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Myelodysplastic syndromes (MDS) are clonal neoplasms of the hematopoietic stem cell that result in aberrant differentiation of hematopoietic lineages caused by a wide range of underlying genetic, epigenetic, and other causes. Despite the myriad origins, a recognizable MDS phenotype has been associated with miRNA aberrant expression. A model of aberrant myeloid maturation that mimics MDS was generated using a stable knockdown of miR-378-3p. This model exhibited a transcriptional profile indicating aberrant maturation and function, immunophenotypic and morphologic dysplasia, and aberrant growth that characterizes MDS. Moreover, aberrant signal transduction in response to stimulation specific to the stage of myeloid maturation as indicated by CyTOF mass cytometry was similar to that found in samples from patients with MDS. The aberrant signaling, immunophenotypic changes, cellular growth, and colony formation ability seen in this myeloid model could be reversed with azacytidine, albeit without significant improvement of neutrophil function.

Modern combinations of therapies for multiple myeloma have led to improvement in survival outcomes with near 100% overall response rate and 25% complete response rates, particularly with autologous hematopoietic cell transplant (AHCT). Minimal residual disease (MRD) assessment with multiparameter flow cytometry is a valid prognostic biomarker for progression-free survival (PFS) and overall survival (OS). However, few data exist regarding whether MRD positivity or negativity will meaningfully influence treatment decisions. We evaluated 433 patients who received induction therapy, followed by AHCT. Participants had MRD assessment by multiparameter flow cytometry before and at days +100 and +365 following AHCT. They also received either lenalidomide, bortezomib, or no maintenance therapy following AHCT. Maintenance treatment with lenalidomide improved MRD negativity at day +365 compared to bortezomib (92.9% vs 41.6%, p = 0.01), or no maintenance therapy (92.9% vs 24.4%, p = 0.012). The median PFS for patients who were MRD negative at day + 365 was 42 vs 17.5 months (p < 0.001) and median OS was 80.6 vs 59 months (p = 0.02). Maintenance therapy following AHCT for multiple myeloma improves the depth of response as assessed by MRD.

The Veterans Health Administration is the largest integrated provider of cancer services in the nation, with approximately 50,000 new cancer cases diagnosed each year. The goal of the VA's National Oncology Program is to establish a System of Excellence in cancer care with the help of tools, resources, programs, and best practices across the enterprise to ensure every veteran, regardless of location, receives the same high level of cancer care and access to precision diagnostics. This is mediated through a variety of programs, including the National TeleOncology Service and the National Precision Oncology Program which includes standardized clinical pathways for molecular testing, a second opinion service on diagnostic consultations, test result interpretation and treatment recommendations, as well as a molecular oncology tumor board. The VA National Oncology Program and the Pathology and Laboratory Medicine Program offices have collaborated on a recommended guideline for advanced laboratory and molecular testing algorithm in hematologic diagnosis. We aim to offer best practice for all VA providers who must decide the type of ancillary tests at the time of initial clinical presentation in order to gain prognostic information and guide therapy. The recommendations include traditional laboratory tests, such as morphology, flow cytometry, and immunohistochemistry, as well as the use of fluorescent in situ hybridization (FISH), karyotype, polymerase chain reaction (PCR), and next generation sequencing (NGS). An overview of the draft algorithm for acute leukemias and chronic myeloid neoplasms is presented in Table 1; details will be discussed at the annual meeting. Conclusion: The joint effort from VA Oncology and Pathology presents advanced testing algorithms in a variety of hematolymphoid malignancies as a systematic approach to hematopathology diagnosis, classification, as well as offering prognostic and therapeutic information. The recommendations across VA Healthcare would ensure standardized care for all VA patients with hematologic malignancy to benefit from the latest advances in precision medicine. Figure 1 Figure 1. Disclosures Neff: Spring Discovery: Consultancy, Ended employment in the past 24 months; EUSA Pharma: Speakers Bureau; Enzyvant: Consultancy.

Objectives: To determine the effect of iterative refinement of standard ordering protocols on test utilization and results for bone marrow biopsy specimens. Methods: Eighteen months of test utilization and result data were used to revise the protocols that determine cytogenetic and molecular test selection on bone marrow specimens and then compared with data obtained following protocol revision. Results: Revision of protocols resulted in reduction in total tests and associated charges, due to a decrease in tests both concordant and discordant with the protocols. These reductions only occurred in diseases for which revisions were made and were limited to cases in which reflex testing was performed. There was an increase in the fraction of positive tests, which was also limited to reflex testing. Conclusions: Data-driven iterative revision of protocols further improves test utilization and performance, while reducing cost. Analysis of testing data can be used to continuously improve test ordering decisions.

Context.— Detailed diagnostic features of acute myeloid leukemia in Down syndrome are lacking, leading to potential misdiagnoses as standard acute myeloid leukemia occurring in patients with Down syndrome. Objective.— To evaluate diagnostic features of acute myeloid leukemia and myelodysplastic syndrome in patients with Down syndrome. Design.— Diagnostic bone marrow samples from 163 patients enrolled in the Children's Oncology Group study AAML0431 were evaluated by using central morphologic review and institutional immunophenotyping. Results were compared to overall survival, event-free survival, GATA1 mutation status, cytogenetics, and minimal residual disease results. Results.— Sixty myelodysplastic syndrome and 103 acute myeloid leukemia samples were reviewed. Both had distinctive features compared to those of patients without Down syndrome. They showed megakaryocytic and erythroid but little myeloid dysplasia, and marked megakaryocytic hyperplasia with unusual megakaryocyte morphology. In acute myeloid leukemia cases, megakaryoblastic differentiation of blasts was most common (54 of 103, 52%); other cases showed erythroblastic (11 of 103, 11%), mixed erythroid/megakaryoblastic (20 of 103, 19%), or no differentiation (10 of 103, 10%). Myelodysplastic syndrome and acute myeloid leukemia cases had similar event-free survival and overall survival. Leukemic subgroups showed interesting, but not statistically significant, trends for survival and minimal residual disease. Cases with institutional diagnoses of French American British M1-5 morphology showed typical features of Down syndrome disease, with survival approaching that of other cases. Conclusions.— Myelodysplastic syndrome and acute myeloid leukemia in Down syndrome display features that allow discrimination from standard cases of disease. These distinctions are important for treatment decisions, and for understanding disease pathogenesis. We propose specific diagnostic criteria for Down syndrome–related subtypes of acute myeloid leukemia and myelodysplastic syndrome.

