ALK gene products in anaplastic large cell lymphomas and Hodgkin's disease

The translocation t(2;5)(p23;q35), discovered in CD30+ anaplastic large cell (ALC) lymphomas, creates a potentially oncogenic fusion gene, part of which is contributed by a novel tyrosine kinase, ALK. Absence of ALK expression from normal hematolymphoid cells provides a basis for the morphologic assessment of t(2;5). The distribution of the t(2;5) in ALC lymphomas and Hodgkin's disease (HD), as assayed by nonmorphologic methods, is controversial. We used in situ hybridization and/or immunohistology to show ALK gene products in 85 ALC lymphomas, 82 HD cases, 40 other lymphoproliferations, as well as in 6 HD- and 4 ALC lymphoma-derived cell lines. ALK gene products were restricted to t(2;5)-positive ALC lymphoma cell lines and tumor cells of 16 primary non-B cell, common-type ALC lymphomas. These were mainly from young patients with initial lymphonodal disease. ALK expression was not detectable in any other specimen, including all cases of HD and HD-like type ALC lymphoma as well as secondary ALC lymphomas. Full congruence was noted for labeling results obtained with both methods. In agreement with cytogenetic analyses, but at variance with recently published studies, ALK gene expression distinguishes a subset of ALC lymphomas from other CD30+ lymphomas, including HD. The results do not support concepts attributing a significant role to the t(2;5) in the development of HD.     

ALK expression defines a distinct group of T/null lymphomas ("ALK lymphomas") with a wide morphological spectrum

The t(2;5)(p23;q35) translocation associated with CD30-positive anaplastic large cell lymphoma results in the production of a NPM-ALK chimeric protein, consisting of the N-terminal portion of the NPM protein joined to the entire cytoplasmic domain of the neural receptor tyrosine kinase ALK. The ALK gene products were identified in paraffm sections by using a new anti-ALK (cytoplasmic portion) monoclonal antibody (ALKc) that tends to react more strongly than a previously described ALK1 antibody with the nuclei of ALK-expressing tumor cells after microwave heating in 1 mmol/L ethylenediaminetetraacetic acid buffer, pH 8.0. The ALKc monoclonal antibody reacted selectively with 60% of anaplastic large cell lymphoma cases (60 of 100), which occurred mainly in the first three decades of life and consistently displayed a T/null phenotype. This group of ALK-positive tumors showed a wide morphological spectrum including cases with features of anaplastic large cell lymphoma "common" type (75%), "lymphohistiocytic" (10%), "small cell" (8.3%), "giant cell" (3.3%), and "Hodgkin's like" (3.3%). CD30-positive large anaplastic cells expressing the ALK protein both in the cytoplasm and nucleus represented the dominant tumor population in the common, Hodgkin's-like and giant cell types, but they were present at a smaller percentage (often with a perivascular distribution) also in cases with lymphohistiocytic and small cell features. In this study, the ALKc antibody also allowed us to identify small neoplastic cells (usually CD30 negative) with nucleus-restricted ALK positivity that were, by definition, more evident in the small cell variant but were also found in cases with lymphohistiocytic, common, and "Hodgkin's-like" features. These findings, which have not been previously emphasized, strongly suggest that the neoplastic lesion (the NPM-ALK gene) must be present both in the large anaplastic and small tumor cells, and that ALK-positive lymphomas lie on a spectrum, their position being defined by the ratio of small to large neoplastic cells. Notably, about 15% of all ALK-positive lymphomas (usually of the common or giant cell variant) showed a cytoplasm-restricted ALK positivity, which suggests that the ALK gene may have fused with a partner(s) other than NPM. From a diagnostic point of view, detection of the ALK protein was useful in distinguishing anaplastic large cell lymphoma cases of lymphohistiocytic and small cell variants from reactive conditions and other peripheral T-cell lymphoma subtypes, as well as for detecting a small number of tumor cells in lymphohemopoietic tissues. In conclusion, ALK positivity appears to define a clinicopathological entity with a T/null phenotype ("ALK lymphomas"), but one that shows a wider spectrum of morphological patterns than has been appreciated in the past.     

