Molecular genetic evaluation of myeloproliferative neoplasms.

The classical myeloproliferative neoplasms (MPNs) consist of chronic myelogenous leukemia (CML) and the non-CML MPNs, polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Molecular testing plays a crucial role in each of these disease entities. In this review, we discuss the role and caveats of BCR-ABL1 fusion transcript evaluation in CML diagnosis and monitoring, as well as ABL1 kinase mutation testing in the setting of tyrosine kinase inhibitor resistance. We also focus on JAK2, MPL, and CALR mutations in PV, ET, and PMF.

The Impact of EBV Status on Characteristics and Outcomes of Posttransplantation Lymphoproliferative Disorder.

We examined the associations of Epstein-Barr virus (EBV) status with characteristics and outcomes of posttransplantation lymphoproliferative disorder (PTLD) by studying 176 adult solid organ transplant recipients diagnosed with PTLD between 1990 and 2013 (58 [33%] EBV-negative; 118 [67%] EBV-positive). The proportion of EBV-negative cases increased over time from 10% (1990-1995) to 48% (2008-2013) (p < 0.001). EBV-negative PTLD had distinct characteristics (monomorphic histology, longer latency) though high-risk features (advanced stage, older age, high lactate dehydrogenase, central nervous system involvement) were not more common compared to EBV-positive PTLD. In multivariable analysis, EBV negativity was not significantly associated with worse response to initial therapy (adjusted odds ratio, 0.84; p = 0.75). The likelihood of achieving a complete remission (CR) was not significantly different for EBV-negative versus EBV-positive PTLD including when therapy was reduction of immunosuppression alone (35% vs. 43%, respectively, p = 0.60) or rituximab (43% vs. 47%, p = 1.0). EBV negativity was also not associated with worse overall survival (adjusted hazard ratio, 0.91; p = 0.71). Our findings indicate that EBV status is not prognostic or predictive of treatment response in adults with PTLD. The high proportion of EBV-negative disease diagnosed in recent years highlights the need for new strategies for prevention and management of EBV-negative PTLD.

Report from the OECI Oncology Days 2014.

The 2014 OECI Oncology Days was held at the 'Prof. Dr. Ion Chiricuta' Oncology Institute in Cluj, Romania, from 12 to 13 June. The focus of this year's gathering was on developments in personalised medicine and other treatment advances which have made the cost of cancer care too high for many regions throughout Europe.

A robust xenotransplantation model for acute myeloid leukemia.

Xenotransplantation of human acute myeloid leukemia (AML) in immunocompromised animals has been critical for defining leukemic stem cells. However, existing immunodeficient strains of mice have short life spans and low levels of AML cell engraftment, hindering long-term evaluation of primary human AML biology. A recent study suggested that NOD/LtSz-scid IL2Rgammac null (NSG) mice have enhanced AML cell engraftment, but this relied on technically challenging neonatal injections. Here, we performed extensive analysis of AML engraftment in adult NSG mice using tail vein injection. Of the 35 AML samples analyzed, 66% showed bone marrow engraftment over 0.1%. Further, 37% showed high levels of engraftment (>10%), with some as high as 95%. A 2-44-fold expansion of AML cells was often seen. Secondary and tertiary recipients showed consistent engraftment, with most showing further AML cell expansion. Engraftment did not correlate with French-American-British subtype or cytogenetic abnormalities. However, samples with FLT3 mutations showed a higher probability of engraftment than FLT3 wild type. Importantly, animals developed organomegaly and a wasting illness consistent with advanced leukemia. We conclude that the NSG xenotransplantation model is a robust model for human AML cell engraftment, which will allow better characterization of AML biology and testing of new therapies.

Typical levels of airborne fungal spores in houses without obvious moisture problems during a rainy season in Florida, USA.