Objectives Acute myeloid leukemia (AML) is classified in part by recurrent cytogenetic abnormalities, often detected by both fluorescent in situ hybridization (FISH) and karyotype. The goal of this study was to assess the utility of FISH and karyotyping at diagnosis and follow-up. Methods Adult AML samples at diagnosis or follow-up with karyotype and FISH were identified. Concordance was determined, and clinical characteristics and outcomes for discordant results were evaluated. Results Karyotype and FISH results were concordant in 193 (95.0%) of 203 diagnostic samples. In 10 cases, FISH detected an abnormality, but karyotype was normal. Of these, one had a FISH result with clinical significance. In follow-up cases, 17 (8.1%) of 211 showed FISH-positive discordant results; most were consistent with low-level residual disease. Conclusions Clinically significant discordance between karyotype and AML FISH is uncommon. Consequently, FISH testing can safely be omitted from most of these samples. Focused FISH testing is more useful at follow-up, for minimal residual disease detection.

The National Comprehensive Cancer Network (NCCN) defines the following types of acute myeloid leukemia (AML) as favorable-risk: acute promyelocytic leukemia with t(15;17) (APL); AML with core-binding factor (CBF) rearrangements, including t(8;21) and inv(16) or t(16;16) without mutations in KIT (CBF-KITwt); and AML with normal cytogenetics and mutations in NPM1 (NPM1mut); or biallelic mutations in CEBPA (CEBPAmut/mut), without FLT3-ITD. Although these AMLs are categorized as favorable risk by NCCN, clinical experience suggests that there are differences in clinical outcome amongst these cytogenetically and molecularly distinct leukemias. This study compared clinical and genotypic characteristics of 60 patients with favorable-risk AML, excluding APL, and demonstrated significant differences between them. Patients with NPM1mut AML were significantly older than those in the other groups. Targeted next-generation sequencing on DNA from peripheral blood or bone marrow revealed significantly more mutations in NPM1mut AML than the other favorable-risk diseases, especially in genes related to DNA splicing and methylation. CEBPAmut/mut AMLs exhibited more mutations in transcription-related genes. Patients with NPM1mut AML and CEBPAmut/mut AML show significantly reduced overall survival in comparison with CBF-KITwt AML. These findings emphasize that favorable-risk AML patients have divergent outcomes and that differences in clinical and genotypic characteristics should be considered in their evaluation and management.

Objectives: Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing. Methods: We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively. Results: The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power. Conclusions: Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel.

We report the case of a 39-year-old woman with acute myeloid leukemia (AML) with monocytic differentiation showing hemophagocytosis by leukemic blasts. This phenomenon is known to be associated with certain chromosomal changes, including t(8;16), der(8), inv(8), and t(16;21); however, in this case, the patient had a normal female karyotype. To our knowledge, this is the first published case of normal karyotype AML with hemophagocytosis by leukemic blasts. * AML : acute myeloid leukemia HOXA : homeobox A RET : rearranged during transfection WBCs : white blood cells LDH : lactate dehydrogenase PT : prothrombin time CT : computed tomography Flt3 : Fms-like tyrosine kinase 3 NPM1 : nucleophosmin CEBPA : CCAAT/enhancer-binding protein alpha HLA-DR : human leukocyte antigen-D related HiDAC : high-dose cytarabine MRI : magnetic resonance imaging CNS : central nervous system MOZ : monocytic leukemia zinc finger protein CBP : competitive protein binding TNF-α : tumor necrosis factor–alpha

Utilization management in pathology requires the physician to consider whether a test is necessary and appropriate for the current condition of the patient. This has both medical care implications—how best to detect disease with the highest sensitivity and specificity—and fiscal implications. In an era where reimbursement is tied increasingly to bundled incidents of care or to particular quality measures, unnecessary lab costs must be avoided while adhering to the physician imperative to do no harm.

Personalized medicine requires physicians to employ complex and expensive diagnostic analyses. However, without evidence-based standards, there is over-utilization of testing that complicates patient care, diminishes quality, and increases costs. To address this, we implemented the diagnostic management team (DMT), a multi-disciplinary system for development and deployment of diagnostic testing guidelines for hematologic malignancies. The team created evidence-based standard ordering protocols (SOPs) for cytogenetic and molecular testing that were applied by pathologists to bone marrow biopsies on adult patients. Testing on 780 biopsies performed during the 6 months before SOP implementation was compared with 1,806 biopsies performed during the subsequent 12 months. After implementation, there were significant decreases in tests discordant with SOPs, omitted tests, and the estimated cost of testing to payers. The fraction of positive tests increased. Clinicians reported acceptance of the new procedures and perceived time savings. This process is a model for optimizing complex and personalized diagnostic testing.

Kikuchi-Fujimoto lymphadenitis is a self-limited disorder that typically presents in young females as painless cervical lymphadenopathy with fever, anemia, and leukopenia. The clinical manifestations and pathologic findings suggest a viral etiology, yet specific etiologic agents remain unknown. Although there are studies reporting positive associations between Kikuchi-Fujimoto lymphadenitis and parvovirus B19 and herpesviruses, other studies have failed to find an association with these viruses. To our knowledge, this current study is the largest study of Kikuchi-Fujimoto lymphadenitis in Western patients that used polymerase chain reaction testing for 4 different common viral pathogens often implicated as etiologic agents in Kikuchi-Fujimoto lymphadenitis. Archival material from 3 institutions was included, following confirmation of the diagnosis of Kikuchi-Fujimoto lymphadenitis by 2 independent pathologists. Polymerase chain reaction from the paraffin-embedded tissue sections for parvovirus B19, Epstein-Barr virus, human herpesvirus 6, and human herpesvirus 8 was performed. Eighteen cases of Kikuchi-Fujimoto lymphadenitis were analyzed, 12 of which (60%) were cervical lymph nodes. All the cases showed typical geographic necrosis with abundant apoptotic debris, although the degree of necrosis was variable. Polymerase chain reaction revealed a high prevalence of parvovirus B19 in the controls (44%); there were fewer positive cases seen in the Kikuchi-Fujimoto lymphadenitis cases (11%), but this did not reach statistical significance (P = .25).There were no significant differences between cases and controls in the prevalence of Epstein-Barr virus, human herpesvirus 6, and human herpesvirus 8 (P = .50 for all 3). Polymerase chain reaction failed to reveal a positive association between Kikuchi-Fujimoto lymphadenitis and 4 common suspected viral agents. These findings do not support a role for Epstein-Barr virus, human herpesvirus 6, human herpesvirus 8, or parvovirus B19 in the pathogenesis of Kikuchi-Fujimoto lymphadenitis.

