High abundance of circulating megakaryocytic cells in chronic myeloid leukemia in Indian patients. Revisiting George Minot to re-interpret megakaryocytic maturation.

Circulating megakaryocytic cells abound in chronic myeloid leukemia (CML) seen in India and uniquely provide a setting for observing megakaryocytic maturation in the peripheral blood, a milieu not native to megakaryocytes. Peripheral blood megakaryocytic cells were studied in 324 cases of CML (235 chronic, 65 accelerated and 24 blastic phases). Two maturation themes were evident. Megakaryocytic blasts, especially in some cases of blast crisis, precociously make a foray into platelet formation and end up producing huge agranular or poorly granular cytoplasmic lobulated masses, that break off and come to lie in the circulation. This evidence of unsuccessful effort may exist, in a considerably attenuated form in chronic phase, alongside of the second major theme of megakaryocytic maturation centered around the familiar micromegakaryocyte, characteristic of the chronic phase. This cell is regarded as dysplastic, but produces morphologically normal platelets. The possibility that this occurs via a hitherto unstudied alternative path of platelet maturation that plays out in the peripheral blood, and the contrasting disorderly premature attempt of blasts to form platelets, represent exciting maturation processes that need further study. Our observations fortuitously constitute a revisit of the insightful exposition on the subject by George Minot nearly a century ago.

The application of next-generation sequencing-based molecular diagnostics in endometrial stromal sarcoma.

AIMS: Endometrial stromal sarcomas (ESSs) are divided into low-grade and high-grade subtypes, with the latter showing more aggressive clinical behaviour. Although histology and immunophenotype can aid in the diagnosis of these tumours, genetic studies can provide additional diagnostic insights, as low-grade ESSs frequently harbour fusions involving JAZF1/SUZ12 and/or JAZF1/PHF1, whereas high-grade ESSs are defined by YWHAE-NUTM2A/B fusions. The aim of this study was to evaluate the utility of a next-generation sequencing (NGS)-based assay in identifying ESS fusions in archival formalin-fixed paraffin-embedded tumour samples. METHODS AND RESULTS: We applied an NGS-based fusion transcript detection assay (Archer FusionPlex Sarcoma Panel) that targets YWHAE and JAZF1 fusions in a series of low-grade ESSs (n = 11) and high-grade ESSs (n = 5) that were previously confirmed to harbour genetic rearrangements by fluorescence in-situ hybridization (FISH) and/or reverse transcription polymerase chain reaction (RT-PCR) analyses. The fusion assay identified junctional fusion transcript sequences that corresponded to the known FISH/RT-PCR results in all cases. Four low-grade ESSs harboured JAZF1-PHF1 fusions with different junctional sequences, and all were correctly identified because of the open-ended nature of the assay design, using anchored multiplex polymerase chain reaction. Seven non-ESS sarcomas were also included as negative controls, and no strong ESS fusion candidates were identified in these cases. CONCLUSIONS: Our findings demonstrate good sensitivity and specificity of an NGS-based gene fusion assay in the detection of ESS fusion transcripts.

Disheveled proteins promote cell growth and tumorigenicity in ALK-positive anaplastic large cell lymphoma.

Our previous oligonucleotide array studies revealed that ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL) express high levels of the disheveled proteins (Dvls), a family of proteins that is integral to the Wnt signaling pathways. In this study, we assessed whether the Dvls are important in the pathogenesis of ALK(+)ALCL. By Western blotting, Dvl-2 and Dvl-3 were found to be highly expressed in ALK(+)ALCL cell lines and patient samples. The higher molecular weight forms, consistent with phosphorylated/active Dvl proteins, were observed in these lysates. siRNA knock-down of Dvls did not affect the Wnt canonical pathway, as assessed by the ß-catenin protein levels and nuclear localization. In contrast, the same treatment led to changes in the transcriptional activity of NFAT and the phosphorylation status of Src, both of which are known to be regulated by the Wnt non-canonical signaling pathways in other cell types. Coupled with these biochemical changes, there was a significant decrease in cell growth and soft agar colony formation. NPM-ALK, the oncogenic tyrosine kinase characteristic of ALK(+)ALCL, was found to bind to the Dvls and enhance their tyrosine phosphorylation. In conclusion, our data suggest that the Dvls contribute to the pathogenesis of ALK(+)ALCL via signaling in the Wnt non-canonical pathways. To our knowledge, this is the first report demonstrating a physical and functional interaction between the Dvls and an oncogenic tyrosine kinase.

