Development and evaluation of a secondary reference panel for BCR-ABL1 quantification on the International Scale.

Molecular monitoring of chronic myeloid leukemia patients using robust BCR-ABL1 tests standardized to the International Scale (IS) is key to proper disease management, especially when treatment cessation is considered. Most laboratories currently use a time-consuming sample exchange process with reference laboratories for IS calibration. A World Health Organization (WHO) BCR-ABL1 reference panel was developed (MR(1)-MR(4)), but access to the material is limited. In this study, we describe the development of the first cell-based secondary reference panel that is traceable to and faithfully replicates the WHO panel, with an additional MR(4.5) level. The secondary panel was calibrated to IS using digital PCR with ABL1, BCR and GUSB as reference genes and evaluated by 44 laboratories worldwide. Interestingly, we found that >40% of BCR-ABL1 assays showed signs of inadequate optimization such as poor linearity and suboptimal PCR efficiency. Nonetheless, when optimized sample inputs were used, >60% demonstrated satisfactory IS accuracy, precision and/or MR(4.5) sensitivity, and 58% obtained IS conversion factors from the secondary reference concordant with their current values. Correlation analysis indicated no significant alterations in %BCR-ABL1 results caused by different assay configurations. More assays achieved good precision and/or sensitivity than IS accuracy, indicating the need for better IS calibration mechanisms.

Identification and differentiation of Iranian Leishmania species by PCR amplification of kDNA.

We describe the specific identification of Leishmania species in Iran using PCR DNA amplification of kDNA. For this purpose, we designed a pair of primers--upstream 5' TCGCAGAACGCCCCTACC 3' and downstream 5'-AGGGGTTGGTGTAAAATAGGC 3'--specific for conserved sequences of kDNA of Leishmania. Using this primer, we identified 3 different amplified fragments from the kDNA of the WHO reference Leishmania species. Two bands at 620 and 850 bp were identified for L. major (MRHO/IR/64/Nadim-1 strain) and only 1 band at 620 bp was identified for L. major (P strain). Therefore, we could differentiate 2 Leishmania species. Also, 1 band at 830 bp was identified for L. tropica (MHOM/Sudan/58/OD strain). We determined the sequence analysis of 2 DNA bands (620 and 850 bp) obtained from kDNA of L. major (MRHO/IR/64/Nadim-1). A total of 157 bp from the 5' site and 234 bp from the 3' site were sequenced and showed about 28% homology between 620 and 850 bp fragments. This technique could amplify as little as 1 fg of DNA and was used to differentiate kDNA samples isolated from Iranian patients with cutaneous leishmaniasis. These data indicate that the primer used for PCR amplification of kDNA is specific and can be used for diagnostic and epidemiological purposes.

Strategies for the analysis of oncogene overexpression. Studies of the neu oncogene in breast carcinoma.

The development of a consistent strategy for the analysis of oncogene expression at the cellular level is essential for understanding the roles of these genes in the development and progression of human neoplasia. Detection of the neu oncogene products in breast carcinoma was selected as a model for analysis of oncogene expression. Fifty-two primary human breast carcinomas were evaluated by quantitation of neu DNA amplification and mRNA expression and by localization of neu mRNA and protein (p 185) at the cellular level by in situ hybridization (ISH) and immunohistochemistry (IHC). The specificity and sensitivity of the molecular and immunologic probes for neu were established with the use of genetically engineered cell lines that overexpressed either neu or epidermal growth factor receptor (EGFR). Twenty-nine percent of breast carcinomas demonstrated neu DNA amplification and mRNA overexpression, and there was close correlation between the level of neu mRNA expression and detection of neu gene products by ISH and IHC. Thirty-two percent of carcinomas demonstrated neu mRNA overexpression by ISH. The immunohistochemical method using TA1 monoclonal antibody for p185 was exquisitely sensitive in acetone-fixed frozen sections and provided an excellent approach for judging overexpression as confirmed by the various molecular analyses. All areas of nonmalignant breast epithelium stained weakly, and a wide range of staining intensity was observed in malignant breast epithelium, with 31% of carcinomas judged to be p185 overexpressors. Heterogeneous expression of p185 was seen in some carcinomas. This study provides a strategic approach for the evaluation of oncogene expression in human tumors.

Day-night variation in prepro vasoactive intestinal peptide/peptide histidine isoleucine mRNA within the rat suprachiasmatic nucleus.

Neurons within the suprachiasmatic nuclei of the hypothalamus (SCN) appear to function as a circadian clock that controls the timing of many physiological systems. The SCN contain several chemically distinct neuronal subpopulations, including a large group of interneurons within the ventrolateral SCN that exhibit co-localizable immunoreactivity for both vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI). The purpose of the present study was to determine whether VIP/PHI neurons within the rat SCN exhibit rhythmicity in the cellular levels of the messenger RNA encoding the precursor from which both VIP and PHI are derived. Using both quantitative in situ and solution hybridization prepro-VIP/PHI mRNA levels early in the dark phase were demonstrated to be significantly higher than those 5 h after the onset of the daily light period. Since no statistically reliable (P greater than 0.05) day-night variation was observed in the levels of prepro-VIP/PHI mRNA within cortex, these data suggest that the rhythmicity in prepro-VIP/PHI mRNA is an intrinsic property of VIP/PHI-containing SCN neurons, or rhythmically driven by local synaptic events within the SCN.