A case of cutaneous Ki-1 positive anaplastic large cell lymphoma in a hemodialysed patient

In obstruction of both lacrimal ducts at present the most frequently used method is conjunctivo(dacryo)cystorhinostomy using a Jones bypass cannula. The effectiveness of the procedure is 85-90%. The drainage cannulas can be straight or bent and are as a rule made from pyrex glass. The patients should be highly motivated and made familiar with possible complications, i.e. most frequently escape of the cannula. With regard to the necessary postoperative cooperation the operation is not recommended in young children. The authors performed in 1996-1997 a total of 8 conjunctivo(dacryo)cystorhinotomies. The lacrimal sac is opened into the nose under general anaesthesia by the endonasal route using endoscopes. In one instance the authors used a straight silicone cannula which fell out after three weeks. In the remaining patients they used a bent silicone cannula. Seven patients evaluate their condition after surgery as markedly improved and would be willing to have the operation again. One female patient (after escape of a straight cannula and later reinsertion of a bent cannula) would hesitate to have another operation. The authors evaluate their experience with this type of bypass operation as very satisfactory.     

Lymphomatoid papulosis followed by Ki-1 positive anaplastic large cell lymphoma: proliferation of a common T-cell clone

We report a case of lymphomatoid papulosis (LyP) followed by Ki-1 positive anaplastic large cell lymphoma (LCL). A 33-year-old man developed subcutaneous nodules in the left inguinal region and the left thigh after a seven-year-history of self-healing papulonecrotic lesions of LyP. Histological and immunohistochemical examination of the subcutaneous nodules revealed LCL. DNA was isolated from a nodule of the initial stage of LyP in 1988, a subcutaneous nodule of LCL in 1993, and a papule of LyP in 1993 which appeared after chemotherapy for LCL. T cell receptor gene rearrangement analysis demonstrated an identical rearranged pattern in all the three specimens, indicating that a common T cell clone proliferated throughout the course in both the LyP and LCL lesions.     

Cytologic findings in the sarcomatoid variant of large cell anaplastic (Ki-1) lymphoma. A case report

Anaplastic large cell (Ki-1) lymphomas are a recently described subtype of non-Hodgkin's lymphoma. A single case of the sarcomatoid variant has recently been reported. Below we report the fine needle aspiration findings in an additional case of the sarcomatoid variant of Ki-1 lymphoma. The diagnosis was made only in retrospect with the aid of immunostains because the cytologic findings were more in keeping with a sarcoma. Striking vascular structures in the smears suggested angiosarcoma or liposarcoma. Additional features were pleomorphic cells with eccentric nuclei and abundant, tapering cytoplasm; spindle cells; multinucleation; cytoplasmic vacuolization; and prominent nucleoli. The smear pattern was composed of dispersed cells with some cohesive groups within an acute inflammatory background with a virtual absence of lymphoglandular bodies. These findings, atypical of lymphoma, broaden the spectrum of possible cytologic findings in lymphoid malignancies and highlight the possible utility of immunostaining. The distinction of lymphoma from sarcoma or carcinoma is of therapeutic importance.     

Cellular kinetic differences between Hodgkin''s and anaplastic large cell lymphomas: relation to the expression of p34cdc2 and cyclin B-1

Normal human subjects were tested for their ability to discriminate the orientation of a square plaque tilted in depth, using two different tasks: a grasping task and a perceptual matching task. Both tasks were given under separate monocular and binocular conditions. Accuracy of performance was measured by use of an opto-electronic motion analysis system, which computed the hand orientation (specifically, a line joining the tips of the thumb and index finger) as the hand either approached the target during grasping or was used to match the target. In all cases there was a very strong statistical coupling between hand orientation and target orientation, irrespective of viewing conditions. However, the matching data differed from the grasping data in showing a consistent curvature in the hand-target relationship, whereby the rate of change of hand orientation as a function of object orientation was smaller for oblique orientations than for those near the horizontal or vertical. The results are interpreted as reflecting the operation of two different mechanisms for analysing orientation in depth: a visuomotor system (assumed to be located primarily in the dorsal cortical visual stream) and a perceptual system (assumed to be located in the ventral stream). It may be that the requirements of visuomotor control dictate a primary need for absolute orientation coding, whereas those of perception dictate a need for more categorical coding.     