OBJECTIVE: The aim of this study was to determine types and levels of airborne fungal spores in air-conditioned homes built after 1980 without obvious moisture problems during the 2004 summer (rainy season) in central Florida, USA. METHODS: Eighteen single-family homes were selected based on protocol questionnaire and cursory inspection, which revealed no obvious moisture or visible fungal growth. Non-cultured spores were collected with Air-O-Cell cassettes. Three indoor air samples and 2 outdoor air samples were collected from each home. One indoor and 2 outdoor samples were not interpretable. Fifty-three indoor and 34 outdoor air samples were analyzed by optical microscopy. RESULTS: Several spore types were detected in the indoor samples, at levels generally lower than those detected in the outdoor samples. Spores from the Penicillium/Aspergillus group were the most prevalent types indoors, exceeding the absolute levels and relative percentages of these spores outdoors. Ascospores and basidiospores were the most prevalent spore types outdoors. The percentages of other spore types (Cladosporium and Curvularia) were similar in the indoor and outdoor samples. Moisture-indicator fungi (Chaetomium, Stachybotrys, and Ulocladium species) were nearly absent in both indoor and outdoor samples. CONCLUSION: Airborne fungal spores are present in average central Florida homes without obvious moisture problems during the summer, at levels that are lower than those found outdoors. Spores from the Penicillium/Aspergillus group are prevalent in these homes, and moisture-indicator fungi (Chaetomium, Stachybotrys, and Ulocladium species) are nearly absent. Despite climatic differences, airborne fungal spore types and levels in central Florida houses are similar to those found in other geographical locations.

Evaluation of T cell receptor testing in lymphoid neoplasms: results of a multicenter study of 29 extracted DNA and paraffin-embedded samples.

To evaluate current diagnostic methods used for the evaluation of T cell receptor (TCR) gene rearrangements, 24 different laboratories analyzed 29 lymphoid neoplasm samples of extracted DNA and paraffin-embedded tissue and were asked to complete a technical questionnaire related to the testing. Participating laboratories performed Southern blot and polymerase chain reaction (PCR) testing for rearrangements of the TCRbeta chain gene and PCR for the TCRgamma chain gene rearrangements. Of 14 laboratories performing TCRbeta Southern blot analysis, there was complete agreement in 10 of 14 cases, with some false negative results obtained in 4 cases. No false positive results were obtained by Southern blot analysis. TCRbeta PCR analysis was only performed by two laboratories, and only 47.1% of positive samples were detected. Twenty-one laboratory results were obtained for TCRgamma PCR. This method showed an overall detection rate of 77.9% for T cell gene rearrangements with a 4.1% false positive rate, as compared to both TCRgamma Southern blot analysis results and immunophenotyping. The detection rate for TCRgamma PCR, however, significantly differed when extracted DNA samples from frozen tissue were compared to paraffin-embedded tissue (85.4% versus 65.9%; P = 0.0005). Significant differences in true positive results were obtained when laboratories using primers directed against multiple TCRgamma variable regions (V1-8 plus one to three other primer sets) were compared to laboratories that used only a single set of TCR primers directed against the V1-8 (P < 0.0001). Other technical factors significantly affecting results were also identified. These findings provide useful data on the current state of diagnostic TCR testing, highlight the risk of false negative results for TCR testing directed against only portions of the TCRgamma gene, and identify limitations of testing of paraffin-embedded tissues in some laboratories.

Clinical syndromes of transformation in clonal hematologic disorders.

The majority of clonal hematologic syndromes, including lymphoproliferative, myeloproliferative, and myelodysplastic disorders, tend to undergo transformation. However, the frequency of transformation varies widely. For example, transformation is almost invariable in chronic myelogenous leukemia, but it is infrequent in other myeloproliferative disorders. Similarly, transformation occurs in approximately 33% of follicular lymphomas but less commonly in other lower-grade lymphomas. At a genetic level, although some secondary lesions are seen across the spectrum of transformation syndromes (such as loss of function of p53 and p15/p16), there is considerable intra- and interdisease variability, with no common denominator. This review of the literature will discuss these transformations, noting their frequency, pathologic changes observed, clinical syndromes described, underlying genetic correlates, and prognostic and therapeutic implications.

