displayed MSS phenotype, normal MMR protein expression, and no frameshift mutation in target genes. Our observations do not support the possibility that papillary carcinomas are part of HNPCC syndrome.     

Molecular analysis of ANT1, TWINKLE and POLG in patients with multiple deletions or depletion of mitochondrial DNA by a dHPLC-based assay

ANT1, TWINKLE and POLG genes affect mtDNA stability and are involved in autosomal dominant PEO, while mutations in POLG are responsible for numerous clinical presentations, including autosomal recessive PEO, sensory ataxic neuropathy, dysarthria and ophthalmoparesis (SANDO), spino-cerebellar ataxia and epilepsy (SCAE) or Alpers syndrome. In this study, we report on the mutational analysis of ANT1, TWINKLE and POLG genes in 15 unrelated patients, using a dHPLC-based protocol. This series of patients illustrates the large array of clinical presentations associated with mtDNA stability defects, ranging from isolated benign PEO to fatal Alpers syndrome. A total of seven different mutations were identified in six of 15 patients (40%). Six different recessive mutations were found in POLG, one in TWINKLE while no mutation was identified in ANT1. Among the POLG mutations, three are novel and include two missense and one frameshift changes. Seventeen neutral changes and polymorphisms were also identified, including four novel neutral polymorphisms. Overall, this study illustrates the variability of phenotypes associated with mtDNA stability defects, increases the mutational spectrum of POLG variants and provides an efficient and reliable detection protocol for ANT1, TWINKLE and POLG mutational screening.     

Analysis of the 5' noncoding region versus the NS5b region in genotyping hepatitis C virus isolates from blood donors in France

The 5' noncoding region (5' NCR) of the hepatitis C virus (HCV) has become the standard for genotyping even though several reports show that its use can result in classification errors. The purpose of this study was to perform genotyping based on sequence analysis of the NS5b region in a set of 357 HCV strains isolated from blood donors in France in 2002 and 2003. Results were compared with those previously obtained using 5' NCR analysis, and HCV subtype distribution was reevaluated. Twenty-six of 120 strains (approximately 22%) initially identified as genotype 1b by 5' NCR region sequence analysis were reclassified as genotype 1a by NS5b region sequence analysis. Similarly, 14 of 23 strains (approximately 61%) initially identified as 2a/2c were reclassified as non-2a and non-2c subtypes, and 12 of 22 strains (approximately 45%) initially identified as 4c/4d subtypes were reclassified as non-4c and non-4d subtypes. Sequence analysis of the NS5b region also revealed 5 putative new subtype 2 variants and 2 putative new subtype 4 variants. Although these findings demonstrated full agreement between 5' NCR and NS5b sequence analysis with regard to type classification, genotyping based on phylogenetic analysis of the NS5b region is more accurate for subtype determination than genotyping based on analysis of the 5' NCR. Sequence analysis of the NS5b region is mandatory for epidemiologic studies.     

Regulatory dysfunction of the interleukin-7 receptor in CD4 and CD8 lymphocytes from HIV-infected patients--effects of antiretroviral therapy

Despite an increase in plasma IL-7 levels, the CD4 T-cell pool decrease progressively in HIV-infected patients. Here we report on our tests to check the hypothesis that defects in the IL-7 receptor system might be involved in this phenomenon. The cell surface expression of CD127 was measured ex vivo in CD4 and CD8 T lymphocytes drawn from 3 groups of HIV patients. IL-7 function was also followed in vitro by measuring IL-7-driven T-cell proliferation, the induction of the CD25 activation marker, and overexpression of the antiapoptotic molecule Bcl-2. Untreated viremic patients showed a slight but significant decrease in CD127 expression on the surface of their CD4 lymphocytes. By contrast, CD127 expression was substantially altered on the surface of CD8 T lymphocytes taken from untreated viremic patients. IL-7-induced overexpression of the antiapoptotic molecule Bcl-2 was dramatically altered in viremic patients, whereas IL-7-dependent CD25 induction and T-cell proliferation were reduced. Highly active antiretroviral therapy partially corrected these defects in patients with an undetectable viral load and CD4 counts of more than 400 cells/microL. The effects of HAART were less pronounced in patients with undetectable VL but low CD4 counts (<250 cells/microL). The IL-7 receptor is dysfunctional in the CD4 and CD8 lymphocytes of HIV-infected patients. This may be due to abnormal activation of the immune system in HIV-infected patients and may contribute to the reduced CD4 count and the altered function of the CD8 compartment.     

Modeling Hox gene regulation in digits: reverse collinearity and the molecular origin of thumbness

During the development of mammalian digits, clustered Hoxd genes are expressed following a collinear regulatory strategy, leading to both the growth of digits and their morphological identities. Because gene dosage is a key parameter in this system, we used a quantitative approach, associated with a collection of mutant stocks, to investigate the nature of the underlying regulatory mechanism(s). In parallel, we elaborated a mathematical model of quantitative collinearity, which was progressively challenged and validated by the experimental approach. This combined effort suggested a two-step mechanism, which involves initially the looping and recognition of the cluster by a complex including two enhancer sequences, followed by a second step of microscanning of genes located nearby. In this scenario, the respective rank of the genes, with respect to the 5' extremity of the cluster, is primordial, as well as different gene-specific affinities. This model accounts for the quantitative variations observed in our many mutant strains, and reveals the molecular constraint leading to thumbness; i.e., why a morphological difference must occur between the most anterior digit and the others. We also show that the same model applies to the collinear regulation of Hox genes during the emergence of external genitalia, though with some differences likely illustrating the distinct functionalities of these structures in adults.