Measurement of minimal residual disease is routine in diseases such as chronic myelogenous leukemia, precursor B cell acute lymphoblastic leukemia, and acute promyelocytic leukemia because it provides important prognostic information. However, the role of minimal residual disease testing has not been widely adopted in multiple myeloma (MM), with other parameters such as the International Staging System (ISS) and cytogenetic analysis primarily guiding therapy and determination of prognosis. Until recently, achieving a complete response (CR), as defined by the International Myeloma Working Group (IMWG) criteria, was rare in patients with MM. The use of novel agents with or without autologous peripheral blood stem cell transplantation (auto-PBSCT) has significantly increased CR rates, thus increasing overall survival (OS) rates. The majority of patients with MM have persistent levels of residual disease that are below the sensitivity of bone marrow (BM) morphology, protein electrophoresis with immunofixation, and light chain quantitation even after attaining CR and will eventually relapse. Measurement of minimal residual disease by more sensitive methods, and the use of these methods as a tool for predicting patient outcomes and guiding therapeutic decisions, has thus become more relevant. Methods available for monitoring minimal residual disease in MM include PCR and multiparameter flow cytometry (MFC), both of which have been shown to be valuable in other hematologic malignancies; however, neither has become a standard of care in MM. Here, we review current evidence for using minimal residual disease measurement for risk assessment in MM as well as incorporating pretreatment factors and posttreatment minimal residual disease monitoring as a prognostic tool for therapeutic decisions, and we outline challenges to developing uniform criteria for minimal residual disease monitoring.

BACKGROUND Prostatitis, a clinical syndrome characterized by pelvic pain and inflammation, is common in adult males. Although several induced and spontaneous murine models of prostatitis have been explored, the role of genetic background on induction has not been well-defined. METHODS Using a standard methodology for the induction of experimental autoimmune prostatitis (EAP), we investigated both acute and chronic inflammation on several murine genetic backgrounds. RESULTS In our colony, nonobese diabetic (NOD) mice evinced spontaneous prostatitis that was not augmented by immunization with rat prostate extract (RPE). In contrast, the standard laboratory strain Balb/c developed chronic inflammation in response to RPE immunization. Development of EAP in other strains was variable. CONCLUSIONS These data suggest that Balb/c mice injected with RPE may provide a useful model for chronic prostatic inflammation. Prostate 73: 651–656, 2013. © 2012 Wiley Periodicals, Inc.

Fanconi anemia is a rare autosomal recessive disease which is associated with an increased risk of malignancy. Acute myeloid leukemia and myelodysplastic syndrome are the most common malignancies. This case report is on an infant who was diagnosed with Fanconi anemia and developed myelodysplastic syndrome in his first year of life which later likely transformed into T acute lymphoblastic leukemia. These findings were demonstrated by FISH. The case highlights a rare lymphoid neoplasm in an FA patient and also the increased incidence of T lineage neoplasms when MDS transforms into a lymphoid leukemia.

Context.—Infection is a common cause of lymphadenopathy in children and has numerous microbial etiologies. Lymph node biopsy is considered a keystone in arriving at a definite diagnosis. An accurate differential diagnosis from a lymph node biopsy can expedite diagnosis and minimize ancillary testing. Objective.—To assess and compare the histopathologic and epidemiologic features of common and uncommon pediatric bacterial lymphadenitis. Design.—We searched our database for surgical specimens that had a positive identification of bacteria during an 8-year period. The chart was reviewed to assess the pathogen identified and epidemiologic data. The archival tissue sections were reviewed and the histopathologic findings described for each pathogen. Results.—The review of 368 pediatric lymph node biopsies identified 33 cases with a bacterial infection. These comprised 21 cases of Mycobacterium avium complex (60%), 1 of Mycobacterium fortuitum (3%), 7 of Bartonella henselae (20%), 2 of Yersinia enterocolitica (7%), 1 of Francisella tularensis (3%) and 1 of Streptococcus pyogenes (3%). Conclusions.—Each of these infectious lymphadenitides had distinct epidemiologic and histopathologic features that are discussed in this report.

Nuclear factor-κB (NF-κB) inducing kinase (NIK) is a MAP3K that regulates the activation of NF-κB. NIK is often highly expressed in tumor cells, including melanoma, but the significance of this in melanoma progression has been unclear. Tissue microarray analysis of NIK expression reveals that dysplastic nevi (n=22), primary (n=15) and metastatic melanoma (n=13) lesions showed a statistically significant elevation in NIK expression when compared with benign nevi (n=30). Moreover, when short hairpin RNA techniques were used to knock-down NIK, the resultant NIK-depleted melanoma cell lines exhibited decreased proliferation, increased apoptosis, delayed cell cycle progression and reduced tumor growth in a mouse xenograft model. As expected, when NIK was depleted there was decreased activation of the non-canonical NF-κB pathway, whereas canonical NF-κB activation remained intact. NIK depletion also resulted in reduced expression of genes that contribute to tumor growth, including CXCR4, c-MYC and c-MET, and pro-survival factors such as BCL2 and survivin. These changes in gene expression are not fully explained by the attenuation of the non-canonical NF-κB pathway. Shown here for the first time is the demonstration that NIK modulates β-catenin-mediated transcription to promote expression of survivin. NIK-depleted melanoma cells exhibited downregulation of survivin as well as other β-catenin regulated genes including c-MYC, c-MET and CCND2. These data indicate that NIK mediates both β-catenin and NF-κB regulated transcription to modulate melanoma survival and growth. Thus, NIK may be a promising therapeutic target for melanoma.

The development of PTLD is a rare severe adverse event following ASCT. We report on a child with DS who developed PTLD following autologous transplant for relapsed Hodgkins disease. He was successfully treated with cyclophosphamide, prednisone and rituximab. We also present a comprehensive review of the literature of PTLD in pediatric patients following ASCT.

e19008Background: In acute myeloid leukemia (AML) patients, FLT3 mutations (FLT3 mt) are associated with worse prognosis, while NPM1 mutations (NPM1 mt) have better prognosis. There have been confl...

