Acute purpura fulminans-a rare cause of skin necrosis: A single-institution clinicopathological experience.

BACKGROUND: Purpura fulminans, an uncommon syndrome of intravascular thrombosis with hemorrhagic infarction of the skin, is often accompanied by disseminated intravascular coagulation (DIC) and multi-organ failure, and may ultimately lead to death. METHODS: Herein, we document 13 skin biopsies from 11 adult patients with the clinical diagnosis of sepsis and confirmed histopathologic diagnosis of intravascular thrombosis and/or DIC, compatible with acute infectious purpura fulminans (AIPF). Detailed history and clinical examination were performed, and the lesions were correlated with histopathologic findings. Any underlying medical disease was taken into consideration. RESULTS: There were 5 males and 6 females with lower extremity or peri-incisional purpuric skin lesions. The most important comorbidities identified were a history of surgical procedure or neoplasm, although 4 patients had no relevant underlying history. Most skin biopsies revealed focal epidermal ischemia or necrosis and 3 showed full-thickness epidermal necrosis. In the underlying dermis, there were fibrin thrombi in superficial and deep blood vessels with acute inflammation. Changes of an inflammatory destructive vasculitis were identified in 5 cases. No bacteria or fungi were identified on histopathology. CONCLUSIONS: AIPF is a rapidly-progressing medical emergency which may be identified by histopathology in culture-negative cases. Biopsies may show neutrophilic infiltrate without infective organisms.

Concurrent juvenile myelomonocytic leukemia and T-lymphoblastic lymphoma with a shared missense mutation in NRAS.

Single cases of B- and T-lymphoblastic leukemia/lymphoma occurring after remission of JMML, and JMML occurring after remission of B-lymphoblastic leukemia have been reported in the literature. We present a unique case of a child with concurrent JMML and T-lymphoblastic lymphoma in which an identical missense mutation in NRAS was found in both the neoplastic JMML and T-LBL cells. JMML has been considered a stem cell disorder, and our case provides additional molecular evidence for a stem cell lesion underlying the two different disease phenotypes.

Hypoxia-inducible factors in human pulmonary arterial hypertension: a link to the intrinsic myeloid abnormalities.

Pulmonary arterial hypertension (PAH) is a proliferative vasculopathy characterized by high circulating CD34(+)CD133(+) proangiogenic progenitors, and endothelial cells that have pathologic expression of hypoxia-inducible factor 1 α (HIF-1α). Here, CD34(+)CD133(+) progenitor cell numbers are shown to be higher in PAH bone marrow, blood, and pulmonary arteries than in healthy controls. The HIF-inducible myeloid-activating factors erythropoietin, stem cell factor (SCF), and hepatocyte growth factor (HGF) are also present at higher than normal levels in PAH blood, and related to disease severity. Primary endothelial cells harvested from human PAH lungs produce greater HGF and progenitor recruitment factor stromal-derived factor 1 α (SDF-1α) than control lung endothelial cells, and thus may contribute to bone marrow activation. Even though PAH patients had normal numbers of circulating blood elements, hematopoietic alterations in myeloid and erythroid lineages and reticulin fibrosis identified a subclinical myeloproliferative process. Unexpectedly, evaluation of bone marrow progenitors and reticulin in nonaffected family members of patients with familial PAH revealed similar myeloid abnormalities. Altogether, the results show that PAH is linked to myeloid abnormalities, some of which may be related to increased production of HIF-inducible factors by diseased pulmonary vasculature, but findings in nonaffected family suggest myeloid abnormalities may be intrinsic to the disease process.

Utility of Bruton's Tyrosine Kinase Inhibitors in Light Chain Amyloidosis Caused by Lymphoplasmacytic Lymphoma (Waldenström's Macroglobulinemia).