Identification of a novel crosstalk between casein kinase 2α and NPM-ALK in ALK-positive anaplastic large cell lymphoma.

It was previously reported that ß-catenin contributes to the tumorigenesis of ALK-positive anaplastic large cell lymphoma (ALK(+)ALCL), and the oncogenic effects of ß-catenin in these tumors are promoted by NPM-ALK, an abnormal fusion protein characteristic of ALK(+)ALCL. In this study, we hypothesized that NPM-ALK promotes the oncogenic activity of ß-catenin via its functional interactions with the Wnt canonical pathway (WCP). To test this hypothesis, we examined if NPM-ALK modulates the gene expression of various members in the WCP. Using a Wnt pathway-specific oligonucleotide array and Western blots, we found that the expression of casein kinase 2α (CK2α) was substantially downregulated in ALK(+)ALCL cells in response to siRNA knockdown of NPM-ALK. CK2α is biologically important in ALK(+)ALCL, as its inhibition using 4,5,6,7-tetrabromobenzotriazole or siRNA resulted in a significant decrease in cell growth and a substantial decrease in the ß-catenin protein level. Furthermore, CK2α co-immunoprecipitated with NPM-ALK and regulated its level of serine phosphorylation, a feature previously shown to correlate with the oncogenic potential of this fusion protein. To conclude, this study has revealed a novel crosstalk between NPM-ALK and CK2α, and our data supports the model that these two molecules work synergistically to promote the tumorigenicity of these lymphomas.

Blockade of fatty acid synthase triggers significant apoptosis in mantle cell lymphoma.

Fatty acid synthase (FASN), a key player in the de novo synthetic pathway of long-chain fatty acids, has been shown to contribute to the tumorigenesis in various types of solid tumors. We here report that FASN is highly and consistently expressed in mantle cell lymphoma (MCL), an aggressive form of B-cell lymphoid malignancy. Specifically, the expression of FASN was detectable in all four MCL cell lines and 15 tumors examined. In contrast, benign lymphoid tissues and peripheral blood mononuclear cells from normal donors were negative. Treatment of MCL cell lines with orlistat, a FASN inhibitor, resulted in significant apoptosis. Knockdown of FASN expression using siRNA, which also significantly decreased the growth of MCL cells, led to a dramatic decrease in the cyclin D1 level. ß-catenin, which has been previously reported to be upregulated in a subset of MCL tumors, contributed to the high level of FASN in MCL cells, Interesting, siRNA knock-down of FASN in turn down-regulated ß-catenin. In conclusion, our data supports the concept that FASN contributes to the pathogenesis of MCL, by collaborating with ß-catenin. In view of its high and consistent expression in MCL, FASN inhibitors may hold promises for treating MCL.

Constitutive activation of metalloproteinase ADAM10 in mantle cell lymphoma promotes cell growth and activates the TNFα/NFκB pathway.

One of the main functions of A Disintegrin and Metalloproteinase 10 (ADAM10) is to regulate the bioavailability of adhesion molecules and ligands to various cellular-signaling receptors. Constitutive activation of ADAM10 has been implicated in the pathogenesis of several types of solid tumors. In this study, we found that mantle cell lymphoma (MCL) cell lines and all 12 patient samples examined expressed the active/mature form of ADAM10. In contrast, PBMCs from healthy donors (n = 5) were negative. Using immunohistochemistry, ADAM10 was readily detectable in 20 of 23 (87%) MCL tumors, but absent in 5 reactive tonsils. Knockdown of ADAM10 using short interfering RNA (siRNA) in MCL cells significantly induced growth inhibition and cell-cycle arrest, and these changes were correlated with down-regulation of cyclin D1, up-regulation of p21(waf1), and significant reductions in the TNFα production/transcriptional activity of NFκBp65. The addition of recombinant ADAM10 to MCL cells led to the opposite biologic effects. Lastly, down-regulation of ADAM10 using siRNA enhanced the growth-suppressing effects mediated by the proteasome inhibitors MG132 and bortezomib. We conclude that constitutive activation of ADAM10 contributes to the growth of MCL and therefore inhibition of ADAM10 may be a useful strategy to enhance the response of MCL to other therapeutic agents.