Treatment-related acute myeloid leukemia (tAML) is a rarely reported complication of therapy of melanoma. In fact, there are only four cases of tAML or myelodysplastic syndrome (MDS) in chemotherapy treated melanoma patients reported in English literature. Two cases implicated either fotemustine or

Contamination of a paraffin-embedded tissue block with another patient's tissue can lead to an incorrect diagnosis. We report the use of short tandem repeats for the analysis of DNA extracted from microdissected tissue from unstained slides prepared from a decalcified block from a 33-year-old woman who was previously diagnosed with a low-grade B-cell lymphoma. This diagnosis was based on a single fragment of tissue found among bone fragments taken during orthopedic surgery at a referring hospital. Our results confirm that the B-cell lymphoma tissue was not derived from our patient. Furthermore, we suggest that in cases for which the definitive identification of a tissue contaminant can resolve clinically, therapeutically, and prognostically significant questions, short tandem repeat analysis of DNA derived from microdissected surgical pathology samples should be considered to minimize errors and enhance the quality of care.

Vascular endothelial growth factor (VEGF), a major factor in tumor-host interactions, plays a critical role in the aberrant hematopoiesis observed in cancer-bearing hosts. To dissect the roles of VEGF receptor (VEGFR)-1 and VEGFR-2 in cancer-associated hematopoiesis in vivo, we selectively stimulated VEGFR-1 and VEGFR-2 by continuous infusion of receptor-specific ligands or selective blockade with VEGF receptor-specific antibodies in mice infused with recombinant VEGF at levels observed in tumor-bearing animals. We found that the effect of VEGF on the accumulation of Gr1+CD11b+ cells is mediated by VEGFR-2, but that the 2 receptors have opposite effects on lymphocyte development. Pathophysiologic levels of VEGF strongly inhibit T-cell development via VEGFR-2, whereas VEGFR-1 signaling decreases this inhibition. VEGFR-1, and not VEGFR-2, signaling is responsible for the observed increase of splenic B cells. Both receptors are capable of inhibiting dendritic cell function. These data suggest that most of observed aberrant hematopoiesis caused by excess tumor-derived VEGF is mediated by VEGFR-2, and VEGFR-1 alone has very limited independent effects but clearly both positively and negatively modulates the effects of VEGFR-2. Our findings suggest that selective blockade of VEGFR-2 rather than of both receptors may optimally overcome the adverse hematologic consequences of elevated VEGF levels found in malignancy.

Optimizing a clinical flow cytometry panel can be a subjective process dependent on experience. We develop a quantitative method to make this process more rigorous and apply it to B lymphoblastic leukemia/lymphoma (B-ALL) minimal residual disease (MRD) testing.We retrospectively analyzed our existing three-tube, seven-color B-ALL MRD panel and used our novel method to develop an optimized one-tube, eight-color panel, which was tested prospectively.The optimized one-tube, eight-color panel resulted in greater efficiency of time and resources with no loss in diagnostic power.Constructing a flow cytometry panel using a rigorous, objective, quantitative method permits optimization and avoids problems of interdependence and redundancy in a large, multiantigen panel.

Increased use of novel agents and autologous stem cell transplantation has led to a significant improvement in PFS and overall survival in patients with multiple myeloma. Despite improved treatment strategies, most patients eventually relapse due to persistent low levels of disease in the bone marrow. Increasingly sensitive methods to measure or detect such disease have been evaluated, including multi-parametric flow cytometry, PCR, next-generation sequencing and imaging modalities. The following literature review examines current methods for detecting and monitoring minimal or measurable residual disease (MRD) in the post-transplant setting. Improved methods for detecting MRD will refine the current definitions of remission and could guide treatment approaches.

Autoimmune diseases often result from inappropriate or unregulated activation of autoreactive T cells. Traditional approaches to treatment of autoimmune diseases through immunosuppression have focused on direct inhibition of T cells. In the present study, we examined the targeted inhibition of antigen-presenting cells as a means to downregulate immune responses and treat autoimmune disease. Dendritic cells (DCs) are the central antigen-presenting cells for the initiation of T cell responses, including autoreactive ones. A large portion of DCs are derived from hematopoietic progenitors that express FLT3 receptor (CD135), and stimulation of the receptor via FLT3 ligand eitherin vivoorin vitrois known to drive expansion and differentiation of these progenitors toward a DC phenotype. We hypothesized that inhibition of FLT3 signaling would thus produce an inhibition of DC-induced stimulation of T cells, thereby inhibiting autoimmune responses. To this end, we used small-molecule tyrosine kinase inhibitors targeted against FLT3 and examined the effects on DCs and their role in the promulgation of autoimmune disease. Results of our studies show that inhibition of FLT3 signaling induces apoptosis in both mouse and human DCs, and thus is a potential target for immune suppression. Furthermore, targeted inhibition of FLT3 significantly improved the course of established disease in a model for multiple sclerosis, experimental autoimmune encephalomyelitis, suggesting a potential avenue for treating autoimmune disease.

Previous studies established that after inhibition of proteasome activity, tyrosinase could be detected in the cytosol after initial translation in the endoplasmic reticulum (ER), with a molecular weight consistent with that of a full-length, deglycosylated polypeptide. Here we show that most of these molecules are glycosylated, but have been proteolyzed at the carboxyl terminus by a protease that is insensitive to proteasome inhibitors. We also demonstrate the inhibitor-dependent accumulation of a membrane species that appears structurally homologous to the glycosylated and partially proteolyzed cytosolic form. Under some circumstances, cytosolic tyrosinase that had been deglycosylated and not proteolyzed prior to proteasomal degradation could also be detected. The presence of cytosolic tyrosinase was dependent upon glycosylation of the molecule during synthesis in the ER. These results suggest the existence of at least two alternative pathways for degradation of tyrosinase in the cytosol.

