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.

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.

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.

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.

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.

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.

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.

Human papillomavirus genotyping by multiplex pyrosequencing in cervical cancer patients from India.

Cervical cancer is a leading cause of cancer-related deaths among women in India. Human papillomavirus (HPV) infection is the causative agent of cervical cancer; and infection with the high-risk genotypes, predominantly HPV16 and 18,is the biggest risk factor. Vaccines targeting HPV16 and 18 have been found to confer protection in large- scale clinical trials.HPV genotyping has traditionally been carried out to screen the population "at risk" using indirect methods based on polymerase chain reaction (PCR) using consensus primers combined with various DNA hybridization techniques,and often followed by the sequencing of candidate products.Recently,a high-throughput and direct method based on DNA sequencing has been described for HPV genotyping using multiplex pyrosequencing. We present a pilot study on HPV genotyping of cervical cancer and non-malignant cervical samples using multiplex pyrosequencing. Using genomic DNA from cell lines,cervical biopsies,surgical tissues or formalin-fixed,paraffin- embedded tissue samples,we could successfully resolve 6 different HPV types out of the 7 tested,with their prevalence found to be in agreement with earlier reports. We also resolved coinfections with two different HPV types in several samples. An HPV16 genotype with a specific and recurrent sequence variation was observed in 8 cancer samples and one non-malignant sample. We find this technique eminently suited for high-throughput applications,which can be easily extended to large sample cohorts to determine a robust benchmark for HPV genotypes prevalent in India.

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.

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.

Pelger-Huet anomaly: a case report.

Impaired lobulation of neutrophils together with exaggerated clumping of chromatin, characteristic of Pelger-Huet anomaly was seen as an incidentalfinding in a 43-year old man who presented with depression. Peripheral blood and bone marrow findings and cytochemistry of the abnormal cells are described and the disease entity discussed.

Comparison of three different staining techniques for intraoperative assessment of nodal metastasis in breast cancer.

Imprint cytology has increasingly been used for intraoperative assessment of nodal status in breast cancer. We carried out this study to compare the efficacy of Jenner Giemsa (JG), hematoxylin-eosin (H&E), and Papanicolaou (Pap) stains for intraoperative lymph node imprint cytology (IIC) in breast cancer. One hundred and seven cases of stage I-III breast cancer were studied. Overall, IIC was accurate in 95.3% cases and had a sensitivity and specificity of 98.5% and 90.0%, respectively. The accuracy of JG (95.3%) was better than that of H&E (90.6%) and Pap (94.0%), although the differences were not statistically significant. Problems encountered included cell loss and drying artifacts with H&E and Pap and the inability to distinguish between tumor cells and histiocytes confidently in tight cellular clusters that were occasionally seen. Opinion was possible in all JG cases, but not in five and four cases by H&E and Pap, respectively. Although the choice of the stain would vary depending on the experience of the pathologist, our work suggests that JG, because of fewer technical problems and superior accuracy, may be preferable over H&E and Pap.

Metastatic anaplastic oligodendroglioma simulating acute leukemia. A case report.

BACKGROUND: Anaplastic oligodendroglioma (OG) is an uncommon tumor that rarely metastasizes outside the central nervous system. Spread to the bone marrow (BM) is so rare that when it occurs in the course of follow-up of a case of OG, a disseminated second primary tumor may be a more likely possibility unless BM examination provides evidence to the contrary. Potentially misleading cytologic features of metastatic anaplastic OG can be seen in a BM touch preparation. CASE: A 50-year-old man had undergone left frontal lobectomy in September 1999 for anaplastic OG and presented seven months later with evidence, on BM scan, of focal abnormal uptake at multiple sites. Bone marrow biopsy confirmed OG secondaries, which, on the touch preparation, appeared not only in clusters but also as single cells, simulating acute leukemia. CONCLUSION: The morphology of anaplastic OG metastatic to BM simulates acute leukemia, as seen on the BM touch preparation. This is relevant particularly in the context of anaplastic OG on follow-up. This diagnostic pitfall can be heightened if a BM aspirate rather than biopsy is performed. Metastatic OG can be added to the list of tumors that metastasize to BM as single cells.

Sentinel lymph node biopsy assessment using intraoperative imprint cytology in breast cancer patients: results of a validation study.

OBJECTIVE: Sentinel lymph node biopsy (SLNB) in breast cancer patients is emerging as a promising minimally-invasive tool. There has been an exponential increase in the literature related to sentinel lymph nodes (SLN) in breast cancer patients, mainly from Western centres. This study was carried out to address issues relevant to breast cancer patients in developing countries, including the method of SLN detection, the role of imprint cytology in the assessment of SLN, and the role of SLNB in locally advanced breast cancer (LABC). METHODS: This study included 76 women with breast cancer. The blue-dye method was used to identify the sentinel node. Touch imprint smears were prepared from the sectioned node, stained using the Jenner-Geimsa technique, and examined for tumour deposits. RESULTS: Sentinel nodes were identified in 69 of 76 patients. The sensitivity, specificity and accuracy of SLNB in predicting axillary node status were 84.2%, 100% and 91.3%, respectively. The sensitivity, specificity and accuracy of intraoperative imprint cytology were 96.9%, 100% and 98.6%, respectively. CONCLUSIONS: These results prove that high levels of SLN detection can be achieved using the blue-dye method alone. Its role in LABC patients needs further evaluation. In view of promising results, imprint cytology should be used more frequently as an alternative to frozen section for the assessment of sentinel nodes.

Non hodgkin's lymphoma seven years following remission of acute lymphoblastic leukemia.

The authors describe a case of extramedullary relapse in lymph node presenting as lymphoblastic lymphoma seven years following remission of acute lymphoblastic leukemia. To the best of our knowledge, this is the first reported case of an isolated lymph node relapse with hematopoietic remission of leukemia. We have discussed cases of large cell lymphoma and other unusual areas of extramedullary relapse complicating acute lymphoblastic leukemia in hematopoietic remission.

Economical choices in setting up a diagnostic myeloma laboratory.

In setting up a diagnostic myeloma laboratory the popular, highly automated and otherwise excellent choices of equipment and laboratory practices, so exorbitantly raise costs that the sustainability, even in large government hospitals in third world countries may become difficult. Based on our experience in a regional cancer center in India, we offer here, guidelines for carrying out high resolution electrophoresis, densitometry, immunofixation and urine concentration. We show that by simply employing well established techniques and doing them properly, one can get results of excellent quality at minimum cost and minimum dependence on costly imports.