Of the variety of immunoglobulin related amyloidosis (AL), immunoglobulin M (IgM) related AL represents only 6 to 10% of affected patients, and the majority of these cases are associated with underlying non-Hodgkin's Lymphoma including Waldenström's macroglobulinemia (WM). Ibrutinib, acalabrutinib, and zanubrutinib are Bruton tyrosine kinase (BTK) inhibitors approved for certain indolent B cell non-Hodgkin's lymphoma (NHL). BTK is a nonreceptor kinase involved in B-cell survival, proliferation, and interaction with the microenvironment. We retrospectively evaluated the tolerability and effectiveness of BTK inhibitors ibrutinib and acalabrutinib therapy in (n = 4) patients with IgM-related AL amyloidosis with underlying WM. Treatment was well tolerated with both hematologic and organ response in patients with AL amyloidosis in the setting of WM. Atrial fibrillation led to the discontinuation of ibrutinib in one patient, and acalabrutinib caused significant thumb hematoma needing dose reduction in another patient. All patients evaluated had the MYD88 mutation. This may explain the good response to BTK inhibitors therapy in our series. BTK inhibitors should be further investigated in larger prospective studies for treatment of AL amyloidosis in patients with lymphoplasmacytic lymphoma/WM.

Pathology Residents as Testing Personnel in the Hematology Laboratory.

CONTEXT.­: Clinical laboratories and the training of pathology residents are tightly regulated environments. Compliance with regulatory requirements must be addressed when developing entrustable professional activities (EPAs) for pathology residents. OBJECTIVE.­: To describe the development of EPAs for peripheral blood and body fluid review in compliance with Clinical Laboratory Improvement Amendments and College of American Pathologists personnel and testing requirements. To examine the impact of EPA implementation on the workflow in a busy hematology laboratory. DESIGN.­: A training program was designed to prepare pathology residents to function as independent testing personnel in compliance with Clinical Laboratory Improvement Amendments. After a series of lectures, hands-on microscopy sessions, self-assessment quizzes, and achievement of a passing score on a training assessment exam, residents were deemed competent to release certain results independently. The volume and the turnaround time of hematology tests were compared before and after residents were integrated into the laboratory workflow. Faculty and residents were surveyed to assess satisfaction with the training. RESULTS.­: Empowering residents to independently release noncritical results from peripheral blood and body fluid reviews had no adverse impact on test turnaround time. The resident contribution to workflow resulted in a corresponding decrease in the number of cases that required attending pathologist review. Faculty and residents viewed the EPAs as beneficial to service and education. CONCLUSIONS.­: The implementation of the EPAs had a beneficial effect on the laboratory, the trainees, and faculty. Our experience may be helpful to other training programs as EPAs become more widely implemented in residency training.

College of American Pathologists/American College of Medical Genetics proficiency testing for constitutional cytogenomic microarray analysis.

PURPOSE: To evaluate the feasibility of administering a newly established proficiency test offered through the College of American Pathologists and the American College of Medical Genetics for genomic copy number assessment by microarray analysis, and to determine the reproducibility and concordance among laboratory results from this test. METHODS: Surveys were designed through the Cytogenetic Resource Committee of the two colleges to assess the ability of testing laboratories to process DNA samples provided and interpret results. Supplemental questions were asked with each Survey to determine laboratory practice trends. RESULTS: Twelve DNA specimens, representing 2 pilot and 10 Survey challenges, were distributed to as many as 74 different laboratories, yielding 493 individual responses. The mean consensus for matching result interpretations was 95.7%. Responses to supplemental questions indicate that the number of laboratories offering this testing is increasing, methods for analysis and evaluation are becoming standardized, and array platforms used are increasing in probe density. CONCLUSION: The College of American Pathologists/American College of Medical Genetics proficiency testing program for copy number assessment by cytogenomic microarray is a successful and efficient mechanism for assessing interlaboratory reproducibility. This will provide laboratories the opportunity to evaluate their performance and assure overall accuracy of patient results. The high level of concordance in laboratory responses across all testing platforms by multiple facilities highlights the robustness of this technology.

A rare trigger for macrophage activation syndrome.

Macrophage activation syndrome (MAS) is a disorder characterized by increased activation of mononuclear cells leading to phagocytosis of blood cell precursors in the bone marrow. We describe a case of MAS triggered by disseminated histoplasmosis occurring in a patient with Still's disease on long-term treatment with adalimumab.