Fluorescence in situ hybridization for abnormalities common to myelodysplastic syndrome (MDS FISH) is often used with traditional karyotype in the diagnosis and monitoring of myeloid neoplasms. However, its value in these roles has been questioned. To evaluate its utility, we compared MDS FISH results with karyotype in 544 bone marrow specimens obtained for diagnosis (180 cases) or follow-up (364 cases) of myeloid neoplasia. We found excellent concordance between FISH and karyotype, such that FISH is rarely abnormal (1.7% at diagnosis and 3.0% at follow-up) in cases with normal karyotype. Even in the rare discordant cases, the abnormal FISH has little or no clinical value. Thus, we propose that this test should be limited to cases with inadequate karyotype only. Such guidelines could result in significant cost savings with no impact on patient diagnosis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic malignancies characterized by ineffective hematopoiesis. Recently, we identified MDS-associated microRNAs (miRNAs) that are down-regulated in MDS. This study examines possible explanations for that observed down-regulation of miRNA expression in MDS. Since genomic losses are insufficient to explain the down-regulation of all our MDS-associated miRNAs, we explored other avenues. We demonstrate that these miRNAs are predominantly intragenic, and that, in many cases, they and their host genes are expressed in a similar pattern during myeloid maturation, suggesting their co-regulation. This co-regulation is further supported by the down-regulation of several of the host genes in MDS and increased methylation of the shared promoters of several miRNAs and their respective host genes. These studies identify a role of hypermethylation of miRNA promoters in the down-regulation of MDS-associated miRNAs, unifying research on miRNAs in MDS and epigenetic regulation in MDS into a common pathway.

Prostatitis, a clinical syndrome characterized by pelvic pain and inflammation, is common in adult males. Although several induced and spontaneous murine models of prostatitis have been explored, the role of genetic background on induction has not been well-defined.Using a standard methodology for the induction of experimental autoimmune prostatitis (EAP), we investigated both acute and chronic inflammation on several murine genetic backgrounds.In our colony, nonobese diabetic (NOD) mice evinced spontaneous prostatitis that was not augmented by immunization with rat prostate extract (RPE). In contrast, the standard laboratory strain Balb/c developed chronic inflammation in response to RPE immunization. Development of EAP in other strains was variable.These data suggest that Balb/c mice injected with RPE may provide a useful model for chronic prostatic inflammation.

We have purified to homogeneity from the myofibrillar fraction of pig bladder a mammalian heterotrimeric form of PP-1, SMPP-1M. Purified pig bladder SMPP-1M is similar in composition and substrate specificity to avian gizzard PP-1M reported by Alessi et al. (Alessi, D., Macdougall, L. K., Sola, M. M., Ikebe, M., and Cohen, P. (1992) Eur. J. Biochem. 210, 1023-1035) and consists of the catalytic subunit of PP-1 (37 kDa) and two other equimolar subunits of 130 and 20 kDa. The properties of SMPP-1M and the role of its regulatory subunits in the dephosphorylation of myosin and in the initiation of relaxation were characterized both in vitro and in smooth muscle. We show that the relaxant effect of the catalytic subunit in smooth muscle is markedly potentiated by the addition of the regulatory subunits of SMPP-1M. Our findings demonstrate that SMPP-1M is the major phosphatase dephosphorylating myosin in mammalian smooth muscle and that myosin dephosphorylation is regulated in vivo via targeting subunits that specifically alter the substrate specificity of PP-1C toward myosin.

Background: Retrospective studies have shown that achieving minimal residual disease (MRD) by multi-parameter flow cytometry (MFC) is predictive of outcomes in multiple myeloma (MM). Modern induction therapies including bortezomib (V), lenalidomide (R) followed by autologous hematopoietic cell transplant (AHCT) have resulted in substantial improvements in progression free survival (PFS) and overall survival (OS). The impact of bortezomib-based therapy on end of induction chemotherapy MRD status and post-AHCT MRD status is not well defined. We designed a prospective clinical trial (NCT01215344) to study the incidence of MRD negativity at end of induction and following AHCT, and its association with PFS and OS. Patients and Methods: Twenty patients with newly diagnosed, symptomatic MM were enrolled. They received four cycles of VRd induction therapy followed by AHCT. Dose modifications of VRd were controlled during the clinical trial. MRD status by MFC was evaluated at the end of induction chemotherapy and at day 100 post-AHCT. The MFC cut-off to determine MRD negativity was defined as 10-4. Outcomes analyzed included MM disease status, PFS and OS. Results: Three cohorts of patients were identified; patients achieving MRD negative status at the end of induction (cohort 1, 50%), those achieving MRD negative status only after AHCT (cohort 2, 15%) and patients never achieving MRD negative status (cohort 3, 35%). All patients achieving MRD negative status at the end of induction remained MRD negative post-AHCT. There were no significant differences in median age, renal function, disease stage, cytogenetic risk and maintenance chemotherapy post-AHCT between cohorts. The table summarizes characteristics of these cohorts. Patients who never achieved MRD negative status after AHCT had significantly shorter PFS (cohort 3) compared with patients who achieved MRD negative status (cohorts 1 and 2, p=0.008) (figure). The median PFS for cohort 3 was 2.64 years and not yet reached for the other cohorts. There were no significant differences in OS. Median follow-up for survivors was 2.53 years (range, 0.73-4.04). Conclusions: In this study, bortezomib-based therapy resulted in half of patients achieving MRD negative status with induction chemotherapy alone. AHCT improved the depth of response with 30% of patients who were MRD positive after induction therapy converting to MRD negativity following AHCT. Achieving a negative MRD state, pre- and at D100 post-AHCT resulted in improved PFS. These findings were independent of molecular cytogenetics or ISS stage. MRD positive status at day 100 post-AHCT is highly predictive of earlier disease progression, and may help identify patients for alternative management approaches. Delay of AHCT may be considered as a potential management option for patients with MRD negative status at the end of induction therapy, as being studied in the IFM DFCI study (NCT01208662). As some patients with MRD positive disease at the end of induction therapy become MRD negative with AHCT, these patients may benefit from AHCT and this population warrants further investigation. Table. Descriptive analysis Cohort 1 MRD -/- (%) (n=10) Cohort 2 MRD +/- (%) (n=3) Cohort 3 MRD +/+ (%) (n=7) Test Statistic Median Age 55 54 60 0.31 ISS Stage III 40 33 14 0.52 DS Stage III 89 67 60 0.14 Median Serum creatinine 0.90 0.60 0.88 0.10 Serum M-spike 2.0 1.9 2.1 0.80 Bone marrow plasma cells 14 44 14 0.18 Cytogenetics 0.37 Standard risk 40 0 57 Intermediate risk 50 67 43 High risk 10 33 0 Figure 1. Kaplan-Meier curve of progression-free survival comparing patients achieving MRD negative disease with MRD positive disease. Figure 1. Kaplan-Meier curve of progression-free survival comparing patients achieving MRD negative disease with MRD positive disease. Disclosures Cornell: Prothena: Research Funding. Jagasia:Takeda Inc: Research Funding.