Chronic myelogenous leukemia: laboratory diagnosis and monitoring.

Rapid developments have occurred both in laboratory medicine and in therapeutic interventions for the management of patients with chronic myelogenous leukemia (CML). With a wide array of laboratory tests available, selecting the appropriate test for a specific diagnostic or therapeutic setting has become increasingly difficult. In this review, we first discuss, from the point of view of laboratory medicine, the advantages and disadvantages of several commonly used laboratory assays, including cytogenetics, fluorescence in situ hybridization (FISH), and qualitative and quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). We then discuss, from the point of view of clinical care, the test(s) of choice for the most common clinical scenarios, including diagnosis and monitoring of the therapeutic response and minimal residual disease in patients treated with different therapies. The purpose of this review is to help clinicians and laboratory physicians select appropriate tests for the diagnosis and monitoring of CML, with the ultimate goal of improving the cost-effective usage of clinical laboratories and improving patient care.

SHP-1 expression by malignant small B-cell lymphomas reflects the maturation stage of their normal B-cell counterparts.

SHP-1 is a protein phosphotyrosine phosphatase that plays an important role in modulating intracellular signaling, which regulates cell activation, proliferation, differentiation, and migration. It is a negative regulator of signal transduction induced by a number of cell receptors. Our immunohistochemical examination of paraffin-embedded reactive lymph nodes and lymphoid tissues revealed that B lymphocytes in follicle germinal centers do not express SHP-1. A weak staining of the B cells in the germinal center light zones was detected when an ultrasensitive amplification system was used. In contrast, normal B cells in mantle and marginal zones as well as interfollicular B lymphocytes and plasma cells displayed strong immunoreactivity. This pattern of SHP-1 expression was repeated in small B-cell lymphomas. All cases of mantle cell lymphoma (12 of 12), marginal zone lymphoma (10 of 10), and chronic lymphocytic leukemia/small lymphocytic lymphoma (13 of 13) expressed SHP-1 protein. However, only 1 of 30 cases of grade 1 follicle center cell lymphoma expressed SHP-1. Our observations highlight the biologic functions of SHP-1 and demonstrate that the SHP-1 expression pattern by small B-cell lymphomas reflects the maturation stage of their normal cell counterparts. These results indicate that determination of SHP-1 expression may help in the differential diagnosis of small B-cell lymphomas.

Molecular pathology of leukemia and lymphoma.

Molecular dissection of physiologic and pathologic genetic phenomena in hematologic malignancy has provided the pathologist with a broad menu of new assays. By integrating the data gleaned from these techniques we can formulate more rational and biologically based diagnoses, which should lead to the ultimate goal of targeted therapy for these specific entities. We summarize some of the more relevant molecular genetic assays and present an overview of those genetic mechanisms usually evaluated in the current practice of hematopathology. The usefulness of such assays extends beyond refining diagnoses in that they also provide relevant prognostic information. Moreover, since most are based on the polymerase chain reaction and reverse transcriptase polymerase chain reaction, we are more sensitively able to monitor for residual disease after attempts at curative therapy, and our definition of remission has been dramatically altered. However, molecular genetic tests are not without limitations, and we must remain cognizant of their cost effectiveness and be aware of current deficiencies in standardization. The challenge will be to meaningfully and economically harness and integrate the information we obtain from these and future technologies into appropriate clinical practice.

Antisense raf oligodeoxyribonucleotide is a radiosensitizer in vivo.

