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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.

A 40-year-old woman with hypertrophic obstructive cardiomyopathy presents to the hematology clinic for a second opinion regarding a history of headaches and fatigue for the past 10 years. She has been diagnosed with idiopathic erythrocytosis, presumed to be due to polycythemia vera. She periodically

Introduction/Objective Entrustable professional activities (EPAs) are defined as specialty-specific tasks representing a unit of professional practice that can be entrusted to unsupervised performance by a trainee following attainment of sufficient task-specific competence. EPAs and periodic competency assessments also provide a framework for evaluating relevant ACGME milestones. We describe our experience creating EPAs for peripheral blood smear (PBS) and body fluid (BF) analysis through which residents became qualified to act as laboratory testing personnel. Methods Training occurred during a 6 week “boot camp” for PGY2 and PGY4 residents in July-August 2018 and July-August 2019. Training for PBS included didactic lectures in automated hematology and RBC morphology (2 hr) and WBC and platelet morphology (2 hr); faculty-guided microscope reviews of RBC (2 hr) and WBC morphology (2 hr) using a training checklist; completion of RBC and WBC self-assessment quizzes; and a 40 question graded exam that covered cell identification, lab protocols, and case scenarios. Training for BF included didactic lectures (2 hr); faculty-guided microscope review of BF slides using a training checklist; completion of a BF self-assessment quiz; and a 40 question graded exam that covered cell identification, lab protocol, and case scenarios. Following successful completion of the graded exam residents were deemed competent to perform unsupervised review of cases initially flagged as abnormal by laboratory technologists; they were required to obtain attending review prior to release of results in defined situations. Formal competency assessments according to CLIA standards were done at 6 and 12 months after initial training. Impact on laboratory workflow and turnaround time was assessed before and after training. Conclusion We successfully created EPAs for PBS and BF analysis through which residents became qualified to act as laboratory testing personnel. There was no adverse impact on laboratory turnaroud time, and the number of PBS and BF cases requiring attending pathologist review decreased. Residents appreciated this tangible opportunity to gain graduated responsibility that prepared them for future practice. Periodic competency assessments provide an opportunity to evaluate relevant ACGME milestones. Our training and assessment program and EPAs can serve as a template for other residency programs.

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 ( R 2 = .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.

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.

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.

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.

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.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.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.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.

It is unclear how often and in what setting fluorescence in situ hybridization (FISH) panels for myelodysplastic syndrome (MDS)/acute myeloid leukemia (AML) provide additional information over metaphase cytogenetics alone. Furthermore, the usefulness of peripheral blood vs bone marrow FISH has also not been directly compared. We prospectively compared metaphase cytogenetics and FISH for -5/5q-, -7/7q-, +8, and 20q- in 433 cases of suspected MDS/AML. FISH testing was abnormal in 6 (14%) of 43 and 10 (19%) of 54 cases with fewer than 20 normal metaphases or no growth, respectively. FISH was only rarely abnormal in cases with 20 normal metaphases obtained (6/222 [2.7%]). Comparison of peripheral blood and bone marrow results in 48 cases showed abnormal peripheral blood FISH results in 18 (69%) of 26 cases with abnormal bone marrow FISH results and in 5 (23%) of 22 cases with normal bone marrow FISH results. These findings, the largest published comparison of FISH vs metaphase cytogenetics in MDS/AML, provide a rational strategy for FISH testing in peripheral blood and bone marrow.

TP53 mutations in myeloid malignancies are either homozygous or hemizygous due to copy number-neutral loss of heterozygosity or deletion of 17p

Fludarabine and 200 cGy TBI are commonly used for reduced-intensity conditioning preceding allogeneic hematopoietic SCT (HSCT). However, graft rejection and disease relapse are significant causes of treatment failure with this regimen. We modified this regimen by escalating the TBI dose to 400 cGy in 40 patients with hematologic malignancies. Thirty-four patients achieved complete donor T-cell chimerism at a median of 40 days following HSCT. The incidences of grades II-IV and III-IV acute GVHD were 40 and 15%, respectively, whereas that of limited and extensive chronic GVHD were 12 and 20%, respectively. Two patients rejected their grafts and 12 relapsed. The 100-day mortality was 18%, 2-year transplant-related mortality 20% and overall survival was 58% at a median follow-up of 16 months. There were no significant survival differences between patients with lymphoid compared to myeloid malignancies. A dose of 400 cGy TBI administered with fludarabine is well tolerated and further study is needed to determine whether outcomes are superior to those with 200 cGy TBI.

