Establishment of a novel human myeloid leukaemia cell line (FKH-1) with t(6;9)(p23;q34) and the expression of dek-can chimaeric transcript

Calcein-labeled B16 melanoma (B16M) cells were injected intraportally, and in vivo video microscopy was used to study the distribution and damage of cancer cells arrested in the liver microvasculature over a period of 4 hours. The contribution of glutathione (GSH)-dependent antioxidant machinery to the possible oxidative stress-resistance mechanism of B16M cell was determined by in vitro incubation with the selective inhibitor of GSH synthesis L-buthionine (S,R)-sulphoximine (BSO) before B16M cell injection in untreated and 0.5-mg/kg lipopolysaccharide (LPS)-treated mice. In addition, untreated and LPS-treated isolated syngeneic hepatic sinusoidal endothelial cells (HSE) were used to determine in vitro their specific contribution to B16M cell damage. Trauma inherent to intrasinusoidal lodgement damaged 35% of B16M cells in both normal and LPS-treated mouse liver. The rest of the arrested B16M cells remained intact in normal liver for at least 4 hours, although their damaged cell percentage significantly (P < .05) increased since the second hour in normal mice injected with BSO-treated cells and since the first hour in LPS-treated mice given untreated cells. Recombinant human interleukin-1 receptor antagonist (rHuIL-1-Ra) given to mice 15 minutes before LPS significantly (P < .05) abrogated B16M cell damage. On the other hand, 40% of the B16M cells co-cultured with unstimulated HSE and 70% of the co-cultured with LPS-treated HSE became sensitive to endothelial cell-mediated damage after BSO treatment. These results demonstrate that a high intracellular level of GSH protects B16M cells from possible in vivo and in vitro sinusoidal cell-mediated oxidative stress, contributing to the mechanism of metastatic cell survival within the hepatic microvasculature.     

Acute transformation of chronic myelomonocytic leukemia with t(1;3) (p36;q21) abnormality

A 66-year-old man was given a peripheral blood test because of low grade fever. Leukocytosis was detected, and the blood and bone marrow findings were consistent with those of chronic myelomonocytic leukemia. Three months later the hematological findings were: WBC 58,800/microliter (19% blastoid cells, 22% monocytes), Hb 9.0 g/dl, and a platelet count of 116 x 10(4)/microliters. A bone marrow examination revealed the presence of 52.6% blastoid cells and dysmegakaryocytopoiesis, including micromegakaryocytes. Serum and urinary lysozyme levels were elevated. Karyotypic analysis detected t(1; 3) (p36;q21), but not major bcr/abl mRNA. The patient was given a diagnosis of acute transformation of chronic myelomonocytic leukemia. Despite treatment, he died about 3 months later. t(1;3) is occasionally observed in cases of myelodysplastic syndrome (MDS) and leukemia. Patients with t(1;3) often exhibit dysmegakaryocytopoiesis; furthermore, acute leukemia develops more readily in those who also have MDS. Cases of long-term survival are rare.     

Fetal t(5p;21q) misdiagnosed as monosomy 21: a plea for in situ hybridization studies

We report on a girl having congenital chloride diarrhea (CCD) who has been followed for 7 years and 6 months sequentially. Dilated intestinal loops, marked enlargement of the abdominal circumference of the fetus and hydramnios were noted by ultrasound examination at 31 weeks of gestation. After delivery by cesarean section for hydramnios, she excreted profuse watery yellow green stools with marked abdominal distension. At 4 months of age, hypochloremia, hyponatremia and a high concentration of chloride in the stool were identified. She was diagnosed as having CCD. Because it was difficult to administer a large volume of potassium chloride (KCl), and sodium chloride (NaCl), we decided to administer spironolactone. After administration of spironolactone, we could generate correct serum electrolytes using less amounts of KCl. At 7 years and 6 months of age, her body size was within normal limits and her intellectual, mental and physical development had been normal. In spite of normal serum electrolytes, blood pH and the presence of chloriduria, secondary hyperaldosteronism was noted. We consider that spironolactone may be useful to decrease the amount of KCl administration in the neonatal period, but frequent measurements of renin, angiotensin and aldosterone would be necessary for adequate control in CCD cases.     