Raf-1, a cytosolic protein serine/threonine kinase, plays important roles in cell growth, proliferation, transformation, and cell survival. The aim of the present study was to evaluate the radiotherapeutic efficacy of a fully phosphorothioated and well-characterized antisense raf oligodeoxyribonucleotide (ODN) corresponding to the 3'-untranslated region of human c-raf-1 mRNA (ISIS 5132/5132). Using our recently developed liposome encapsulation of ODN approach, we first compared the pharmacokinetic parameters of a liposomal formulation of 5132 (LE-5132) and 5132. The peak plasma concentrations 5 minutes after ODN administrations (30 mg/kg i.v.) were 28.5 microg/ml and 13.5 microg/ml for LE-5132 and 5132, respectively. The decrease in plasma concentration of LE-5132 and 5132 followed a biexponential pattern, with initial distribution half-lives (t1/2alpha) of 34.8 minutes and 21.6 minutes, respectively. The terminal half-lives (t1/2beta) with LE-5132 and 5132 were 14.5 hours and 4.3 hours, respectively. The area under the plasma concentration-time curve (AUC) was 5.8 times higher with LE-5132 than with 5132. Significantly higher intact ODN levels could be measured in most organs within 48 hours of administration of LE-5132 compared with 5132 (liver 18.4-fold, spleen, 31-fold, heart 3-fold, lungs 1.5-fold). In kidneys, the level was lower with LE-5132 (0.77-fold). LE-5132 composition, unlike 5132, did not affect clotting time in vitro. Significant decline in the level of Raf-1 protein was observed in vitro in relatively radioresistant human laryngeal squamous cell carcinoma cells (SQ-20B) treated with LE-5132 compared with SQ-20B cells treated with equimolar concentration of 5132 or liposome-encapsulated mismatched 5132 (0.5 microM LE-5132, 71.3%+/-22.5%; 1.0 microM LE-5132, 79.6%+/-16.7%). In addition, LE-5132 appeared to be a more potent antitumor compound than 5132 (p < 0.001). These data established the suitability of LE-5132 for in vivo radiotherapeutic efficacy studies. Intravenous administration of LE-5132 into SQ-20B tumor-bearing athymic mice inhibited Raf-1 expression in tumor tissue compared with blank liposome-treated or untreated control groups. LE-5132 or ionizing radiation (IR) treatment alone caused significant but transient inhibition of SQ-20B tumor growth but not tumor regression. Remarkably, a combination of LE-5132 and IR treatments led to significant and sustained tumor regression for at least 27 days after the last treatment (< 0.001). Histopathologic examination of tumor samples revealed a significant proportion of cells containing fragmented chromatin in the LE-5132 + IR treatment group as compared with single agent and untreated control groups. These in vivo data support the notion that Raf-1 has proliferative and survival functions and advance the scientific and technologic bases for the use of antisense raf ODN in the management of radioresistant malignancies.

Posttherapy surveillance of B-cell precursor acute lymphoblastic leukemia. Value of polymerase chain reaction and limitations of flow cytometry.

Flow cytometric immunophenotypic analysis is critical in diagnosis and classification of acute leukemia and has been used after therapy to monitor for minimal residual disease. However, the presence of normal B-cell precursors, hematogones, particularly in the context of treated pediatric B-cell precursor acute lymphoblastic leukemia (BP-ALL), may confound such evaluation. In this study, the value of more specific genotypic markers (polymerase chain reaction evaluation of 2 antigen receptor genes) was assessed to resolve this issue. Flow cytometric analysis of enriched mononuclear cells revealed 1% to 20% precursor B cells (PBCs), based on expression of 1 or more pan-B cell antigens in addition to CD10, CD34, and terminal deoxynucleotidyl transferase in all 14 patients studied. Inasmuch as this mimicked the immunophenotype of the original leukemic clone, PBCs, in isolation, were considered suspicious for minimal residual disease. However, 11 of the 14 posttherapy specimens (79%) revealed no monoclonally rearranged antigen receptor genes, and 7 of these 11 patients had trackable genotypic markers at presentation. Accordingly, by PCR these 7 patients had complete molecular remission, supported by clinical follow up of 16 to 73 months. Among the remaining 4 patients with PCR-negative disease, 3 continue in remission, confirming the interpretation of false-positive flow cytometric analysis. In conclusion, flow cytometric monitoring of posttherapy bone marrow specimens from patients with BP-ALL may be misleading, if considered in isolation, in falsely suggesting the presence of minimal residual disease. Rather, PCR for antigen receptor gene rearrangements is a valuable and specific tool, helpful in differentiating hematogones from minimal residual disease in patients with treated BP-ALL whose bone marrow harbors increased PBCs.