Treatment of adipocytes with insulin or phorbol 12-myristate 13-acetate (PMA) results in transient activation of mitogen-activated protein kinase kinase (MEK) (Tmax = 90 s) and mitogen-activated protein kinase (MAPK) (Tmax = 300 s). We have identified a novel insulin-stimulated MEK kinase (I-MEKK) in the 100,000 x g infranatant that shows rapid phasic kinetics that temporally precede that of MEK. Maximal activation of I-MEKK occurs within 20 +/- 5 s (S.D., n = 3) followed by complete inactivation by 30 +/- 10 s (S.D., n = 3). I-MEKK was characterized by anion-exchange and gel filtration chromatography and separated into two distinct activities of approximately 56 kDa that phosphorylated and activated MEK. I-MEKKs did not co-elute on anion exchange with c-Raf or 73-kDa MEK kinase (MEKK), suggesting they are distinct enzymes. Protein phosphatase 2A inactivated both I-MEKKs in vitro and in the intact cell okadaic acid blocked inactivation in the presence of insulin. These results suggest activation of I-MEKK involves phosphorylation on serine or threonine residues. I-MEKK was not activated by PMA, suggesting that in adipocytes the enzyme represents a divergence point between signal transduction pathways mediated by insulin and those activating protein kinase C.

We used flow cytometry to analyze the cell cycle, DNA damage, and apoptosis in hematopoietic subsets in MDS marrow. Subsets were assigned using CD45, side scatter, CD34, and CD71. Cell cycle fractions were analyzed using DRAQ 5 (DNA content) and MPM-2 (mitoses). DNA damage was assessed using p-H2A.X, and apoptosis using Annexin V. Compared to controls, MDS patients demonstrated no increased mitoses in erythroid, myeloid, or CD34+ cells. Myeloid progenitors demonstrated increased G2 cells, which with no increased mitoses suggested delayed passage through G2. Myeloid progenitors demonstrated increased p-H2A.X, consistent with DNA damage causing this delay. Annexin V reactivity was equivalent in MDS and controls. Results for each parameter varied among hematopoietic compartments, demonstrating the need to analyze compartments separately. Our results suggest that peripheral cytopenias in MDS are due to delayed cell cycle passage of marrow progenitors and that this delayed passage and leukemic progression derive from excessive DNA damage.

The natural killer (NK)-cell neoplasms are rare, representing less than 1% of non-Hodgkin lymphoma, except in Asia and Latin America, where they represent 3% to 6%. NK-cell neoplasms include immature acute leukemias; a blastic NK-cell lymphoma, which is obsolete because of its plasmacytoid dendritic-cell origin; and mature NK neoplasms, comprising extranodal NK/T-cell lymphoma (ENKL), nasal-type; aggressive NK-cell leukemia; and chronic NK-cell lymphoproliferative disorders, which are often reactive. Epstein-Barr virus is usually detected in tumor cells of ENKL and aggressive NK-cell leukemia. The latter two mature NK neoplasms are relatively chemoresistant because of the frequent expression of P-glycoprotein. Early radiation is advocated for localized nasal ENKL. Stem cell transplantation is recommended for advanced disease, owing to a poor prognosis. Novel agents, including chemotherapy, inhibitors of molecular pathways, and monoclonal antibodies, are under investigation.

The mature (postthymocyte; peripheral) T/natural killer (NK) lymphomas/leukemias represent 5–15% of all non-Hodgkin lymphoma (NHL) and vary according to geography. Peripheral T cell lymphoma, not otherwise specified (PTCL, NOS), is the most common type worldwide. There are more nodal presentations in Europe and North America, where the second most common types in each region are angioimmunoblastic T cell lymphoma (AITL) and anaplastic large cell lymphoma (ALCL), respectively. There is more extranodal disease in Asia, due to Epstein–Barr virus related NK/T lymphoma and human T cell leukemia virus (HTLV)-1 associated adult T cell leukemia/lymphoma (ATLL). With the exceptions of the indolent mycosis fungoides (MF) and the chemo-sensitive anaplastic lymphoma kinase (ALK)-positive ALCL, the prognosis in most peripheral T/NK neoplasms is poor, with a 5-year survival less than 30%. (Table 25.1)

Laboratory medicine offers attractive opportunities for individuals who have MD and PhD degrees and advanced training in medicine and the underlying basic biomedical sciences, and these individuals have much to contribute to the field. The modern era of basic biomedical sciences has produced a wealth of genomic, postgenomic, and proteomic knowledge. As a bridge discipline, a major challenge and opportunity for laboratory medicine is to bring these advances to the diagnostic, prognostic, and therapeutic care of patients. The authors believe that, for many reasons, the field of laboratory medicine represents an excellent, although underrecognized, career choice for graduates of MD/PhD programs.

Although multiple components of the class I MHC processing pathway have been elucidated, the participation of nonproteasomal cytosolic enzymes has been largely unexplored. In this study, we provide evidence for multiple cytosolic mechanisms in the generation of an HLA-A*0201-associated epitope from tyrosinase. This epitope is presented in two isoforms containing either Asn or Asp, depending on the structure of the tyrosinase precursor. We show that deamidation of Asn to Asp is dependent on glycosylation in the endoplasmic reticulum (ER), and subsequent deglycosylation by peptide-N-glycanase in the cytosol. Epitope precursors with N-terminal extensions undergo a similar process. This is linked to an inability of ER aminopeptidase 1 to efficiently remove N-terminal residues, necessitating processing by nonproteasomal peptidases in the cytosol. Our work demonstrates that processing of this tyrosinase epitope involves recycling between the ER and cytosol, and an obligatory interplay between enzymes involved in proteolysis and glycosylation/deglycosylation located in both compartments.