Detection of bcr/abl fusion transcripts by semiquantitative multiplex RT-PCR combined with a colormetric assay in Ph positive leukemia

OBJECTIVES: The EORTC Invasive Fungal Infections Cooperative Group (IFICG) conducted a prospective survey by questionnaire of all cases of invasive aspergillosis (IA) in cancer patients to ascertain current diagnostic and therapeutic approaches. METHODS: All members of the IFICG were asked prospectively to complete a detailed questionnaire for each IA case identified in their institution over a 12-month period. RESULTS: One hundred and thirty questionnaires were returned. All cases were independently evaluated (DWD & JC) and 123 were eligible. Cases came from 20 hospitals in eight countries and the number of cases per institution varied from 1-21. Acute myeloid leukaemia (AML) (60, 49%), acute lymphoblastic leukaemia (ALL) (21, 17%) and lymphoma (11, 9%) were the most frequent underlying diseases, and 16 (12%) patients had received an allogeneic bone marrow transplant. Pulmonary involvement was present in 87%, infection of sinuses/nose in 16% and brain in 8%. The chest radiograph was initially normal in 9% of those with primary pulmonary disease. The diagnosis was confirmed in 50%, probable in 31% and possible in 19%. The evidence for IA was on the basis of clinical and radiological features alone in 28%, with culture or histology in another 31% and 9%, respectively, and with both culture and histology in 29%. In three (2%) patients with diagnosis was based on culture or histology alone. Treatment was given to 120 patients (98%)-amphotericin B 75%, lipid-associated amphotericin B 36%, itraconazole 40%, flucytosine 12%, growth factors 33%, lobectomy 5%. At 3 months after diagnosis or first suspicion of IA, 44 (36%) patients were alive and 79 (64%) dead. Outcome was best in those with AML (30% death and 46% with a complete antifungal response or cure). Growth factors (mostly granulocyte colony stimulating factor) appeared not to influence outcome (P = 0.99). CONCLUSION: IA remains a considerable diagnostic and therapeutic challenge. No single diagnostic procedure was universally successful and a multifaceted approach including surgery is necessary. There was no discernable difference in outcome between initial therapy with amphotericin B, itraconazole or lipid-associated amphotericin B, although numbers are limited and the study was retrospective.     

Human plasminogen activator inhibitor-1 (PAI-1) deficiency: characterization of a large kindred with a null mutation in the PAI-1 gene

Plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of tissue- and urokinase-type plasminogen activators, is considered a critical regulator of the fibrinolytic system. We previously reported a child with abnormal bleeding and complete PAI-1 deficiency caused by a frame-shift mutation in exon 4 of the PAI-1 gene. The purpose of this study was to provide genetic and clinical data on the extended pedigree of the original proband to better define the phenotype associated with PAI-1 deficiency. Allele-specific oligonucleotide hybridization was used to genotype individuals, and serum PAI-1 antigen was measured by enzyme-linked immunosorbent assay. By this approach we have identified 19 individuals who are heterozygous for the PAI-1 null allele and 7 homozygous individuals with complete PAI-1 deficiency. Clinical manifestations of PAI-1 deficiency were restricted to abnormal bleeding, which was observed only after trauma or surgery in homozygous affected individuals. A spectrum of bleeding patterns was observed, including intracranial and joint bleeding after mild trauma, delayed surgical bleeding, severe menstrual bleeding, and frequent bruising. Fibrinolysis inhibitors, including epsilon-aminocaproic acid and tranexamic acid, were effective in treating and preventing bleeding episodes. Other than abnormal bleeding, no significant developmental or other abnormalities were observed in homozygous PAI-1-deficient individuals. Heterozygous PAI-1 deficiency was not associated with abnormal bleeding, even after trauma or surgery. These observations define the clinical spectrum of PAI-1 deficiency and provide additional evidence to support the hypothesis that the primary function of plasminogen activator inhibitor-1 in vivo is to regulate vascular fibrinolysis.     

CD30-positive anaplastic large cell lymphoma (ALCL) of T-cell lineage in a 14-month-old infant with perinatally acquired HIV-1 infection