Low dose poly I:C prevents diabetes in the diabetes prone BB rat.

Poly I:C, an inducer of IFN-alpha and other cytokines, has been used to study the development of diabetes in both the BioBreeding (BB) diabetes prone rat and non-obese diabetic (NOD) mouse animal models of insulin-dependent diabetes mellitus (IDDM). Surprisingly, poly I:C accelerates the disease in the BB rat while inhibiting it in the NOD mouse. Since cytokines can have dose related opposing effects on immune responses, we hypothesized that the paradoxical effect of polyinosinic polycytidylic acid (poly I:C) on diabetes in the two animal models is dose related. Accordingly, we compared the incidence of diabetes and degree of insulitis in diabetes prone BB rats administered saline and poly I:C at doses (0.05 microg/g body weight and 0.1 microg/g body weight) up to 100-fold lower than doses (poly-5 microg/g) previously found to accelerate diabetes. In addition, the non-specific suppressor activity of mononuclear splenocytes from BB rats administered low dose (poly-0.05 microg/g body weight), high dose (poly-5 microg/g body weight), and saline were compared. The development of diabetes was inhibited in rats treated with each dose of poly I:C. The degree of insulitis in poly-I:C treated animals was also less severe. The total white blood cell count and proportion of RT6+ T-cells and each T-cell subset were unaltered by poly I:C. When compared to splenocytes of control animals, splenocytes from poly I:C (0.05 microg/g body weight) treated rats suppressed responder cell proliferation to concanavalin A and alloantigen. However, spleen cells from high dose poly-I:C did not suppress responder cell proliferation to alloantigen. In adoptive transfer studies, the administration of spleen cells from poly-0.05 treated rats decreased the development of diabetes in recipient BB rats. In vitro studies also demonstrated that poly-I:C inhibits the proliferative response of BB rat spleen cells to concanavalin A. The administration of poly-0.05, but not poly-5.0, decreased TNF-alpha mRNA and IL-10 mRNA content in spleen cells. We conclude that poly I:C, at a dose 100 times lower than that required to accelerate diabetes prevents the development of diabetes in BB rates by interfering with the development of insulitis. The induction of suppressor cell activity induced by low dose poly-I:C in vivo and the inhibition of T-cell responses by poly-I:C in vitro suggests that the diabetes sparing activity of poly I:C is mediated by augmented immunoregulatory cell activity. Further studies with poly I:C may be important in increasing our understanding of the pathogenesis of IDDM and provide a means to prevent it.

Gene expression by single Reed-Sternberg cells: pathways of apoptosis and activation.

Although Hodgkin's disease is highly responsive to treatments that cause apoptosis, it remains resistant to the physiological mechanisms intended to cause cell death. Presumably, the Reed-Sternberg cell defies endogenous apoptosis, persists, accumulates, and manifests the malignant disorder seen clinically. The Reed-Sternberg cell expresses several members of the tumor necrosis factor receptor superfamily. This family of receptors is involved in both activation and proliferation of cells, as well as either protection from or initiation of apoptosis in cells expressing these surface proteins. Signals from these receptors affect transcription. We reasoned that the activation state and resistance to apoptosis of Reed-Sternberg cells might be attributable to dysregulation of genes controling these processes. To determine gene expression by Reed-Sternberg cells, we developed a method of micromanipulation, global reverse transcription, and the reverse transcription-polymerase chain reaction and applied it to 51 single Reed-Sternberg cells and their variants from six cases of Hodgkin's disease. This report analyzes the gene expression of bcl-xs, bcl-xl, bax-alpha, bax-beta, fadd, fas, fas ligand (fas L), ice, TNF-alpha, TNF-beta, TNFR1, TNFR2, TRAF1, TRAF2, TRAF3, cIAP2, and tradd at the level of mRNA in the single Reed-Sternberg cells and their variants. The findings here suggest a molecular mechanism for the activated state and in vivo survival occurring in untreated Reed-Sternberg cells of Hodgkin's disease.