In the Gleason grading system for prostatic cancer only the two most prevalent patterns are reported, although a third (tertiary) pattern grade may be present. We compared the pathologic stage of 227 radical prostatectomies with tertiary pattern 5 to the pathologic stage of 604 radical prostatectomies lacking a tertiary component. Gleason score 3 + 4 tumors with a tertiary pattern of 5 were more likely to present with higher stage disease than those Gleason score 3 + 4 tumors without a tertiary component (P = 0.012) and at a stage similar to Gleason score 3 + 5 tumors. Gleason score 4 + 3 tumors with a tertiary pattern of 5 were less likely to be organ-confined than Gleason score 4 + 3 tumors (P = 0.02) and less likely to have lymph node metastases than Gleason score 4 + 4 tumors (P = 0.027). However, Gleason score 4 + 4 with a tertiary pattern of 5 were indistinguishable from Gleason score 4 + 4 tumors. The relative effects of a tertiary pattern of 5 were greatest when the primary and secondary stages were low but become obscured by the already aggressive nature of advanced primary and secondary patterns. Therefore, except for very high-grade tumors, tertiary scoring of prostatic adenocarcinoma at radical prostatectomy should be reported as it has prognostic significance.

8605 Background: MRD assayed by multi-parameter flow cytometer (MFC), has prognostic significance after HDT/ASCT for MM (Paiva et. al. 2008). The frequency of MRD negativity (-) after induction therapy using novel agents such as immunomodulatory drugs like lenalidomide (IMiDs), and proteasome inhibitors like bortezomib, is unknown. The impact of HDT/ASCT on MRD status in this patient group has not been studied. Methods: We performed a retrospective study of all MM patients undergoing HDT/ASCT (January 2010 - December 2012) in our institution. No restrictions on inclusion were made based on the International Myeloma Working Group response criteria. All patients had novel agents as part of their initial induction regimen. Statistical analysis was by SPSS software (V 12.0). MRD status was determined by MFC on bone marrow samples pre- HDC/ASCT [M1] and post- HDC/ASCT (D100 [M2] and I year [M3]). MFC was done with antibodies against CD45, CD19, CD138, CD38, CD20, CD56, and anti-k and l cytoplasmic antibodies. Results: MRD status was available on 91 patients pre-transplant. Of these patients, 80 had MFC recorded at M2 and 17 patients had MFC recorded at M3. Fifty-eight percent were male and 76% were Caucasian. Forty percent received IMiDs, while 60% got proteasome based therapies. Of the 91 patients with MRD pre-HDC/ASCT, 58% (53/91) were MRD (-), and of these patients 89% (41/46) remained MRD (-) at M2. 48 patients were MRD positive (+) pre-HDC/ASCT, 58% (20/34) became MRD (-) at M2. Age, cytogenetic risk, disease stage, number of chemotherapy cycles or immunofixation status had no impact on MRD status. There were only 6 relapses in the cohort, thus the impact of MRD status on progression-free survival could not be studied. Conclusions: Novel agents improve depth of response pre-transplant. HDC/ASCT increases MRD negativity post-transplant. MRD status could aid better timing of HDC/ASCT or adoption of a risk-adapted strategy for high-risk patients. MRD status validation in a prospective cohort is underway at our center (NCT01215344). With future follow-up, the impact of MRD on progression-free survival in the era of novel agents will be determined.

Context: Pathologists grade follicular lymphoma (FL) cases by selecting 10, random high power fields (HPFs), counting the number of centroblasts (CBs) in these HPFs under the microscope and then calculating the average CB count for the whole slide. Previous studies have demonstrated that there is high inter-reader variability among pathologists using this methodology in grading. Aims: The objective of this study was to explore if newly available digital reading technologies can reduce inter-reader variability. Settings and Design: In this study, we considered three different reading conditions (RCs) in grading FL: (1) Conventional (glass-slide based) to establish the baseline, (2) digital whole slide viewing, (3) digital whole slide viewing with selected HPFs. Six board-certified pathologists from five different institutions read 17 FL slides in these three different RCs. Results: Although there was relative poor consensus in conventional reading, with lack of consensus in 41.2% of cases, which was similar to previously reported studies; we found that digital reading with pre-selected fields improved the inter-reader agreement, with only 5.9% lacking consensus among pathologists. Conclusions: Digital whole slide RC resulted in the worst concordance among pathologists while digital whole slide reading selected HPFs improved the concordance. Further studies are underway to determine if this performance can be sustained with a larger dataset and our automated HPF and CB detection algorithms can be employed to further improve the concordance.

Abstract 2073 Introduction: Molecular and cytogenetic testing is critical in diagnosis and management of hematologic malignancies. These tests are expensive, complex, and frequently inappropriately ordered. There are no comprehensive guidelines to assist in the disease- and patient-specific selection of these tests. Traditionally, most tests are ordered before morphologic evaluation of the specimen, which could help guide test selection. We hypothesize that this leads to significant over-ordering of tests unnecessary for diagnosis or monitoring, as well as under-ordering of necessary tests. As part of the hematopathology diagnostic management team (DMT) effort, we evaluated this hypothesis by establishing evidence-based rules for molecular and cytogenetic test ordering that are applied by the pathologist after review of clinical history and bone marrow aspirate smears. Methods: Using published evidence and best clinical practices, a team of clinical hematologists and hematopathologists developed rules, termed standard operating procedures (SOPs), for selecting cytogenetic and molecular tests on bone marrow biopsies from adult patients performed for myelodysplastic syndrome/acute myeloid leukemia (MDS/AML), plasma cell myeloma, lymphoma, myeloproliferative neoplasms, lymphoblastic leukemia, and bone marrow failure. The rules specify testing in five clinical scenarios: diagnosis or follow-up with morphologically overt disease, bone marrow staging (for lymphoma), routine follow-up without overt disease, evaluation prior to stem cell transplant (pre-SCT), and follow-up after stem cell transplant (post-SCT). To determine the effect that application of these SOPs would have had on test ordering and results, bone marrow biopsies from adults (>18 years) were evaluated retrospectively over a six-month time period. Each test was ruled concordant or discordant with the corresponding SOP. Omitted tests, those recommended by the SOPs, but not performed, were also identified. Statistical analysis of concordant and discordant tests was performed using the Chi-square test. Results: A total of 3,007 tests (769 karyotypes, 1,790 FISH, and 448 molecular tests) were performed on 804 adult bone marrows. Of these, 1,080 (36%) were discordant with the SOPs. Discordant tests were most frequent in myeloma (471), MDS/AML (231), and lymphoma (215). The most common clinical settings for discordant tests were routine follow-up (403) and post-SCT (417). To determine if these tests were necessary, we determined the positive rate of both concordant and discordant tests. The positive rate was significantly higher in the concordant (490/1,929; 25%) than the discordant tests (38/1,078; 4%) (P Conclusions: Retrospective analysis of cytogenetic and molecular test ordering demonstrates that more than one-third of cytogenetic or molecular tests ordered on bone marrow specimens at a large tertiary care institution were discordant with rules (SOPs) based on published literature and best clinical practices. Furthermore, the vast majority (>99%) of these discordant tests were negative for any abnormality, redundant, or potential false positives, suggesting that the exclusion of these tests would have little negative clinical impact. In addition, there were a large number of omitted tests, seen in 18% of all marrows. This analysis predicts that institution of rules-based morphology-influenced test ordering would decrease uninformative testing, while increasing appropriate informative tests, leading to more cost-effective, accurate, and complete diagnosis and monitoring of hematolymphoid malignancies. We have therefore instituted a rules-based, morphology-directed approach at our institution. Disclosures: No relevant conflicts of interest to declare.