Characterization of chromosome arm 20q abnormalities in myeloid malignancies using genome-wide single nucleotide polymorphism array analysis.

Deletion of the long arm of chromosome 20 is a common abnormality associated with myeloid malignancies. We characterized abnormalities of chromosome 20 as defined by metaphase cytogenetics (MC) in patients with myeloid neoplasms to define commonly deleted regions (CDR) and commonly retained regions (CRR) using genome-wide, high resolution single nucleotide polymorphism array (SNP-A) analysis. We reviewed the MC results of a cohort of 1,162 patients with myeloid malignancies, including myelodysplastic syndromes (MDS), MDS/myeloproliferative neoplasia (MDS/MPN), and acute myeloid leukemia (AML). We further analyzed a subcohort of 532 patients by SNP-A using the Affymetrix Genome-Wide Human SNP Array 6.0 and GeneChip Human Mapping 250K Nsp arrays. By MC, 5% (54/1,162) harbored a deletion of 20q; in 30% (16/54), del(20q) was the sole cytogenetic abnormality. By SNP-A analysis, we identified del(20q) in 23 patients, 3 not detected by MC. In four cases, monosomy 20 with a marker chromosome by MC was proven to be an interstitial deletion of 20q by SNP-A. We defined 2 CDR and 2 CRR on chromosome arm 20q: CDR1 spanned 2.5 Mb between bands 20q11.23 and 20q12, while CDR2 encompassed 1.8 Mb within 20q13.12. CRR1 spanned 1.9 Mb within 20q11.21 and CRR2 encompassed 2.5 Mb within 20q13.33. In contrast to other chromosomes frequently affected by deletions, no somatic copy neutral loss of heterozygosity (CN-LOH) was detected. Our data suggest that SNP-A is useful for the detection of cryptic aberrations of chromosome 20q and allows for a more precise characterization of complex karyotypes. Furthermore, SNP-A allowed definition of a CDR on 20q.

High-resolution genomic arrays facilitate detection of novel cryptic chromosomal lesions in myelodysplastic syndromes.

OBJECTIVE: Unbalanced chromosomal aberrations are common in myelodysplastic syndromes and have prognostic implications. An increased frequency of cytogenetic changes may reflect an inherent chromosomal instability due to failure of DNA repair. Therefore, it is likely that chromosomal defects in myelodysplastic syndromes may be more frequent than predicted by metaphase cytogenetics and new cryptic lesions may be revealed by precise analysis methods. METHODS: We used a novel high-resolution karyotyping technique, array-based comparative genomic hybridization, to investigate the frequency of cryptic chromosomal lesions in a cohort of 38 well-characterized myelodysplastic syndromes patients; results were confirmed by microsatellite quantitative PCR or single nucleotide polymorphism analysis. RESULTS: As compared to metaphase karyotyping, chromosomal abnormalities detected by array-based analysis were encountered more frequently and in a higher proportion of patients. For example, chromosomal defects were found in patients with a normal karyotype by traditional cytogenetics. In addition to verifying common abnormalities, previously cryptic defects were found in new regions of the genome. Cryptic changes often overlapped chromosomes and regions frequently identified as abnormal by metaphase cytogenetics. CONCLUSION: The results underscore the instability of the myelodysplastic syndromes genome and highlight the utility of array-based karyotyping to study cryptic chromosomal changes which may provide new diagnostic information.

Detection of cryptic chromosomal lesions including acquired segmental uniparental disomy in advanced and low-risk myelodysplastic syndromes.