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.

Children with Down syndrome who develop transient myeloproliferative disorder or acute megakaryoblastic leukemia harbor somatic mutations in theGATA1 gene, a phenomenon specific to this syndrome. Heald and colleagues report the first case of a stillborn fetus with Down syndrome-associated acute megakaryoblastic leukemia/transient myeloproliferative disorder and a GATA1mutation. The authors discuss the management of fetuses with suspected or confirmed diagnosis of Down syndrome and highlight the need for genetic screening and counseling based on maternal age. 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.

Single nucleotide polymorphism arrays complement metaphase cytogenetics in detection of new chromosomal lesions in MDS

The diagnosis of myelodysplastic syndrome (MDS) is difficult to establish based on morphologic features alone because dysplasia may not always be detectable and the presence of dysplasia is not itself evidence of clonal disorder. As a result, the detection of a clonal cytogenetic abnormality has a major role in difficult cases in regard to diagnosis and the recognition of morphological cytogenetic correlates. In an attempt to assess the frequency and characteristic type of abnormal clones when it is not clear whether or not a hematological condition is neoplastic, cytogenetics have been analyzed necessarily in 159 patients with unexplained cytopenia or suspected MDS. We found 14 patients (8.8%) with cytogenetic abnormalities in the absence of concomitant dysplastic features of the marrow at diagnosis. The cytogenetic changes were characteristic of those reported for myeloid malignancies: 3 del(20q), 2 Y chromosome losses, 2 del(5q), 2 11q23 abnormalities, and one each of t(3;5), i(7q), trisomy 8, and del(13q). One case of ring chromosome 4 was also found. A few months later, 3 of these patients showed marrow changes consistent with MDS. Our data demonstrated that a significant proportion of otherwise uncertain diagnoses presented abnormal clones. Long-term follow-up will be required to help determine the malignant potential of these clones.

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.

We observed increased ferritin levels in newly diagnosed MDS-RARS patients without transfusional iron-overload. Hence, we hypothesized RARS patients may harbor hemochromatosis-related mutations, which could contribute to the pathophysiology of this myelodysplastic syndromes (MDS) subset. We studied a cohort of 140 MDS patients: 42 with RARS, 10 with increased ringed sideroblasts, and 96 with other forms of MDS (43 RA, 27 RAEB, 17 RAEB-T, 8 MDS/MPD, 1 CMML). Patients were genotyped using restriction fragment length polymorphism, designed to detect C282Y and H63D mutations of the HFE gene. We found significantly higher frequency of heterozygosity for C282Y mutation in RARS patients compared with a large control population of matched race individuals (21 vs. 9.8% in controls, P = 0.03); H63D genotype was not significantly increased. Frequency of HFE variation in other MDS subtypes failed to differ significantly from controls. Within this group, we included patients with a rare form of MDS, provisionally subclassified by WHO as RARS with thrombocytosis (RARSt). 10/14 RARSt patients were carriers of either C282Y or H63D allele significantly increased compared with the combined prevalence in a healthy population (71 vs. 33%, P < 0.01). We found expected distribution of mutant HFE alleles in patients with other forms of MDS (9.1 vs. 9.8%, P = 0.82). Increased prevalence of HFE gene mutations is not a generalized feature of MDS, but some subgroups of MDS, especially those characterized by excessive accumulation of ringed sideroblasts, exhibit C282Y mutations at a higher frequency than in other forms of MDS and healthy controls.