Reed-Sternberg cell: survival in a hostile sea.

In contrast to the non-Hodgkin's lymphomas, little is known regarding the origin, genetics, and function of the Reed-Sternberg cell of Hodgkin's disease. Unlike other cancers, the neoplastic cell of Hodgkin's disease, the Reed-Sternberg cell, is vastly outnumbered by a surrounding intense inflammatory infiltrate. How this rare neoplastic cell originates, persists, and disseminates in a presumably hostile cellular environment has remained a mystery. Understanding the biology of the Reed-Sternberg cell has been impeded by the rarity of the cell in tumor tissue. Herein, we describe how the application of single-cell genetic analysis has revealed a clonal and, possibly, germinal center B-cell origin of the Reed-Sternberg cell. By phenotype and function, Reed-Sternberg cells are highly interactive with their cellular microenvironment through cell-cell adhesion, expression of members of the tumor necrosis factor receptor superfamily, and elaboration of cytokines. Perhaps by their mimicry of immune system cells with antigen-presenting function, Reed-Sternberg cells mediate the unusual clinical and pathologic features of Hodgkin's disease: intense tissue inflammatory infiltrate, fibrosis, and constitutional symptoms.

Development of rheumatoid arthritis after treatment of large granular lymphocyte leukemia with deoxycoformycin.

The association of T-cell large granular lymphocyte (LGL) leukemia and rheumatoid arthritis is well described and it is now recognized that these patients and patients with Felty's syndrome represent different aspects of a single disease process. Most patients have rheumatoid arthritis at the time of diagnosis of LGL leukemia. This is the first detailed report of the development of rheumatoid arthritis after the diagnosis and treatment of LGL leukemia as well as the first report of rheumatoid arthritis that occurred in association with deoxycoformycin treatment. It is likely that the beneficial sustained normalization of neutrophil counts as a result of deoxycoformycin treatment played a significant role in the development of this complication. Hematological improvement occurred despite molecular genetic evidence of persistence of the abnormal T-cell clone. The role of the clonally expanded T cells in the pathogenesis of neutropenia and rheumatoid arthritis is discussed.

Rearrangement, hypermutation, and possible preferential use of a VH5 gene, VH32, in a Hodgkin's cell line.

Nonrandom use of immunoglobulin variable (V) gene segments is a feature of some B-cell neoplasms, possibly as a consequence of antigen selection. In Hodgkin's disease, the primary tissues, cell lines, and even single Reed-Sternberg cells can carry immunoglobulin gene rearrangements. Here, we examined the immunoglobulin heavy-chain genes of a well-characterized Hodgkin's-derived cell line, L428, and found a hypermutated VH32 gene involving a conventional V(N)D(N)J4-C gamma 4 rearrangement. VH32 is one of two rearranging members of the VH5 family that is also rearranged preferentially in some B-cell neoplasms and familial CLL. Unexpectedly, the closest known rearranged sequence match for the rearranged VH gene of L428 was found in the single Reed-Sternberg cells of lymphocyte-predominance Hodgkin's disease, and is a mutated VH251, the only other rearranging member of the VH5 family. Assuming random usage of the human VH pool, the chance of coincident VH5 family gene rearrangement in the two cases of Hodgkin's disease is only about 10(-3). Biased use of VH genes suggests a B-cell target that is either selected by antigen or vulnerable to transformation at an early antigen-independent, developmental stage. These findings raise the question whether similar processes operate in Hodgkin's disease.