OBJECTIVES: Using metaphase cytogenetics (MC), chromosomal defects can be detected in 40% to 60% of patients with myelodysplastic syndromes (MDS); cytogenetic results have a major impact on prognosis. We hypothesize that more precise methods of chromosomal analysis will detect new/additional cryptic lesions in a higher proportion of MDS patients. METHODS: We have applied single nucleotide polymorphism microarrays (SNP-A) to perform high-resolution karyotyping in MDS to determine gene copy number and detect loss of heterozygosity (LOH). RESULTS: Using this method, chromosomal defects were found in 82% of MDS patients vs 50% as measured by MC; lesions were present in 68% of patients with normal MC, while in 81% of those with abnormal MC, new aberrations were found. In addition to gains or losses of chromosomal material, areas of LOH due to segmental uniparental disomy were found in 33% of patients. CONCLUSION: SNP-A findings demonstrate that chromosomal lesions are present in a much higher proportion of patients than predicted by traditional cytogenetics. These lesions may reflect an underlying generalized chromosomal instability in MDS. Additional previously cryptic defects may explain the clinical variability of MDS. New lesions may have important prognostic implications, suggesting that, in the future, SNP-A-based karyotyping may complement MC in laboratory evaluation of MDS.

Quantification of the Effectiveness of a Residency Program Using the Resident In-Service Examination.

This study describes a quantitative tool in the assessment of residency programs, in which national ranking of residents after the resident in-service examination in postgraduate year 4 is compared to that in postgraduate year 1. The relationship between training and changes in ranking, resident in-service examination results before and after training in specific areas are also compared. To illustrate the use of this novel approach, data from a large residency program were analyzed. The 70 residents were ranked as a postgraduate year 1 group at the 50th national percentile. As postgraduate year 4 residents, they were ranked at the 59th percentile, a significant (P < .003) improvement. There was moderate correlation between performance in postgraduate year 1 and that in postgraduate year 4 (0.61); however, initial ranking was no indication of the final (R2 = .34), with the exception of high performers. Training in specific areas improved ranking, demonstrating association between training and performance. In conclusion, the effectiveness of training provided by a residency program can be quantified using the resident in-service examination. This should provide a quantitative tool in the assessment of postgraduate programs.

Severe TMD/AMKL with GATA1 mutation in a stillborn fetus with Down syndrome.

BACKGROUND: A 34-year-old woman was referred for evaluation of a recent stillborn male fetus, gestational age 27 6/7 weeks, found to have congenital myeloid leukemia at autopsy. Autopsy findings included high weight for gestational age, hepatomegaly, and extensive intravascular leukemic infiltrates in the placenta, heart, liver, thymus, lung, kidneys, and brain. Genetic consultation and examination of photographs of the fetus revealed dysmorphic features. INVESTIGATIONS: Immunoperoxidase staining of placental tissue, fluorescence in situ hybridization of paraffin-embedded sections of the placenta using probes for t(12;21)(p13;q22), t(8;21)(q22;q22) and t/del(11q23), cytogenetic analysis of fetal tissue (tendon), sequence analysis of GATA1 in placental leukemic cells, and parental chromosome studies. DIAGNOSIS: Down syndrome with in utero onset of GATA1 mutation-positive severe transient myeloproliferative disorder/acute megakaryoblastic leukemia. MANAGEMENT: Genetic counseling for the recurrence risk of Down syndrome on the basis of maternal age.

Initial Diagnostic Workup of Acute Leukemia: Guideline From the College of American Pathologists and the American Society of Hematology.

CONTEXT: - A complete diagnosis of acute leukemia requires knowledge of clinical information combined with morphologic evaluation, immunophenotyping and karyotype analysis, and often, molecular genetic testing. Although many aspects of the workup for acute leukemia are well accepted, few guidelines have addressed the different aspects of the diagnostic evaluation of samples from patients suspected to have acute leukemia. OBJECTIVE: - To develop a guideline for treating physicians and pathologists involved in the diagnostic and prognostic evaluation of new acute leukemia samples, including acute lymphoblastic leukemia, acute myeloid leukemia, and acute leukemias of ambiguous lineage. DESIGN: - The College of American Pathologists and the American Society of Hematology convened a panel of experts in hematology and hematopathology to develop recommendations. A systematic evidence review was conducted to address 6 key questions. Recommendations were derived from strength of evidence, feedback received during the public comment period, and expert panel consensus. RESULTS: - Twenty-seven guideline statements were established, which ranged from recommendations on what clinical and laboratory information should be available as part of the diagnostic and prognostic evaluation of acute leukemia samples to what types of testing should be performed routinely, with recommendations on where such testing should be performed and how the results should be reported. CONCLUSIONS: - The guideline provides a framework for the multiple steps, including laboratory testing, in the evaluation of acute leukemia samples. Some aspects of the guideline, especially molecular genetic testing in acute leukemia, are rapidly changing with new supportive literature, which will require on-going updates for the guideline to remain relevant.