Allograft dendritic cell (DC) content has been identified as a predictor of relapse and event-free survival after allogeneic bone marrow transplantation. However, the prognostic importance of DCs has not been evaluated in the setting of autologous hematopoietic stem cell transplantation (HSCT). We prospectively determined pre-transplant and post transplant DC levels, including DC1 and DC2 subset levels, in 53 patients with diffuse large B-cell non-Hodgkin's lymphoma (DLBC NHL) undergoing autologous HSCT. Pre-transplant DCs were measured in the collected stem cell products and were therefore indicative of cell numbers infused directly into patients; post transplant analysis of DCs was performed on the peripheral blood of patients 6 weeks after the infusion of autologous stem cells. Higher pre-transplant levels of DC1 cells and total DCs were significantly associated with improved survival. Similarly, greater post transplant levels of total DCs and both subsets were significantly associated with survival. These findings suggest a relationship between DC reconstitution and survival following autologous HSCT for DLBC NHL. Strategies to increase autograft DC content or accelerate DC recovery after autologous HSCT might improve outcomes in this setting.

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.

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.

Dendritic cells (DCs) are antigen-presenting cells that are critical to the generation of immunologic tumor responses. Myeloid DCs (DC1) express myeloid antigen CD11c; lymphoid DCs (DC2) express CD123(+) and are CD11c(-). Analysis of DC subsets from peripheral blood progenitor cells (PBPC) collected from normal donors mobilized with G-CSF shows a predominance of DC2 cells. Whether PBPCs mobilization by chemotherapy yields different subsets of DCs has not been studied. We analyzed DC subsets in apheresis products from 44 patients undergoing autologous stem cell transplantation from 6/00 to 5/01. Patients received either G-CSF alone (10 microg/kg per day, n=11) or etoposide (2 g/m(2)) plus G-CSF (n=33) for progenitor cell mobilization. The patients were apheresed for 2-10 days (median 3) to reach a minimum of 2.0 x 10(6) CD34(+) cells/kg. Patients receiving G-CSF alone mobilized significantly more total DC2s than did those receiving etoposide plus G-CSF (median 6.2 x 10(6)/kg vs 2.9 x 10(6)/kg, P=0.001). The DC2/DC1 ratio was also significantly different in the two groups, with the G-CSF group having a higher ratio (median 1.2 vs 0.4, P0.001). We conclude that the combination of chemotherapy plus G-CSF yields different mobilized dendritic cell subsets than does G-CSF alone.

We used spectral karyotyping (SKY) to study 29 adults with acute myeloid leukemia and a complex karyotype containing one to nine abnormalities that were not fully identifiable by G-banding. SKY showed the origin of rings and unidentified material in unbalanced translocations in all cases and the origin of markers in most, allowing reinterpretation of 136 aberrations and discovery of three aberrations hidden in normal chromosomes. SKY confirmed 10 and refined the interpretation of three balanced aberrations recognized by G-banding and identified another nine balanced aberrations, including a novel translocation involving the RUNX1 gene. Eleven of 32 deletions found by G-banding were shown to be cryptic translocations or insertions, including three of four chromosome 3 deletions, two of three del(7q), and two of 12 del(5q). Of the 92 chromosomes deemed lost entirely by G-banding, 63 (68%) were shown to be involved in structural aberrations. This was especially true for −21 (eight of eight patients), −5 (five of six patients), −20 (seven of nine patients), and −18 (six of 12 patients). Unexpectedly, SKY uncovered a hidden overrepresentation of segments from at least one chromosome in 21 patients. The most frequently overrepresented was 21q, found in eight patients, including four with high-level 21q amplification. Fluorescence in situ hybridization showed that the RUNX1 gene was not the target of amplification in seven of these patients. Also frequently gained were 11q (in seven patients, including three with high-level MLL gene amplification) and 22q (in seven patients). We conclude that SKY considerably enhances the accuracy of karyotype interpretation, and that amplification of chromosomal material may play a greater role in leukemogenesis than has been recognized. © 2002 Wiley-Liss, Inc.