Rearrangement of the human CDC5L gene by a t(6;19)(p21;q13.1) in a patient with multicystic renal dysplasia

Alpha-2-adrenergic receptor (alpha2-AR) agonists potently inhibit the activity of noradrenergic neurons of the locus coeruleus (LC), an effect that may be mediated by the A- and/ or C-subtypes of alpha2-AR (alpha2A- and alpha2C-AR). To gain insight into the functional significance of these alpha2-AR subtypes in the LC, we have examined their ultrastructural localization by using subtype-specific antibodies. We recently demonstrated that alpha2A-ARs are localized prominently in axon terminals and catecholaminergic dendrites in the LC. In the present study, we sought to identify the subcellular substrates underlying alpha2C-AR actions in the LC by analyzing the ultrastructural distribution of alpha2C-AR immunoreactivity (alpha2C-AR-IR) in sections that were dually labeled for the catecholamine-synthesizing enzyme tyrosine hydroxylase (TH). Alpha-2C-AR-IR was predominantly localized in dendrites, most of which also contained immunolabeling for TH. Within such dendrites, alpha2C-AR-IR was associated with the plasma membrane and occasionally Golgi cisternae and tubulovesicles. The vast majority of dendrites containing alpha2C-AR-IR received asymmetric (excitatory) contacts from unlabeled axon terminals that often contained dense core vesicles. Alpha-2C-AR-IR was observed in some unmyelinated axons and astrocytic processes that were apposed to TH-immunoreactive dendrites but was rarely associated with axon terminals. These results provide the first ultrastructural evidence that alpha2C-ARs (1) are localized postsynaptically in catecholaminergic neurons of the LC and (2) may be strategically situated to modulate the activation of LC neurons by excitatory inputs.     

Biological characteristics of chronic eosinophilic leukemia cells with a t(2;5)(p23;q35) translocation

We studied the biological features of eosinophils in a patient with chronic eosinophilic leukemia and a unique t(2:5)(p23;q35) translocation. Microscopic and cytochemical studies revealed no particular abnormalities, although more than 90% of the peripheral eosinophils had a density lighter than 1.080 g/ml. Clonogenic assay disclosed that myeloid progenitor cells possessed the translocation, although in vitro eosinophilopoiesis seemed normal, and there were also hematopoietic cells with a normal karyotype. In a surface marker study, EG1 was positive on 34.0% of the eosinophils, while EG2 positivity was only 0.5%. Eosinophilopoietic growth factors and adhesion molecules were virtually absent with the exception of GM-CSF and CD11b. Functional studies showed that chemotaxis for C5a was normal, although that for IL-2 or FMLP was attenuated. In addition, leukotriene C4 production was decreased while O2- production was intact. These findings indicated that our patient's eosinophils were not in an activated state despite their extreme hypodensity, and suggested that the leukemic eosinophils had slight defects of cellular function. These characteristics may have saved the patient from the multiple organ damage which occurs in typical hypereosinophilic syndrome.     

A lymphohistiocytic variant of anaplastic large cell lymphoma with demonstration of the t(2;5)(p23;q35) chromosome translocation

Although a seemingly simple concept, sample volume and the reaction vessel size are important considerations when undertaking an alkaline hydrolysis of a phosphoprotein, particularly if the phosphoamino acid of interest is either phosphohistidine or phosphotyrosine. It should be noted that the experiments conducted in this article used large concentrations of both phosphotyrosine and the most stable form of phosphohistidine (8), which highlights the problems that may be faced during phosphoamino acid analysis of a protein sample that contains only small amounts of either phosphoamino acid. Although not tested, it is likely that similar hydrolysis effects may occur for phospholysine. If the reaction volume is to be kept to a minimum and the alkaline digestion is from either a membrane blot or in solution, then the use of a mineral oil overlay should be considered to prevent concentration of the alkali and hydrolysis of the phosphate moiety.     

