Coexistence of lymphoproliferative and myeloproliferative neoplasms with simultaneous CALR and JAK2 V617F mutations.

BACKGROUND: Hematopoietic malignancies are a group of blood cell disorders characterized by abnormal hematopoietic proliferation. OBJECTIVE: The identification of specific clinicopathologic characteristics and tumor-related gene status provides critical information on potential therapeutic targets. METHODS: The specimens were tested with immunohistochemistry, flow cytometry, RT-PCR and fragment analysis. RESULTS: In this study, a patient with a long history of tobacco use was reported with a diagnosis of simultaneous low-grade B-cell lymphoproliferative disorder (LPD) and myeloproliferative neoplasm (MPN). Mutational analysis revealed that JAK2 V617F mutation and CALR mutation with 52bp deletion were present in this patient. CONCLUSION: These results suggest that lymphoproliferative and myeloproliferative neoplasms may coexist, although the pathogenetic mechanism of coexisting hematologic requires further investigation. Additionally, the data indicate that JAK2 V617F and CALR mutations are not mutually exclusive and the actual frequency of simultaneous JAK2 V617F and CALR mutations is unknown. Whether the coexistence of these mutations imposes any biological or clinical significance awaits further investigation.

Gain of copy number and amplification of the RET gene in lung cancer.

RET rearrangement represents a unique molecular subset of lung cancer. The identification of specific clinicopathologic characteristics and RET gene status would provide critical information on targeted therapeutics. In this study, we investigated the patterns of RET gene in a series of lung carcinomas. Of one hundred and sixteen tumors, a low frequency (1.7%) of RET translocation was identified. Only two specimens of lung adenocarcinomas displayed the rearrangement of RET in 54% and 78% of tumor cells respectively. A high incidence of gain of copy number (3-4 copies) and amplification (≥ 5 copies) of the RET gene was observed in 52% and 12% of all 116 samples. An association between increased copy number of RET and EGFR mutation was statistically significant (p < 0.05) in these lung carcinomas. This study sheds light on the unique molecular characteristics of the RET gene in lung carcinomas.

Diagnosis of paroxysmal nocturnal hemoglobinuria in peripheral blood and bone marrow with six-color flow cytometry.

AIM: Identification of paroxysmal nocturnal hemoglobinuria (PNH) by detecting a glycophosphatidylinositol-anchored defect by flow cytometry is presently the standard method of choice for diagnosing PNH. However, the selection of suitable markers will be critical and significantly affect the determination and quantification of PNH clones in various cell lineages. MATERIALS & METHODS: In this study, we investigated the performance of various immunophenotypic markers including CD59, GPHA (a clustered antigen, CD235a), CD33, CD15 and fluorescent aerolysin (FLAER) combined with CD16, CD24 and CD14 in a PNH panel using six-color flow cytometry. RESULTS: The results strongly indicate that these markers can collectively and effectively identify and quantify PNH clones in erythrocyte, granulocyte and monocyte populations derived from peripheral blood and bone marrow (BM). A sensitivity threshold as low as 0.01% in identifying PNH clones in erythrocyte and granulocyte populations from peripheral blood is achieved by this panel in a series dilution assay. In addition, a direct side-by-side comparison between BM and peripheral blood from the same patients suggests that the FLAER PNH test is capable of identifying to PNH clones in BM specimens. CONCLUSION: The data support the premise that a six-color flow cytometry PNH panel using the combination of CD59, CD235a, CD33, CD15, FLAER, CD16, CD24 and CD14 can enhance and improve the current methods used in diagnosis and management of PNH by specifically identifying PNH clones in the erythrocyte, granulocyte and monocyte population.

Risk stratification of plasma cell neoplasm: insights from plasma cell-specific cytoplasmic immunoglobulin fluorescence in situ hybridization (cIg FISH) vs. conventional FISH.