PURPOSE: To prospectively compare cytogenetics and reverse transcriptase–polymerase chain reaction (RT-PCR) for detection of t(8;21)(q22;q22) and inv(16)(p13q22)/t(16;16)(p13;q22), aberrations characteristic of core-binding factor (CBF) acute myeloid leukemia (AML), in 284 adults newly diagnosed with primary AML. PATIENTS AND METHODS: Cytogenetic analyses were performed at local laboratories, with results reviewed centrally. RT-PCR for AML1/ETO and CBFβ/MYH11 was performed centrally. RESULTS: CBF AML was ultimately identified in 48 patients: 21 had t(8;21) or its variant and AML1/ETO, and 27 had inv(16)/t(16;16), CBFβ/MYH11, or both. Initial cytogenetic and RT-PCR analyses correctly classified 95.7% and 96.1% of patients, respectively (P = .83). Initial cytogenetic results were considered to be false-negative in three AML1/ETO-positive patients with unique variants of t(8;21), and in three CBFβ/MYH11-positive patients with, respectively, an isolated +22; del(16)(q22),+22; and a normal karyotype. The latter three patients were later confirmed to have inv(16)/t(16;16) cytogenetically. Only one of 124 patients reported initially as cytogenetically normal was ultimately RT-PCR–positive. There was no false-positive cytogenetic result. Initial RT-PCR was falsely negative in two patients with inv(16) and falsely positive for AML1/ETO in two and for CBFβ/MYH11 in another two patients. Two patients with del(16)(q22) were found to be CBFβ/MYH11-negative. M4Eo marrow morphology was a good predictor of the presence of inv(16)/t(16;16). CONCLUSION: Patients with t(8;21) or inv(16) can be successfully identified in prospective multi-institutional clinical trials. Both cytogenetics and RT-PCR detect most such patients, although each method has limitations. RT-PCR is required when the cytogenetic study fails; it is also required to determine whether patients with suspected variants of t(8;21), del(16)(q22), or +22 represent CBF AML. RT-PCR should not replace cytogenetics and should not be used as the only diagnostic test for detection of CBF AML because of the possibility of obtaining false-positive or false-negative results.

We have analyzed the survival motor neuron gene (SMN1) dosage in 100 parents of children with homozygous SMN1 deletions. Of these parents, 96 (96%) demonstrated the expected one-copy SMN1 carrier genotype. However, four parents (4%) were observed to have a normal two-copy SMN1 dosage. The presence of two intact SMN1 genes in the parent of an affected child indicates either the occurrence of a de novo mutation event or a situation in which one chromosome has two copies of SMN1, whereas the other is null. We have separated individual chromosomes from two of these parents with two-copy SMN1 dosage by somatic cell hybridization and have employed a modified quantitative dosage assay to provide direct evidence that one parent is a two-copy/ zero-copy SMN1 carrier, whereas the other parent had an affected child as the result of a de novo mutation. These findings are important for assessing the recurrence risk of parents of children with spinal muscular atrophy and for providing accurate family counseling.

The associations of cytogenetics with complete remission (CR) rates, overall survival (OS), and outcomes after CR were studied in 609 previously untreated AML patients younger than 56 years old in a clinical trial comparing 3 intensive postremission therapies: intensive chemotherapy, autologous transplantation (ABMT), or allogeneic bone marrow transplantation (alloBMT) from matched related donors. Patients were categorized into favorable, intermediate, unfavorable, and unknown cytogenetic risk groups based on pretreatment karyotypes. CR rates varied significantly (P

TEL gene rearrangement due to the 12;21 chromosome translocation is believed to be the most common molecular genetic abnormality in childhood acute lymphoblastic leukemia (ALL). A study was conducted to investigate the frequency and prognostic significance of TEL/AML-1 fusion gene resulting from a cryptic t(12;21)(p13;q22). Bone marrow samples from 86 patients diagnosed over the past 5 years at Columbus Children's Hospital were analyzed by fluorescence in situ hybridization (FISH) technique for TEL/AML-1 fusion gene, using LSI((R)) DNA probes. The positive cases were analyzed for clinical outcome. Patients in this study received treatment according to Children's Cancer Group (CCG) protocols. Fifteen of the 86 cases (17%) were positive for the fusion gene. All were B-cell lineage and except for one, all were CD10 positive. TEL/AML-1 was not found in any T-cell ALL. The mean overall survival (OS) following diagnosis for the TEL/AML-1-positive group was significantly longer than for the TEL/AML-1-negative group by log-rank = 7.84, P = 0.005. Similarly, the event-free survival (EFS) after remission for the positive group (median 94.5 months) was longer than the negative group (median 57 months) by log-rank = 7.19, P = 0.007. This study confirms that the TEL/AML-1 fusion gene may be the most common genetic event in childhood ALL, occurring in 17% of the patients. It appears restricted to the B-cell lineage. In this study, the presence of a TEL/AML-1 fusion gene was statistically significant in predicting both OS and EFS, indicating a favorable clinical outcome for these patients. Screening for TEL/AML-1 should become routine at diagnosis and a useful biological variable for risk stratification in future clinical trials.