p21 (WAF1/CIP1) expression is induced in newly nondividing cells in diverse epithelia and during differentiation of the Caco-2 intestinal cell line

The incidence of brain metastases secondary to small cell lung cancer (SCLC) is about 35% and the treatment strategy of brain irradiation with respect to dose and fractionation is controversial. In order to evaluate treatment outcome of brain irradiation in SCLC patients with brain relapse, we retrospectively evaluated all patients treated with brain irradiation in the eastern part of Denmark from 1988 to 1992 (PCI patients excluded). During this 5-year period, 101 evaluable patients were included (44 females, 57 males) (median age 61 years; range, 39-75 years). Forty-four patients, of whom 43 were in extracerebral complete remission (CR), received extended course (EC) brain irradiation (> 45 Gy, treatment schedule > 4 weeks). Fifty-seven patients received short course (SC) brain irradiation (< 30 Gy, treatment schedule < 1 week). Among the SC treated patients, 14 were in CR, 20 had partial remission or stable disease and 23 had progressive extracerebral disease. The median survival (from diagnosis of brain metastases) in the group receiving irradiation with EC (44 patients) was 160 days (range, 74-2021 days), while the 57 patients treated with SC had a median survival of 88 days (range, 20-948 days) (P = 0.00001, Log-Rank analysis). In a subgroup of 14 patients in extracerebral CR, receiving SC irradiation, the median survival was 83 days (range, 15-948 days). When the latter patients were compared to the 43 patients in CR in the group treated with EC, a statistically significant difference was shown (P = 0.034, Log-Rank analysis). Using Cox-hazard regression analysis with backward elimination, liver metastases and poor performance status were adverse prognostic signs, although the only significant parameters of survival were gender (female vs. male, relative risk of dying 1 and 1.52, P = 0.05) and schedule of brain irradiation (extended course vs. short course, relative risk of dying, 0.36 and 1, P < 0.001). Extended course irradiation of brain relapse secondary to SCLC seems in general to be of limited value, although a significant prolonged survival at approximately 7 weeks, was obtained. The prolongation of survival does not seem worthwhile considering the length of treatment time (5-6 weeks) compared to SC treatment (1 week). However, the data do not permit evaluation of the quality of life of the patients. This retrospective evaluation suggests the need for randomized trials with carefully planned quality-of-life assessments.     

Analysis of the t(2;5) (p23;q35) translocation in CD30+ primary cutaneous lymphoproliferative disorders and Hodgkin''s disease

To observe the expression of p16, pRb, cdk4 and cyclinD1 in non-small cell lung cancers, 104 cases of resected lung cancers were collected, which included squamous cell carcinomas, adenocarcinomas and large cell carcinomas. Immunohistochemistry assay was carried out. The results showed that 67% of squamous cell carcinomas and 46% of adenocarcinomas expressed p16, 64% of squamous cell carcinomas and 85% of adenocarcinomas expressed pRb and 66% of cancers expressed p16 or pRb. About 70% of the tumors expressed cyclinD1. More than 90% of the tumors expressed cdk4 and there was an increased trend with decreasing differentiation of both squamous cell carcinomas and adenocarcinomas. Sixty-seven percent of the highly differentiated and 100% of the poorly differentiated squamous cell carcinomas expressed cdk4. The aberrant p16 and pRb gene product expression played a significant role in the development and histological subtype of lung cancers by conditioning the biological behavior of NSCLC. cdk4 was an important factor in histological differentiation.     