UNLABELLED: We directly compared the results of routine fluorescence in situ hybridization (FISH) and plasma cell-specific cytoplasmic immunoglobulin (cIg) FISH from 75 paired samples for myeloma risk stratification. CIg FISH improves test specificity and sensitivity and tends to eliminate borderline results. It proves that most plasma cells (PCs) consistently carry the abnormality in myelomas with an IGH translocation, whereas routine FISH detects these cells only at variably low levels. BACKGROUND: Routine cytogenetic analysis of plasma cell neoplasms (PCNs) has a low sensitivity. Conventional fluorescence in situ hybridization (FISH) is not plasma cell (PC) specific and results are diluted by other cells in the sample. Although PC-specific FISH testing has been recommended for multiple myeloma (MM) risk stratification, eg, by combining cytoplasmic immunoglobulin (cIg) staining with FISH, the benefits of cIg FISH have never been directly demonstrated in a controlled study. PATIENTS AND METHODS: Seventy-five samples from patients with PCNs were analyzed by concomitant conventional FISH and cIg FISH with probes for t(4;14), t(11;14), t(14;16), -13, 17p-, and +3. The results were compared for their reliability, specificity, and consistency. RESULTS: Apart from marginally improving detection threshold in samples with low PC burden, cIg FISH identified more abnormal cases (50 vs. 47 cases) and more chromosome abnormalities (113 vs. 103 events) than did conventional FISH. It differentiated del(13q) in myelodysplasia from MM. Remarkably, cIg FISH consistently identified a high percentage of abnormal PCs in all cases. It detected IGH translocation in 78% to 100% of PCs in all but 2 positive cases, whereas conventional FISH detected 0% to 46% in these cases (median, 91% vs. 9%). The abnormal cells found in patients with 17p- were 19% to 96% by cIg FISH vs. 0% to 13% by conventional FISH (median, 54% vs. 9%). Cases with insufficient PCs for cIg FISH had only normal conventional FISH results. CONCLUSION: CIg FISH improves reliability of FISH testing for PCNs by eliminating borderline results. In myelomas with an IGH translocation, myeloma cells invariably carry the abnormality.

pRb-mediated control of epithelial cell proliferation and Indian hedgehog expression in mouse intestinal development.

BACKGROUND: Self-renewal of the epithelium of the small intestine is a highly regulated process involving cell proliferation and differentiation of stem cells or progenitor cells located at the bottom of the crypt, ending ultimately with extrusion of the terminally differentiated cells at the tip of villus. RESULTS: Here, we utilized the Cre/loxP system to investigate the function of the retinoblastoma protein, pRb in intestinal epithelium. pRb null mice displayed a profoundly altered development of the intestine with increased proliferation and abnormal expression of differentiation markers. Loss of pRb induces cell hyperproliferation in the proliferative region (crypt) as well as in the differentiated zone (villi). The absence of pRb further results in an increase in the population of enterocytes, goblet, enteroendocrine and Paneth cells. In addition, differentiated enteroendocrine cells failed to exit the cell cycle in the absence of pRb. These proliferative changes were accompanied by increased expression of Indian hedgehog and activation of hedgehog signals, a known pathway for intestinal epithelial cell proliferation. CONCLUSION: Our studies have revealed a unique function of pRb in intestine development which is critical for controlling not only the proliferation of a stem cell or progenitor cell population but that of terminally differentiated cells as well.

Cellular senescence requires CDK5 repression of Rac1 activity.

Cellular senescence is a tumor-suppressive process characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated beta-galactosidase (SA-beta-Gal). We report here a role for CDK5 in induction of senescent cytoskeletal changes. CDK5 activation is upregulated in senescing cells. The increased activity of CDK5 further reduces GTPase Rac1 activity and Pak activation. The repression of the activity of the GTPase Rac1 by CDK5 is required for expression of the senescent phenotype. CDK5 regulation of Rac1 activity is necessary for actin polymerization accompanying senescent morphology in response to expression of pRb, activated Ras, or continuous passage. Inhibition of CDK5 attenuates SA-beta-Gal expression and blocks actin polymerization. These results point to a unique, nonneuronal role for CDK5 in regulation of Rac1 activity in senescence, illuminating the mechanisms underlying induction of senescence and the senescent shape change.