Interleukin 22 signaling promotes cell growth in mantle cell lymphoma.

Mantle cell lymphoma (MCL) is a specific type of aggressive B-cell non-Hodgkin lymphoma. We recently found that IL-22RA1, one of the two subunits of the interleukin 22 (IL-22) receptor, is expressed in MCL cell lines but not benign lymphocytes. In view of normal functions of IL-22 signaling, we hypothesized that the aberrant expression of IL-22RA1 may contribute to the deregulation of various cell signaling pathways, thereby promoting cell growth in MCL. In this study, we first demonstrated the expression of IL-22RA1 in all three MCL cell lines and eight frozen tumors examined using reverse transcription-polymerase chain reaction and Western blot analysis. In support of the concept that IL-22 signaling is biologically important in MCL, we found that MCL cells treated with recombinant IL-22 had a significant increase in cell growth that was associated with STAT3 activation. To investigate the mechanism underlying the aberrant expression of IL-22RA1, we analyzed the gene promoter of IL-22RA1, and we found multiple binding sites for NF-κB, a transcriptional factor strongly implicated in the pathogenesis of MCL. Pharmacologic inhibition of NF-κB resulted in a substantial reduction in the level of IL-22RA1 protein expression in MCL cells. To conclude, IL-22RA is aberrantly expressed in MCL, and we have provided evidence that IL-22 signaling contributes to the pathogenesis of MCL.

ß-catenin is constitutively active and increases STAT3 expression/activation in anaplastic lymphoma kinase-positive anaplastic large cell lymphoma.

BACKGROUND: The role of ß-catenin in cancer has been most studied in tumors of epithelial cell origin. The functional status and biological significance of this protein in anaplastic lymphoma kinase (ALK)-positive anaplastic large cell lymphoma is unknown. DESIGN AND METHODS: ALK-positive anaplastic large cell lymphoma cell lines and patients' tumor samples were examined for status of ß-catenin expression and signaling. The subcellular localization of ß-catenin was assessed using immunohistochemistry, sub-cellular fractionation and confocal microscopy, while its transcriptional activity was studied using the TOPFlash/FOPFlash luciferase reporter assay. To examine the biological significance of ß-catenin, short interfering RNA was used to knock-down its expression; the resulting biological effects were studied using trypan-blue exclusion and MTS assay, and the impact on its various downstream targets was assessed using quantitative real-time polymerase chain reaction and western blots. RESULTS: ß-catenin was transcriptionally active in three of three ALK-positive anaplastic large cell lymphoma cell lines, and this finding correlates with the nuclear localization of ß-catenin in these cells and the neoplastic cells identified in most of the patients' tumor samples. ß-catenin is biologically significant in ALK-positive anaplastic large cell lymphoma, since down-regulation of ß-catenin resulted in a significant reduction in their cell growth. Down-regulation of ß-catenin led to a marked reduction in both the total protein level and the activated/phosphorylated form of STAT3, another signaling protein previously shown to be important in the pathogenesis of ALK-positive anaplastic large cell lymphoma. In contrast to some of the oncogenic tyrosine kinases, modulation of nucleophosmin-anaplastic lymphoma kinase expression did not result in any detectable change in the protein level, nuclear localization or tyrosine phosphorylation of ß-catenin; however, inhibition of nucleophosmin-anaplastic lymphoma kinase expression significantly down-regulated the transcriptional activity of ß-catenin. CONCLUSIONS: ß-catenin signaling is constitutively active in ALK-positive anaplastic large cell lymphoma and represents a previously unknown mechanism by which the high levels of STAT3 expression and activation in these tumors are sustained. Our results suggest that the interaction between oncogenic tyrosine kinases and various cell signaling proteins may be more complex than previously believed.