We have identified a gene at 11q23, telomeric to MLL , that encodes a guanine nucleotide exchange factor (GEF). This gene is transcribed into a 9.5-kb mRNA containing a 4.6-kb ORF. By Northern analysis, it was found to be expressed in all human tissues examined including peripheral blood leukocytes, spleen, prostate, testis, ovary, small intestine, colon, and minimally in thymus. Analysis of the predicted protein sequence indicates that it has strong homology to several members of the family of Rho GEFs that includes such oncogenes as Dbl , Vav , Tiam , and Bcr . A patient with primary acute myeloid leukemia (AML) and a karyotype of 51,XY,+8,+19,+3mar was found to have the 5′ end of MLL at exon 6 fused in-frame with the 3′ end of almost the entire ORF of this gene, which we named LARG for leukemia-associated Rho GEF. Transcriptional orientation of both genes at 11q23 is from centromere to telomere, consistent with other data that suggest the MLL-LARG fusion resulted from an interstitial deletion rather than a balanced translocation. LARG does not appear to have any homology with other MLL partner genes reported thus far. Thus, LARG represents an additional member of the GEF family and a novel MLL fusion partner in acute myeloid leukemia.

We describe a five-generation kindred with familial eosinophilia (FE; MIM131400), characterized by the occurrence of sustained eosinophilia of unidentifiable cause in multiple relatives. The inheritance pattern is consistent with an autosomal dominant pattern. Among 52 related subjects studied, 19 were affected and 33 were unaffected. Ten unaffected spouses were also evaluated. Four subjects with sustained eosinophilia were diagnosed with cardiac abnormalities and two of them also had neurologic symptoms. In comparison with the unaffected or spouses, evaluation of complete blood counts showed that the affected relatives had, as expected, significantly higher white cell (P < 0.005) and absolute eosinophil counts (P < 0.001) and lower red cell counts (P < 0.05). Evaluation of serum cytokine levels (IL-5, IL-3, and granulocyte-macrophage colony-stimulating factor (GMCSF) and serology for parasitic helminth infection demonstrated no differences between the affected and unaffected individuals; no individuals studied had serologic evidence for parasitic infection. There were also no differences in anti-nuclear antibody, serum cobalamin (vitamin B12) level, immunoglobulin level, leukocyte alkaline phosphatase, rheumatoid factor, HLA analysis, and stool findings for ova and parasites. Among eight affected persons who had peripheral blood or bone marrow karyotype analysis, two carried the same chromosome abnormality, a pericentric inversion of chromosome 10, inv (10) (p11.2q21.2). A gene mapping study is currently underway to study the underlying genetic mechanism(s) of this syndrome. Am. J. Med. Genet. 76:229–237, 1998. © 1998 Wiley-Liss, Inc.

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.

Two structurally unrelated chemicals, aflatoxin B1 and propane sultone, transformed human foreskin cells to a stage of anchorage-independent growth. Isolation from agar and repopulation in monolayer culture of these transformed cells was followed by transfection with a cDNA library, which resulted in cells that exhibited an altered epithelioid morphology. Chemically transformed/nontransfected cells and transfected normal cells did not undergo a significant morphological change. These epithelioid-appearing, transfected cells, when inoculated into nude mice, form progressively growing tumors. The tumors are histopathologically interpreted as carcinomas. All of the first generation tumors in the surrogate hosts exhibited characteristic rates of growth similar to those of transplants of spontaneous human tumors. In the second generation of tumor xenografts, the progressively growing tumors derived from the transfected cells exhibited a more rapid rate of growth. Southern analysis and reverse transcription PCR confirmed that a 1.3-kb genetic element was integrated into the genome and was actively being transcribed. Examination of the metaphase chromosomes in normal human cells revealed that the genetic element responsible for this conversion was located at site 31-32 of the q arm of chromosome 7. The DNA sequence of this 1.3-kb genetic element contains a coding region for 79 amino acids and a long 3'-untranslated region and appears to be identical to CATR1.3 isolated from tumors produced by methyl methanesulfonate-converted, nontransplantable human tumor cells.