We characterized the effect of ten days of training on lipid metabolism in 6 [age 37.2 (2.3) years] sedentary, obese [BMI 34.4 (3.0) kg x m(-2)] males with normal glucose tolerance. An oral glucose tolerance test was performed prior to and at the end of the 10 d of training period. The duration of each daily exercise session was 40 min at an intensity equivalent to approximately 75% of the age predicted maximum heart rate. Blood measurements were performed after an overnight fast, before and at the end of the 10 d period. Plasma triacylglycerol was significantly (p < 0.05) reduced following exercise training (2.15+/-0.29 vs. 1.55+/-0.28 mmol x l(-1)). Very low density lipoprotein-triacylglycerol was also significantly (p < 0.05) reduced (1.82+/-0.3 vs. 1.29+/-0.29 mmol x l(-1)). No significant changes in high density lipoprotein-cholesterol were observed as a result of training. Following training fasting plasma glucose and fasting plasma insulin were significantly reduced [Glucose: 5.9 (0.2) mmol x l(-1) vs. 5.3 (0.22) mmol x l(-1) (p < 0.05); Insulin 264.3 (53.8) rho x mol x l(-1) vs. 200.9 (30.1) rho x mol x l(-1), p=0.05]. The total area under the glucose curve during the OGTT decreased significantly (p < 0.05). These preliminary data suggest that short-term exercise, without concomitant loss of body mass, induces favorable changes in plasma triacylglycerol, and very low density lipoprotein-triacylglycerol and glucose tolerance but has no effect on high density lipoprotein-cholesterol.     

Expression of retinoblastoma gene product (pRb) in mantle cell lymphomas. Correlation with cyclin D1 (PRAD1/CCND1) mRNA levels and proliferative activity

Mantle cell lymphomas (MCLs) are molecularly characterized by bcl-1 rearrangement and constant cyclin D1 (PRAD-1/CCND1) gene overexpression. Cyclin D1 is a G1 cyclin that participates in the control of the cell cycle progression by interacting with the retinoblastoma gene product (pRb). Inactivation of the Rb tumor suppressor gene has been implicated in the development of different types of human tumors including some high grade non-Hodgkin's lymphomas. To determine the role of the retinoblastoma gene in the pathogenesis of MCLs and its possible interaction with cyclin D1, pRb expression was examined in 23 MCLs including 17 typical and 6 blastic variants by immunohistochemistry and Western blot. Rb gene structure was studied in 13 cases by Southern blot. Cytogenetic analysis was performed in 5 cases. The results were compared with the cyclin D1 mRNA levels examined by Northern analysis, and the proliferative activity of the tumors was measured by Ki-67 growth fraction and flow cytometry. pRb was expressed in all MCLs. The expression varied from case to case (mean, 14.1% of positive cells; range, 1.3 to 42%) with a significant correlation with the proliferative activity of the tumors (mitotic index r = 0.85; Ki-67 r = 0.7; S phase = 0.73). Blastic variants showed higher numbers of pRb-positive cells (mean, 29%) than the typical cases (10%; P < 0.005) by immunohistochemistry and, concordantly, higher levels of expression by Western blot. In addition, the blastic cases also had an increased expression of the phosphorylated protein. No alterations in Rb gene structure were observed by Southern blot analysis. Cyclin D1 mRNA levels were independent of pRb expression and the proliferative activity of the tumors. These findings suggest that pRb in MCLs is normally regulated in relation to the proliferative activity of the tumors. Cyclin D1 overexpression may play a role in the maintenance of cell proliferation by overcoming the suppressive growth control of pRb.     

Expression of the ERBB2/neu and neurofibromatosis type 1 gene products in reactive and neoplastic schwann cell proliferation

In the present study we investigated the expression of the ERBB2 protein and neurofibromin in human benign and malignant Schwann cell tumors, traumatic neuromas and peripheral nerves without pathological findings. By immunohistochemistry and Western analysis ERBB2 expression was not detectable in normal nerves but in proliferating Schwann cells of traumatic neuromas. While none of the malignant schwannomas exhibited ERBB2 expression, weak expression was seen in a small proportion of the benign schwannomas. Low levels of neurofibromin were detectable in normal nerves but not in traumatic neuromas or tumors. These data indicate an inverse expression pattern of ERBB2 and neurofibromin in human non-neoplastic Schwann cells, but not in neurinoma cells.     