The Philadelphia chromosome as a secondary abnormality in inv(3)(q21q26) acute myeloid leukemia at diagnosis: confirmation of p190 BCR-ABL mRNA by real-time quantitative polymerase chain reaction.

The Philadelphia chromosome (Ph) as a secondary cytogenetic abnormality is a rare event. It is observed mostly as an additional, late-appearing cytogenetic change during the evolution of acute leukemia and its presentation as a secondary change at the onset of disease is much rarer. We describe here a patient with acute myelogenous leukemia (AML) who had Ph as a secondary chromosome abnormality at diagnosis. Cytogenetic analysis showed an abnormal karyotype, 45,XY,inv(3)(q21q26),-7[4]/45,idem, t(9;22)(q34;q11.2). The p190 variety of BCR-ABL rearrangements was confirmed by a real-time reverse-transcriptase polymerase chain reaction using fluorescent probes. To our knowledge, the minor BCR-ABL fusion gene involving a secondary Ph superimposed on inv(3) and monosomy 7 has not been reported in AML at diagnosis. Along with the identification of more cases, it will be possible to understand the exact role of this secondary Ph in a multistep leukemogenesis.

Frequencies and characterization of cytogenetically unrelated clones in various hematologic malignancies: seven years of experiences in a single institution.

Cytogenetically unrelated clones are uncommon findings in hematologic disorders. In the present study, among the 1,110 various hematologic malignancies analyzed during the past seven years, 27 (2.4%) patients had karyotypically unrelated clones: 3.5% (7/202) of acute myeloid leukemias, 5.3% (11/206) of myelodysplastic syndromes, none of 40 acute lymphoblastic leukemias, 0.4% (1/233) of myeloproliferative disorders, and 2.6% (8/306) lymphoproliferative disorders with clonal chromosomal abnormalities. Twenty-five patients showed two unrelated clones and two had three unrelated clones. The most consistent chromosome abnormalities were del(5q) (seven cases), +8 (six cases), del(20q) (five cases), and del(7q), +12, +21, and -22 (three cases each). Of interest, the high frequency of different numeric or structural abnormalities affecting the same chromosomes in such clones supports the hypothesis that these karyotypically unrelated clones originate from the common malignant clone through submicroscopic molecular genetic changes and evolutionary processes.

Thrombocytosis and STAT5 activation in chronic myelogenous leukaemia are not associated with JAK2 V617F or calreticulin mutations.

AIMS: Marked thrombocytosis is uncommon in chronic myelogenous leukaemia (CML) but may be associated with mutation of JAK2 V617F, calreticulin (CALR) and/or phospho-STAT5 (p-STAT5) activation in other myeloproliferative neoplasms (MPNs), particularly essential thrombocythaemia (ET). We investigated the JAK2 V617F, CALR and STAT5 activation status in patients with CML and thrombocytosis (CML-T) that mimicked ET, trying to identify a common mechanism for thrombocytosis in MPN. METHODS: Blood and bone marrow morphological findings were reviewed from seven CML-T, four otherwise typical CML and one CML in blast phase. All cases were analysed for BCR-ABL1, JAK2 V617F and CALR exon 9 mutation and p-STAT5 expression. RESULTS: Four of seven cases of CML-T had marked thrombocytosis (>1000×10(9)/L). Eleven of 12 cases had megakaryocyte morphology typical for CML. All cases were BCR-ABL1 positive. Eleven of 12 cases were negative for JAK2 V617F, while STAT5 was activated in six of seven CML-T and in four of five CML cases. No case had a detectable CALR exon 9 mutation. One case of CML developed ET-like morphology and had JAK2 V617F detected while in molecular remission for CML. CONCLUSIONS: Detection of BCR-ABL1 is critical in the distinction of ET from CML. Thrombocytosis and STAT5 activation in CML-T are not consistently associated with CALR exon 9 or JAK2 V617F mutation.