A cell line, SCC83-01-82, derived from a human oral squamous carcinoma, was non-tumorigenic in nude mice, a characteristic of premalignant cells. Conversion of these cells to a tumorigenic phenotype with chemical mutagens did not increase mutations in hot spots or other conserved regions of p53 or H-ras genes. Investigation of the tumorigenic conversion using an expression library resulted in isolation of a previously unidentified gene, CATR1, located on the long arm of chromosome 7 at band approximately q31-32. Evidence for the involvement of this gene in conversion to tumorigenicity was demonstrated by introduction of a eukaryotic expression CATR1 construct into SCC83-01-82 cells. Transfection with the antisense construct reduced the expression of CATR1 in tumors formed by the transfected cells, suggesting that the antisense suppression of endogenous CATR1 expression appeared to be sufficient for tumorigenic conversion. These results are consistent with previous reports of cytogenetic analyses of tumors, that 7q31-32 contains a gene(s) with tumor suppressor activity; CATR1 is a candidate for this putative suppressor gene.

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.

C.B-17 SCID mice were inoculated with human peripheral blood leukocytes (PBLs) from normal Epstein-Barr virus (EBV)-seropositive and -seronegative donors. Confirmation of a functioning human immune response was demonstrated by the detection of human antibody after inoculation with rotavirus, tetanus toxoid, or EBV. One group of animals inoculated with PBLs from an EBV-seropositive donor developed immunoblastic lymphomas approximately 9 weeks after transplantation. Confirmation of the species and sex of origin of the tumor cells was established using a spontaneous cell line prepared from the tumor. At passage I, the tumor-cell line (AGTI) showed 15% of the metaphases with a translocation involving chromosomes 5 and 14. A lymphoblastoid cell line (AGLCL) established from the same PBLs from the same donor at the time of inoculation of the mice had a normal female karyotype. The AGLCL and a clone of AGTI cells were analyzed for rearrangement of immunoglobulin heavy chain (IgH) genes; both cell lines showed rearrangement of both IgH alleles. The results outlined in this report suggest that a spontaneous chromosomal translocation involving chromosome 14 occurred in normal PBLs in the SCID mouse.

The 8;21 translocation is one of the most common chromosomal translocations in acute myelogenous leukemia (AML), accounting for 40% of pediatric AML with French-American-British (FAB)-M2 morphology. The chromosomal breakpoints have recently been identified at the molecular level and shown to involve the AML1 gene on chromosome 21 and the ETO gene on chromosome 8. Translocation results in the consistent fusion of these genes on the der(8) chromosome, resulting in the production of a novel chimeric gene and message. Using oligonucleotide primers derived from the AML1 and ETO cDNAs, we were able to amplify a specific fusion transcript from 26 of 26 patients with t(8;21) by a reverse transcriptase polymerase chain reaction (PCR) approach. DNA fragments of identical size were generated from each case including two with complex translocations. Studies on the sensitivity and specificity of this approach show that PCR analysis can be used as a rapid, accurate, and sensitive means for detecting this chromosomal abnormality, and for following the patients' response to therapy.

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.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma of childhood and accounts for 10% of all solid tumors in children. There are three different histologic forms of this tumor: embryonal (RMS-E), alveolar (RMS-A), and primitive (RMS-P). Among these, the embryonal form has responded well to chemotherapy. Identification of the correct subtype is important for both the management and treatment of this malignancy. However, the histopathologic classification of RMS is sometimes difficult and distinguishing between the embryonic and primitive forms can present a diagnostic dilemma. Chromosomal abnormalities have been observed in all subtypes. We present the cytogenetic findings in six cases of RMS or related sarcoma. All four cases with RMS-A had both numerical and structural abnormalities in the tumor and involved bone marrow specimens. Three patients had a common marker, t(2;13)(q37;q14), and one patient had a variant marker involving 13q14, t(1;13) (p36;q14), and double minutes (dmin). The single embryonal RMS patient had modal chromosome numbers in the hypertriploid range and extensive structural abnormalities; the t(2;13) was not present, but translocation of 13q to both 1q and 2p was observed, der(1)t(1;13)(q21;q14) and der(2)t(2;13)(p25;q14). The patient with primitive type RMS had a hypodiploid line with several markers, including a complex translocation involving chromosomes 5 and 13 with a breakpoint at 13q14, and t(11;12)(q24;q12), a chromosome marker heretofore found only in Ewing's sarcoma and related tumors. This patient had atypical RMS with mixed neural and myogenic elements. The significance of these chromosomal markers and their importance in the characterization of childhood tumors are discussed, along with a review of the literature.