Nuclear import, virion incorporation, and cell cycle arrest/differentiation are mediated by distinct functional domains of human immunodeficiency virus type 1 Vpr

The vpr gene product of human immunodeficiency virus type 1 (HIV-1) is a virion-associated protein that is essential for efficient viral replication in monocytes/macrophages. Vpr is primarily localized in the nucleus when expressed in the absence of other viral proteins. Vpr is packaged efficiently into viral particles through interactions with the p6 domain of the Gag precursor polyprotein p55gag. We developed a panel of expression vectors encoding Vpr molecules mutated in the amino-terminal helical domain, leucine-isoleucine (LR) domain, and carboxy-terminal domain to map the different functional domains and to define the interrelationships between virion incorporation, nuclear localization, cell cycle arrest, and differentiation functions of Vpr. We observed that substitution mutations in the N-terminal domain of Vpr impaired both nuclear localization and virion packaging, suggesting that the helical structure may play a vital role in modulating both of these biological properties. The LR domain was found to be involved in the nuclear localization of Vpr. In contrast, cell cycle arrest appears to be largely controlled by the C-terminal domain of Vpr. The LR and C-terminal domains do not appear to be essential for virion incorporation of Vpr. Interestingly, we found that two Vpr mutants harboring single amino acid substitutions (A30L and G75A) retained the ability to translocate to the nucleus but were impaired in the cell cycle arrest function. In contrast, mutation of Leu68 to Ser resulted in a protein that localizes in the cytoplasm while retaining the ability to arrest host cell proliferation. We speculate that the nuclear localization and cell cycle arrest functions of Vpr are not interrelated and that these functions are mediated by separable putative functional domains of Vpr.     

Differentiation of promonocytic U937 subclones into macrophagelike phenotypes regulates a cellular factor(s) which modulates fusion/entry of macrophagetropic human immunodeficiency virus type 1

Monocytes/macrophages (M/M) and CD4+ T cells are two important targets of human immunodeficiency virus (HIV) infection. Different strains of HIV-1 vary markedly in their abilities to infect cells belonging to the M/M lineage. Macrophagetropic (M-tropic) HIV-1 strains replicate well in primary lymphocytes as well as in primary macrophages; however, they generally infect T-cell lines poorly, if at all. Although promonocytic cell lines such as U937 have been used as in vitro models of HIV-1 infection of M/M, these cell lines are susceptible to certain T-cell-tropic (T-tropic) HIV-1 strains but are resistant to M-tropic HIV-1. In this study, we demonstrate that (i) certain U937 clones ("plus" clones), which are susceptible only to T-tropic HIV-1, become highly susceptible to M-tropic HIV-1 upon differentiation with retinoic acid (RA); (ii) other U937 clones ("minus" clones), which are resistant to both T- and M-tropic HIV-1, remain resistant to both viruses; and (iii) RA treatment induces expression of CCR5, a fusion/entry cofactor for M-tropic HIV-1 in both types of U937 clones, and yet enhances the fusogenicity of the plus clones, but not the minus clones, with M-tropic Env's. These results indicate that the major restriction of M-tropic HIV-1 infection in promonocytic cells occurs at the fusion/entry level, that differentiation into macrophage-like phenotypes renders some of these cells highly susceptible to infection with M-tropic HIV-1, and that CD4 and CCR5 may not be the only determinants of fusion/entry of M-tropic HIV-1 in these cells.     

Inhibition of cyclic AMP-dependent kinase by expression of a protein kinase inhibitor/enhanced green fluorescent fusion protein attenuates angiotensin II-induced type 1 AT1 receptor mRNA down-regulation in vascular smooth muscle cells

Expression of the angiotensin II type 1 receptor (AT1-R) mRNA in vascular smooth muscle cells (VSMC) is down-regulated by a variety of agonists, including growth factors, agonists of Galphaq protein-coupled receptors, and activators of adenylyl cyclase. To determine whether cAMP-dependent protein kinases (PKA) participates in AT1-R mRNA down-regulation controlled by multiple classes of receptors, a PKA inhibitor peptide (PKIalpha) was developed and expressed in rat VSMC as a fusion with the enhanced green fluorescent protein (eGFP). PKA activity elicited both by forskolin and angiotensin II is suppressed in cells expressing this fusion protein (PKIalpha-eGFP), but platelet-derived growth factor-BB does not stimulate PKA activity in this preparation. PKIalpha-eGFP expression fully inhibits the forskolin-stimulated down-regulation of AT1-R mRNA levels and blocks 50% of the effect elicited by angiotensin II. This indicates that PKA plays a substantial role in angiotensin II-stimulated AT1-R mRNA down-regulation. However, inhibition of PKA has no effect on AT1-R mRNA down-regulation caused by platelet-derived growth factor-BB. These findings show how agonists such as angiotensin II that are not normally considered as activators of PKA can use PKA-dependent processes to modulate gene expression. These findings also provide definitive evidence that PKA-dependent pathways are involved in modulation of AT1-R mRNA levels in VSMC.