Alpha 1 antitrypsin deficiency in children with chronic liver disease in North India.

OBJECTIVE: We attempted to determine the role of alpha-1-antitrypsin (AAT) deficient variants as an etiologic factor for chronic liver disease in North Indian children. DESIGN: This study investigated 1700 children (682 retrospectively and 1018 prospectively) (840 CLD, 410 neonatal cholestasis and 450 without liver disease) for AAT deficiency. SETTING: Tertiary referral center, All India Institute of Medical Sciences, New Delhi. PATIENTS: Of 1250 liver disease patients, 98 (7.8%) were suspected to be AAT deficient on the basis of screening tests (low serum AAT levels and/or absent/faint alpha-1-globulin band on serum agarose electrophoresis and/or diastase resistant PAS positive granules on liver biopsy). MAIN OUTCOME MEASURES: AAT deficient Z or S allele in suspected patients. RESULTS: Z or S allele was not observed on phenotyping (1700 subjects), or with PCR-RFLP, SSCP and sequencing done in 50 of 98 suspected AAT deficient patients. A novel mutation G-to-A at position 333 in exon V was found in two siblings having positive immunohistochemistry for AAT on liver biopsy, both of whom had significant liver disease with portal hypertension. CONCLUSION: In conclusion, AAT deficiency as an etiologic factor for chronic liver disease in childhood appeared to be uncommon in North India.

The tyrosine 343 residue of nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK) is important for its interaction with SHP1, a cytoplasmic tyrosine phosphatase with tumor suppressor functions.

The cytoplasmic tyrosine phosphatase SHP1 has been shown to inhibit the oncogenic fusion protein nucleophosmin (NPM)-anaplastic lymphoma kinase (ALK), and loss of SHP1 contributes to NPM-ALK-mediated tumorigenesis. In this study, we aimed to further understand how SHP1 interacts and regulates NPM-ALK. We employed an in vitro model in which GP293 cells were transfected with various combinations of NPM-ALK (or mutants) and SHP1 (or mutants) expression vectors. We found that SHP1 co-immunoprecipitated with NPM-ALK, but not the enzymatically inactive NPM-ALK(K210R) mutant, or the mutant in which all three functionally important tyrosine residues (namely, Tyr(338), Tyr(342), and Tyr(343)) in the kinase activation loop (KAL) of ALK were mutated. Interestingly, whereas mutation of Tyr(338) or Tyr(342) did not result in any substantial change in the NPM-ALK/SHP1 binding (assessed by co-immunoprecipitation), mutation of Tyr(343) abrogated this interaction. Furthermore, the NPM-ALK/SHP1 binding was readily detectable when each of the remaining 8 tyrosine residues known to be phosphorylated were mutated. Although the expression of SHP1 effectively reduced the level of tyrosine phosphorylation of NPM-ALK, it did not affect that of the NPM-ALK(Y343F) mutant. In soft agar clonogenic assay, SHP1 expression significantly reduced the tumorigenicity of NPM-ALK but not that of NPM-ALK(Y343F). In conclusion, we identified Tyr(343) of NPM-ALK as the crucial site for mediating the NPM-ALK/SHP1 interaction. Our results also support the notion that the tumor suppressor effects of SHP1 on NPM-ALK are dependent on its ability to bind to this oncogenic protein.

Biological and clinical significance of GSK-3beta in mantle cell lymphoma--an immunohistochemical study.