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.

Cytogenetic aberrations identified by metaphase cytogenetics (MC) have important diagnostic, prognostic and therapeutic roles in myelodysplastic syndromes (MDS). Fluorescence in situ hybridization (FISH) complements MC by the ability to evaluate large numbers of both interphase and metaphase nuclei. However, clinically practical FISH strategies are limited to detection of known lesions. Single nucleotide polymorphism array (SNP-A)-based karyotyping can reveal unbalanced defects with superior resolution over MC and FISH and identify segmental uniparental disomy (UPD) undetectable by either method. Using a standardized approach, we focused our investigation on detection of −5/del(5q), −7/del(7q), trisomy 8 and del(20q) in patients with MDS (N = 52), MDS/myeloproliferative overlap syndromes (N = 7) and acute myeloid leukemia (N = 15) using MC, FISH and SNP-A karyotyping. The detection rate for del(5q) was 30, 32 and 32% by MC, FISH, and SNP-A, respectively. No single method detected all defects, and detection rates improved when all methods were used. The rate for detection of del(5q) increased incrementally to 35% (MC + FISH), 38% (MC + SNP-A), 38% (FISH + SNP-A) and 39% (all three methods). Similar findings were observed for −7/del(7q), trisomy 8 and −20/del(20q). We conclude that MC, FISH and SNP-A are complementary techniques that, when applied and interpreted together, can improve the diagnostic yield for identifying genetic lesions in MDS and contribute to the better description of abnormal karyotypes.

UPD1p indicates the presence of MPL W515L mutation in RARS-T, a mechanism analogous to UPD9p and JAK2 V617F mutation

We evaluated patients with newly diagnosed ALL treated at the Cleveland Clinic during the years 1996 through 2005. Cox proportional hazards analysis was used to identify univariate and multivariate correlates of complete remission, overall survival and progression-free survival. On univariate analysis, significant prognostic factors included: age at diagnosis (per 10-year increase), poor risk cytogenetics, time to white blood count recovery, and time from induction chemotherapy (IC) to post-remission therapy (PRT). In patients age60 years without poor risk cytogenetics, time from IC to PRT (per week increase) was a significant prognostic factor by multivariate analysis and was associated with a decreased progression-free survival [HR 1.27, CI (1.04-1.55), p = 0.019] and decreased overall survival [HR 1.34, CI (1.08-1.67), p = 0.009]. Delayed time from IC to PRT (or =6.6 weeks) was associated with a statistically worse progression-free and overall survival.

Achievement of complete donor chimerism (CDC) after allogeneic nonmyeloablative hematopoietic stem cell transplantation (NMHSCT) is important for preventing graft rejection and for generating a graft-vs-malignancy effect. The alloreactivity of NK cells and some T-cell subsets is mediated through the interaction of their killer immunoglobulin-like receptors (KIRs) with target cell HLA/KIR ligands. The influence of KIR matching on the achievement of T-cell CDC after NMHSCT has not been previously described. We analyzed 31 patients undergoing T-cell replete related donor NMHSCT following fludarabine and 200 cGy TBI. Recipient inhibitory KIR genotype and donor HLA/KIR ligand matches were used to generate an inhibitory KIR score from 1 to 4 based upon the potential number of recipient inhibitory KIRs that could be engaged with donor HLA/KIR ligands. Patients with a score of 1 were less likely to achieve T-cell CDC (P=0.016) and more likely to develop graft rejection (P=0.011) than those with scores greater than 1. Thus, patients with lower inhibitory KIR scores may have more active anti-donor immune effector cells that may reduce donor chimerism. Conversely, patients with greater inhibitory KIR scores may have less active NK cell and T-cell populations, which may make them more likely to achieve CDC.