Abstract 1754 Poster Board I-780 Introduction Definitive diagnosis and classification of MDS are often difficult because of variable presence of diagnostic criteria and imprecision and ambiguities of interpretation of both morphologic and ancillary data. An objective criterion that reliably distinguishes MDS from normal marrow would greatly facilitate diagnosis, and might contribute to subclassification of MDS. Gene expression profiling has identified marked (7 fold) down-regulation of myeloid nuclear differentiation antigen (MNDA) message in cases of MDS (Hofmann WK, et al, Blood 2002;100:3553-60), and we have previously shown down-regulation of MNDA protein expression in random MDS cases using immunohistochemistry and flow cytometry (Briggs RC, et al. Cancer Research 2006;66:4645-51). To continue our previous analyses, we evaluated MNDA expression in myeloid progenitors using quantitative flow cytometric analysis in MDS and normal control marrow samples. Patients and Methods The study included 20 MDS patients receiving only supportive care undergoing bone marrow sampling for clinical purposes, and 19 frequency age-matched normal controls undergoing orthopedic surgery with no antecedent primary hematologic abnormalities. MDS diagnosis was based on 2008 WHO criteria. Quantitative flow cytometric analysis was performed with an FC500 flow cytometer (Beckman Coulter, Fullerton, CA). Cells were treated with CD45-PE and CD34-ECD (Beckman Coulter), washed in PBS with 2% FCS, and permeabilized with PermiFlow. MNDA-Alexa-488 was added at a granulocyte-monocyte specific concentration, with analysis using Winlist 5.0 software (Verity Software, Topsham, ME) with DDE links to ModFitLT 3.0 using modifications of published methods. Differentiating myeloid progenitors were identified as high side scatter/intermediate CD45/CD34-negative cells, with lymphocytes (low side scatter/high CD45/CD34-negative cells) serving as an internal dim MNDA control in each sample. Results MDS cases consisted of 12 patients with refractory cytopenia with multilineage dysplasia (RCMD), 3 with refractory anemia with excess blasts-1(RAEB-1), 4 with RAEB-2, and 1 with therapy-related MDS. In myeloid progenitors in MDS patients, the median percent of MNDA-dim cells was 67.4% (range 0.7-97.5%, interquartile range 44.9-82.7%). The analogous median percent of MNDA-dim cells in control patients was 1.2% (range 0.2-13.7%, interquartile range 0.6-2.7%). The area under the ROC curve was 0.96 (p = 9×10-7), indicating almost complete discrimination between cases and controls. 19 of 20 MDS patients demonstrated bimodal distribution of MNDA expression comprising a distinct MNDA-dim population and a separate MNDA-normal population, suggesting an admixture of MDS and normal cells. 18 of 19 control patients demonstrated a single population of MNDA-normal cells without evidence of a bimodal distribution. The single MDS patient with normal MNDA expression had prior clinical and laboratory features suggestive of refractory anemia with ringed sideroblasts. The single pediatric MDS patient had reduced MNDA expression similar to other high grade MDS samples. Patients showed trends for MDS subtype (p = 0.21) and IPSS score (p = 0.07) versus percent MNDA-dim myeloid progenitors. These data demonstrate remarkable sensitivity and specificity for use of MNDA expression to detect MDS. Conclusions Quantitative flow cytometric analysis of MNDA expression in marrow myeloid progenitors is a promising objective test for diagnosis of MDS, demonstrating a striking difference of MNDA expression in myeloid progenitors in MDS versus control patients. Our results require elaboration with analysis of low grade cases. A single possible low grade MDS case in this series demonstrated MNDA expression identical to normal control samples. Our results also require elaboration in cases constituting the differential diagnosis of MDS to evaluate the clinical utility of MNDA evaluation. The biological significance of down-regulation of MNDA in MDS is uncertain, although MNDA has been implicated in regulation of programmed cell death. Our results suggest a mix of normal and abnormal myelopoiesis in MDS patients, as predicted by cytogenetics in many cases of MDS. In summary, testing of MNDA expression in myeloid progenitors shows great promise as an objective test for diagnosis of MDS in marrow samples. Disclosures No relevant conflicts of interest to declare.

Summary Rationale Mounting data suggest that immune cell abnormalities participate in the pathogenesis of pulmonary arterial hypertension (PAH). Objective To determine whether the T lymphocyte subset composition in the systemic circulation and peripheral lung is altered in PAH. Methods Flow cytometric analyses were performed to determine the phenotypic profile of peripheral blood lymphocytes in idiopathic PAH (IPAH) patients ( n =18) and healthy controls ( n =17). Immunocytochemical analyses of lymphocytes and T cell subsets were used to examine lung tissue from PAH patients ( n =11) and controls ( n =11). Measurements and main results IPAH patients have abnormal CD8+ T lymphocyte subsets, with a significant increase in CD45RA+ CCR7− peripheral cytotoxic effector-memory cells ( p =0.02) and reduction of CD45RA+ CCR7+ naive CD8+ cells versus controls ( p =0.001). Further, IPAH patients have a higher proportion of circulating regulatory T cells (T reg ) and 4-fold increases in the number of CD3+ and CD8+ cells in the peripheral lung compared with controls ( p Conclusions Alterations in circulating T cell subsets, particularly CD8+ T lymphocytes and CD4+ T reg , in patients with PAH suggest that a dysfunctional immune system contributes to disease pathogenesis. A preponderance of CD3+ and CD8+ T lymphocytes in the peripheral lung of PAH patients supports this concept.