Localization of vasoactive intestinal peptide and peptide histidine isoleucine immunoreactivity and mRNA within the rat suprachiasmatic nucleus.

The distribution of vasoactive intestinal peptide (VIP) and peptide histidine isoleucine (PHI) mRNA within the suprachiasmatic nucleus (SCN) of rats was evaluated by immunocytochemistry and in situ hybridization. The pattern of VIP and PHI immunoreactivity corresponded closely to the distribution of VIP/PHI mRNA within the ventrolateral SCN. Clear hybridization signal was observed within the SCN of rats killed 5 h after light onset and in rats killed 2 h after the onset of the dark phase of the light-dark cycle. Visual examination of the grain density within the autoradiographs suggested that VIP/PHI mRNA may occur in higher concentrations shortly after the onset of darkness than 5 h after the onset of the light phase.

Thyroid hormone regulates TRH biosynthesis in the paraventricular nucleus of the rat hypothalamus.

Thyroid hormone is important in the regulation of synthesis and secretion of thyroid-stimulating hormone (TSH) in the anterior pituitary, but its role in the control of hypothalamic thyrotropin-releasing hormone (TRH) is controversial. To determine whether thyroid hormone regulates the function of TRH in the hypothalamic tuberoinfundibular system, a study was made of the effect of hypothyroidism on thyrotropin-releasing hormone messenger RNA (proTRH mRNA) and TRH prohormone in the rat paraventricular nucleus. Extracts of rat hypothalamic paraventricular nucleus were examined by quantitative Northern blot analysis, and coronal sections of rat brain were examined by in situ hybridization histochemistry and immunocytochemistry. A nearly twofold increase in proTRH mRNA was observed in hypothyroid animals; this increase could be obliterated by levothyroxine treatment, suggesting an inverse relation between circulating thyroid hormone and proTRH mRNA. In situ hybridization showed that this response occurred exclusively in medial parvocellular neurons of the paraventricular nucleus. A simultaneous increase in proTRH mRNA and immunoreactive TRH prohormone in this region suggests that hypothyroidism induces both transcription and translation of the TRH prohormone in the paraventricular nucleus.

Hypertrophy of pheochromocytoma cells treated with nerve growth factor and activators of adenylate cyclase.

PC12 pheochromocytoma cells treated with nerve growth factor (NGF) in combination with high concentrations of the activators of adenylate cyclase, forskolin or cholera toxin, become more neuron-like in size than cells treated with NGF or with activators of adenylate cyclase alone. Cells treated simultaneously with NGF plus forskolin or cholera toxin paradoxically show less process outgrowth than cells treated with NGF alone. Addition of forskolin or cholera toxin to cells pretreated with NGF, however, produces enlarged cells with intact processes that are indistinguishable from cultured neurons. One possible implication of these findings is that NGF might act in concert with agents that increase intracellular cyclic AMP to cause neuronal maturation during embryogenesis, and that the proper sequence of exposure to these signals is necessary for normal development. Specific activity of acetylcholinesterase is increased by NGF but is unaffected or slightly decreased by forskolin, suggesting that individual aspects of the developing neuronal phenotype are subject to different types of control.

Monoclonal antibody specific for an activated RAS protein.

Activated RAS transforming genes that encode proteins (p21s) with amino acid substitutions at positions 12, 13, or 61 have been detected in 10-20% of human neoplasms. This report describes a monoclonal antibody (DWP) raised against a synthetic peptide corresponding to amino acids 5-16 of a mutated RAS gene encoding Val instead of Gly at position 12. DWP reacted in competition assays with peptides containing Val or Cys at position 12, but did not react with peptides containing Gly, Arg, Ser, Ala, Asp, or Glu at position 12. Immunoblot analysis of transformed NIH cells and human carcinoma cell lines showed that DWP reacts specifically with activated RAS proteins containing Val at position 12 and not with normal p21s or p21s activated by other amino acid substitutions at positions 12 and 61. Immunohistochemical studies showed that DWP-labeled transformed NIH cells and human carcinoma cells contained p21s with either Val or Cys at position 12 but not normal or other activated p21s. In contrast to the specificity seen with human carcinoma cell lines, analysis of formalin-fixed, primary carcinoma specimens indicated that positive immunoperoxidase staining with DWP did not necessarily correlate with immunoblot and transfection assays for the presence of activated RAS proteins. Immunohistochemical studies did show, however, that DWP preferentially binds human carcinoma cells.

Anti-lymphocyte antibody Leu-7 (HNK-1) recognizes a constituent of neuroendocrine granule matrix.

Anti-lymphocyte monoclonal antibody HNK-1 (Leu-7) reacts with the cell surfaces of natural killer (NK) lymphocytes and with myelin-associated glycoprotein (MAG). This antibody reacts intensely with normal and neoplastic adrenal medullary cells. A small proportion of normal pancreatic islet cells, anterior pituitary, and gastroenteropancreatic endocrine cells also show Leu-7 immunoreactivity. In adrenal medulla, ultrastructural immunocytochemical studies and immunoblot analyses reveal that Leu-7 reacts with an intracellular protein of MW 75 KD which is localized within the matrices of the chromaffin granules. The MW of this protein differs from those of MAG and chromogranin A. The findings suggest that Leu-7 immunoreactivity might be a new marker for specific subsets of secretory granules.