GSK-3beta, a biologically important signalling protein, is regulated by the Wnt canonical and the PI3K/Akt pathways. We recently reported that mantle cell lymphoma (MCL) frequently shows evidence of GSK-3beta inactivation, since GSK-3beta is phoshorylated at its functionally critical serine 9 residue in all MCL cell lines and the majority of MCL tumors examined. To further assess the clinical and biological significance of GSK-3beta inactivation in MCL, we employed immunohistochemistry to assess the expression of the phosphorylated/inactive form of GSK-3beta (pGSK-3beta) in 83 paraffin-embedded tumors, and correlated its expression with various biological and clinical parameters. Dichotomizing pGSK-3beta into 2 groups produced twenty-seven (32.5%) tumors assessed as negative and fifty-six (67.5%) as positive. Positive pGSK-3beta expression correlated significantly with positive nuclear expression of beta-catenin and high expression of cyclin D1 (p = 0.0025, 0.0032 Fisher's exact, respectively), both of which have been previously shown to be regulated by GSK-3beta regarding their expression levels and/or sub cellular localization in-vitro. However, no significant correlation was found between pGSK-3beta and Ki67. Of the clinical parameters, continuous pGSK-3beta status had a significant correlation with absolute lymphocyte count in blood (p = 0.0011, Spearman) and negative pGSK-3beta expression was significantly correlated with a longer overall survival (p= 0.045, HR = 1.89), but not with age at diagnosis, clinical stage or the international prognostic index. To conclude, our results support the concept that GSK-3beta inactivation, found in approximately two-thirds of MCL tumors, is biologically and clinically important in MCL.

IL-21 contributes to JAK3/STAT3 activation and promotes cell growth in ALK-positive anaplastic large cell lymphoma.

Interleukin (IL)-21 has been reported to both stimulate cell growth and promote survival in benign lymphoid cells and several types of hematopoietic neoplasms. It induces JAK3/STAT3 signaling, a biologically important cellular pathway activated in most cases of anaplastic lymphoma kinase (ALK)-expressing anaplastic large cell lymphoma (ALK(+)ALCL). Therefore, we hypothesize that IL-21 may contribute to JAK3/STAT3 activation and cell growth in ALK(+)ALCL. By reverse transcription-PCR, we found consistent expression of IL-21 receptor (IL-21R) in all ALK(+)ALCL cell lines and frozen tumors examined. IL-21 was also consistently expressed in ALK(+)ALCL tumors, although its mRNA was detectable in only one of three cell lines tested. By immunohistochemistry, we examined 10 paraffin-embedded ALK(+)ALCL tumors; all cases were positive for both IL-21 and IL-21R in these neoplastic cells. IL-21 signaling is biologically significant in ALK(+)ALCL since the addition of recombinant IL-21 enhanced the activation of JAK3/STAT3 and significantly increased cell growth in ALK(+)ALCL cell lines. However, small interfering RNA down-regulation of IL-21R significantly decreased both STAT3 activation and cell growth. IL-21R expression is not linked to nucleophosmin-ALK since forced expression of nucleophosmin-ALK and small interfering RNA down-regulation of nucleophosmin-ALK did not significantly change the expression of either IL-21R or IL-21. Our findings thus support the enhancement of JAK3/STAT3 activation and cell growth in ALK(+)ALCL via IL-21 signaling. These results further support the concept that constitutive activation of STAT3 in these tumors is multifactorial.

Plasma cell leukemia--a study of 28 cases from India.

Plasma cell leukemia (PCL) is a rare neoplasm that has not been comprehensively reported in an Indian population. We report the clinico-pathological features of 28 cases studied during 1999-2008. Organomegaly and bleeding tendency was common in primary PCL but not in secondary. Misdiagnosis as acute leukemia or the leukemic phase of lymphoma on the initial peripheral blood smear examination was frequent (31.4% cases) in the primary form of PCL. This is best addressed by an emphasis on the morphological appearances and confirmation by simple serum electrophoresis rather than by more sophisticated testing that may not be widely available. Response to treatment is poor and PCL has a poor prognosis, a situation that may be amenable to improvement by a better understanding of the biology of the disease.

Glanzmann's thrombasthenia in North Indians: sub classification and carrier detection by flow cytometry.

Thirty-three patients of Glanzmann's thrombasthenia (GT) and their families were assessed for the expression of alphaIIbbeta3 on platelet surface, by flow cytometry, to determine the common subtypes in North Indians as well as to assess the carrier status in family members of GT patients. GT was diagnosed in patients with bleeding manifestations accompanied by absent/reduced platelet aggregation, secondary to adenosine-di-phosphate, adrenaline, arachidonic acid and collagen. Based on alphaIIbbeta3 levels, 21 patients (64%) were classified as type I (as alphaIIbbeta3 was absent), 4 patients (12%) as type II and 8 patients (24%) as type III. Eight out of 20 fathers, 10 out of 20 mothers and 20 out of 31 siblings were found to have reduced alphaIIbbeta3 levels. Reduced alphaIIbbeta3 expression was seen in 63% of parents and 65% of siblings. It is possible that low alphaIIbbeta3 levels in family members may reflect their carrier status. It is postulated that flow cytometry estimation of alphaIIbbeta3 in parents/siblings may detect carrier status in GT. It is also revealed that type I GT is the commonest subtype in North Indians.