ERM proteins and Cdk5 in cellular senescence.

Cellular senescence is a tumor-suppressive process instigated by proliferation in the absence of telomere replication, by cellular stresses such as oncogene activation, or by activation of the retinoblastoma tumor suppressor protein, pRb. This process is characterized by an irreversible cell cycle exit, a unique morphology, and expression of senescence-associated-beta-galactosidase (SA-beta-gal). Despite the potential biological importance of cellular senescence, little is known of the mechanisms leading to the senescent phenotype. We have recently discovered that expression of active pRb induces expression and altered localization of the ERM family member ezrin, an actin-binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of cdk5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells. Cdk5 activity increases in senescing cells and is required for expression of SA-beta-gal and for actin polymerization accompanying acquisition of the senescent morphology. These results begin to illuminate the mechanisms underlying induction of senescence and the senescent shape change and describe new pathways that may contribute to the ability of senescent cells to influence tumor growth.

pRb inactivation in senescent cells leads to an E2F-dependent apoptosis requiring p73.

Senescent cells in which pRb is inactivated undergo apoptosis on attempted reinitiation of DNA synthesis. To further explore the cell death resulting from loss of pRb function in senescent cells, we employed a temperature-sensitive pRb mutant protein (tspRb). We found that tspRb inactivation results in rapid E2F reactivation and subsequent S-phase reentry associated with the up-regulation of E2F target gene expression and cyclin E-dependent kinase activity. Total inhibition of cyclin-dependent kinase 2 activity results in a cell cycle arrest on pRb loss and a nearly complete suppression of apoptosis. Furthermore, blocking of E2F activity with a dominant-negative DP1 inhibits S-phase reentry and cell death following tspRb inactivation. Finally, inhibition of p73 activity abolishes apoptosis but not S-phase entry on pRb inactivation, suggesting that activation of E2F in senescent cells can result in the use of p73 as a cell death effector. Interestingly, senescent cells rescued from apoptosis maintain their altered shape and express senescence-associated beta-galactosidase despite loss of pRb function. Thus, maintenance of the terminal cell cycle arrest of senescent cells requires continuous pRb-mediated inactivation of E2F activity, the reappearance of which in these irrevocably altered cells triggers a cell death program instead of an inappropriate resumption of cell cycling.

Role of the retinoblastoma protein in differentiation and senescence.

The retinoblastoma protein pRb is functionally inactivated in most human cancers. Numerous studies in cell culture and animal models suggest that pRb has a unique ability to encourage and enforce permanent cell cycle withdrawal, consistent with its role as a tumor suppressor protein. This cell cycle withdrawal has a generic component involving repression of transcription of genes required for proliferation. In addition, numerous studies hint at additional specific roles for pRb in differentiation of certain tissue types. Further, pRb appears to play a central role in the process of cellular senescence, a tumorsuppressive process characterized by proliferative arrest and phenotypic changes. Both differentiation and senescence pathways influenced by pRb involve direct and indirect interactions with the core machinery involved in cell-type-specific differentiation and cell shape control. This review focuses on pRb's role as an participant in osteoblast differentiation illustrative of a broader role in terminal differentiation. In addition, novel pathways activated by pRb in its role as an inducer of cellular senescence will be discussed.

Increased ezrin expression and activation by CDK5 coincident with acquisition of the senescent phenotype.

Passage of normal cells in culture leads to senescence, an irreversible cell cycle exit characterized by biochemical changes and a distinctive morphology. Cellular stresses, including oncogene activation, can also lead to senescence. Consistent with an anti-oncogenic role for this process, the tumor suppressor pRb plays a critical role in senescence. Reexpression of pRb in human tumor cells results in senescence-like changes including cell cycle exit and shape changes. Here we show that senescence is accompanied by increased expression and altered localization of ezrin, an actin binding protein involved in membrane-cytoskeletal signaling. pRb expression results in the stimulation of CDK5-mediated phosphorylation of ezrin with subsequent membrane association and induction of cell shape changes, linking pRb activity to cytoskeletal regulation in senescent cells.