Detection of mature T-cell leukemias by flow cytometry using anti-T-cell receptor V beta antibodies.

A broad array of antibodies directed against the variable (V) region of the T-cell receptor (TCR) beta (V beta) chain has become available in a directly conjugated multicolor format that permits assessment of 19 of 25 V beta families, covering 70% of the normal circulating T-cell repertoire. These antibodies were used to detect expanded T-cell populations in 43 peripheral blood samples submitted for suspected T-cell malignancy. Of 43 samples, 27 were diagnosed as follows: T-cell large granular lymphocyte leukemia, 14 samples; Sézary syndrome, 4 samples; T-cell prolymphocytic leukemia, 5 samples; or T-cell non-Hodgkin lymphoma or T-cell lymphoproliferative disorder not otherwise specified, 4 samples. The remaining 16 samples were determined to be nonneoplastic. All samples were diagnosed before assessment with anti-V beta flow cytometry. By using a cutoff of 1.6 times the upper limit of normal range (ULN) to define malignant restriction of V beta use, pathologic restriction of V beta use was found directly or indirectly in all 27 samples carrying a diagnosis of malignancy and directly in 2 of 16 samples without a diagnosis of malignancy. TCR gene rearrangement studies were used to confirm V beta flow cytometry results. By using a cutoff of 1.6 times the ULN for the detection of malignancy, the antibody panel had a diagnostic sensitivity of 89% for direct detection of pathologic V beta restriction and a specificity of 88%, making it useful for rapid diagnosis of T-cell leukemia.

The prognostic significance of an inv(3)(q21q26.2) in addition to a t(9;22)(q34;q11.2) in patients treated with tyrosine kinase inhibitors.

In chronic myelogenous leukemia, BCR-ABL1 positive detection of cytogenetic abnormalities in addition to the t(9;22) is thought to portend a poor prognosis; however, not all abnormalities associated with the t(9;22) have the same impact. Inv(3) defines a group of aggressive neoplasms with poor response to conventional treatment options. In this study, four cases with the t(9;22) and inv(3) treated with tyrosine kinase inhibitors (TKI) were investigated. In three cases, the inv(3) was not detected at the initial diagnosis and the patients initially responded to TKI therapy; the inv(3) was detected at blast crisis in all three cases, and one case had both abnormalities at the initial presentation, but this case presented as acute myeloid leukemia. In all cases, detection of an inv(3) was associated with a high blast count and a lack of response to treatment regimens including TKI. All patients died within months from the detection of inv(3). This indicates that cases with the t(9;22) and inv(3) have a clinical course similar to that of cases with an inv(3) and no other therapeutically targetable abnormality.

Validation of fluorescence in situ hybridization using an analyte-specific reagent for detection of abnormalities involving the mixed lineage leukemia gene.

CONTEXT: Fluorescence in situ hybridization (FISH) is a molecular cytogenetic assay that is commonly used in laboratory medicine. Most FISH assays are not approved by the US Food and Drug Administration but instead are laboratory-developed tests that use analyte-specific reagents. Although several guidelines exist for validation of FISH assays, few specific examples of FISH test validations are available in the literature. OBJECTIVE: To provide an example of how a FISH assay, using an analyte-specific reagent probe, may be validated in a clinical laboratory. DESIGN: We describe the approach used by an individual laboratory for validation of a FISH assay for mixed lineage leukemia (MLL) gene. RESULTS: Specific validation data are provided illustrating how initial assay performance characteristics in a FISH assay for MLL may be established. CONCLUSIONS: Protocols for initial validation of FISH assays may vary between laboratories. However, all laboratories must establish several defined performance specifications prior to implementation of FISH assays for clinical use. We describe an approach used for assessing performance specifications and validation of an analyte-specific reagent FISH assay using probes for MLL rearrangement in interphase nuclei.