Multiple myeloma presenting with coexisting severe marrow hypoplasia.

A 68-year-old man was referred to us with clinical and bone marrow (BM) features compatible with aplastic anemia. The correct diagnosis, hypoplasia of the BM coexisting with multiple myeloma, became apparent after noting rouleaux in the peripheral blood (PB) and approximately 50% plasma cells in the touch imprint of one of the two BM biopsies done. As standard therapy was precluded, the patient was put on dexamethasone but died within 4 days. This first case of the coexistence of untreated myeloma with aplastic BM shows that even apparently straightforward hypoplasia seen on the BM biopsy should be interpreted in conjunction with the PB smear and the BM touch imprint findings. Among other things, the BM biopsy and imprint should be repeated if the PB has findings such as rouleaux that do not fit with straightforward aplastic anemia. The combination of myeloma and BM aplasia precludes standard therapy and is rapidly fatal.

Constitutive activation of the Wnt canonical pathway in mantle cell lymphoma.

Aberrations of the Wnt canonical pathway (WCP) are known to contribute to the pathogenesis of various types of cancer. We hypothesize that these defects may exist in mantle cell lymphoma (MCL). Both the upstream and downstream aspects of WCP were examined in MCL cell lines and tumors. Using WCP-specific oligonucleotide arrays, we found that MCL highly and consistently expressed Wnt3 and Wnt10. beta-catenin, a transcriptional factor that is a downstream target of WCP, is localized to the nucleus and transcriptionally active in all 3 MCL cell lines examined. By immunohistochemistry, 33 (52%) of 64 MCL tumors showed nuclear localization of beta-catenin, which significantly correlated with the expression of the phosphorylated/inactive form of GSK3beta (p-GSK3beta; P = .011, Fisher). GSK3beta inactivation is directly linked to WCP stimulation, since addition of recombinant sFRP proteins (a naturally occurring decoy for the Wnt receptors) resulted in a significant decrease in p-GSK3beta. Down-regulation of DvL-2 (an upstream signaling protein in WCP) by siRNA or selective inhibition of beta-catenin using quercetin significantly decreased cell growth in MCL cell lines. To conclude, WCP is constitutively activated in a subset of MCL and it appears to promote tumorigenesis in MCL.

Diagnostic considerations in prolymphocytes in pleural fluid: a case report.

BACKGROUND: Prolymphocytes are nucleolated cells that are the defining features of the 2 chronic lymphoproliferative disorders, prolymphocytic leukemia (PLL) and chronic lymphocytic leukemia (CLL) with increased prolymphocytes. Prolymphocytes appear relatively unfamiliar in cytopathology practice, and, particularly when present in body fluids, may resemble blasts or adult T-cell leukemia/ lymphoma (ATLL) cells. CASE: A 32-year-old man, referred to us with a diagnosis of acute leukemia, presented with shortness of breath for 2 months and loss of appetite for 3 months. He had enlarged liver and spleen, 6 and 8 cm, respectively, below the costal margin and pleural effusion. The raised total leukocyte count chiefly comprised prolymphocytes that, especially in the pleural fluid, had prominent nucleoli and significant pleomorphism, raising the possibility of blasts or ATLL. CONCLUSION: Prolymphocytes in body fluids can be misinterpreted as blasts or even ATLL cells. Better awareness among cytopathologists about prolymphocytes and the disease states in which they occur, as well as insistence, in a clinical setting of leukemia, on interpreting the pleural fluid in relation to the clinical and laboratory findings, especially those of the peripheral blood and bone marrow, can prevent misdiagnosis. Equally importantly, immunophenotyping must be done in such situations.