Establishment of a novel human B-cell line (OZ) with t(14;18)(q32;q21) and aberrant p53 expression was associated with the homozygous deletions of p15INK4B and p16INK4A genes

The novel human pre-B cell line OZ was established from a patient with an aggressive form of non-Hodgkin's lymphoma. Karyotypic analysis of both the primary tumour and OZ cells revealed several marker chromosomes, including the t(14;18)(q32;q21) translocation, which involves the Bcl-2 gene, and alterations on chromosome 17p. Southern blot analysis found identical rearrangements in the 5' region of Bcl-2 gene in the primary tumour and OZ cells. Homozygous deletions of the p15INK4B and p16INK4A genes, however, were present only in OZ cells. Western blot analysis detected aberrant small molecular-weight p53 proteins in both cell types. In addition, OZ cells no longer expressed the CD20 antigen. These findings suggest that Bcl-2 gene rearrangement and aberrant p53 expression resulted in the original B-cell tumour. A subsequent transforming event involving the p15INK4B and p16INK4A genes may have generated more immature cells with a growth advantage during in vitro culture. The genetic alterations involving p53, p15INK4B, and p16INK4A may be implicated in the aggressive form of t(14;18)(q32;q21)-bearing tumours and their poor prognosis.     

High incidence of translocations t(11;14)(q13;q32) and t(4;14)(p16;q32) in patients with plasma cell malignancies

Abnormalities involving the 14q32 region are recurrent chromosomal changes in plasma cell malignancies. Recent preliminary molecular analyses found IGH rearrangements in almost 100% of human myeloma cell lines and in 75% of patients. However, no systematic study analyzing the nature of the partner chromosomal regions have been reported thus far. To define the exact incidence of illegitimate IGH rearrangements and the respective incidence of partner genes cloned to date, we analyzed 141 patients with either multiple myeloma (MM, n = 127) or primary plasma cell leukemia (PCL, n = 14) using fluorescence in situ hybridization. The overall incidence of illegitimate recombinations was 57% (80 of 141 patients). Analysis of this incidence according to Durie and Salmon stage, patients' status, i.e., MM versus primary PCL and diagnosis versus relapse, immunoglobulin type and subtype, and beta2-microglobulin value, did not show any correlation. To analyze the nature of the partner chromosomal region, we selected probes specific for the following genes: FGFR3 (4p16), MYC (8q24), CCND1 (11q13), MAF (16q23), and BCL2 (18q21). These probes, combined with differentially labeled 14q32 probes, were used for dual-color fluorescence in situ hybridization on interphase plasma cells. Among the 80 patients with illegitimate IGH rearrangement, we identified 23 IGH-CCND1 fusion cases [i.e., t(11;14)], 17 IGH-FGFR3 fusion cases [i.e., t(4;14)], 3 IGH-MYC fusion cases [i.e., t(8;14)], and only one IGH-MAF fusion case. No IGH-BCL2 fusion case was detected. In 37 of 80 patients, none of these partner genes was involved. Analysis of cases with specific translocations according to their bioclinical features at diagnosis did not show any correlation. This study demonstrated that CCND1 and FGFR3 genes are involved together in about 50% of MM and primary PCL patients with illegitimate IGH rearrangements.     

An alternative route for multistep tumorigenesis in a novel case of hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation

Through allele-segregation and loss-of-heterozygosity analyses, we demonstrated loss of the translocation-derivative chromosome 3 in five independent renal cell tumors of the clear-cell type, obtained from three members of a family in which a constitutional t(2;3)(q35;q21) was encountered. In addition, analysis of the von Hippel-Lindau gene, VHL, revealed distinct insertion, deletion, and substitution mutations in four of the five tumors tested. On the basis of these results, we conclude that, in this familial case, an alternative route for renal cell carcinoma development is implied. In contrast to the first hit in the generally accepted two-hit tumor-suppressor model proposed by Knudson, the familial translocation in this case may act as a primary oncogenic event leading to (nondisjunctional) loss of the der(3) chromosome harboring the VHL tumor-suppressor gene. The risk of developing renal cell cancer may be correlated directly with the extent of somatic (kidney) mosaicism resulting from this loss.     

A new translocation involving chromosomes no. 5 and no. 12 t(5;12) (5q15;12p13)

A new case of translocation between chromosomes No. 5 and No. 12 is described. The translocation t(5;12) (5q15;12p13) is apparently balanced and was found in a child aged 3 1/2 years who was affected with severe psychomotor retardation. On the basis of the clinical picture it is suggested that the phenotype could be not dependent on the chromosomal alteration.