CT-guided bone marrow aspirations and biopsies: retrospective study and comparison with blind procedures.

PURPOSE: To compare the pathology results of CT-guided and blind bone marrow aspirations and biopsies. METHODS: Ninety-eight consecutive CT-guided biopsies and 98 age- and gender-matched blind (non-CT-guided) posterior iliac crest bone marrow aspirations and biopsies performed in 2017 were reviewed for adequacy of core biopsies and aspirate smears. CT procedure images and CT abdomen/pelvis images were reviewed to evaluate anatomic features of the posterior ilium and soft tissues. Statistical analysis was performed using a T test, Fisher exact test, and Kruskal-Wallis test. RESULTS: There was no significant difference in the age and gender of the two groups (p > 0.05). However, the CT-guided group had a higher BMI (p = 0.0049) and posterior soft tissue thickness (p = 0.0016). More CT-guided biopsy samples (CT 93 (95%); blind 77 (79%); p = 0.0006) and aspirate smears (CT 90 (92%); blind 78 (80%); p = 0.042) were categorized as adequate. The CT-guided group had longer core lengths (CT 1.4 ± 0.6 (range 0.3-3.5) cm; blind 1.0 ± 0.60 (range 0-2.6) cm; p = 0.0001). Overall, 131/164 (80%) of the cases had at least one of the described features (slanted posterior ilium (angle > 30°), 30%; rounded posterior ilium, 20%; thick posterior ilium cortex, 13%; focal lesion in posterior ilium, 12%; prior procedure in posterior ilium, 5%; posterior soft tissue thickness > 3 cm, 40%). CONCLUSION: CT-guided bone marrow procedures were more likely to result in both adequate aspirate smears and biopsy samples and longer core lengths when compared with blind procedures.

FOXP3-stained image analysis for follicular lymphoma: Optimal adaptive thresholding with maximal nucleus coverage.

Immunohistochemical detection of FOXP3 antigen is a usable marker for detection of regulatory T lymphocytes (TR) in formalin fixed and paraffin embedded sections of different types of tumor tissue. TR plays a major role in homeostasis of normal immune systems where they prevent auto reactivity of the immune system towards the host. This beneficial effect of TR is frequently "hijacked" by malignant cells where tumor-infiltrating regulatory T cells are recruited by the malignant nuclei to inhibit the beneficial immune response of the host against the tumor cells. In the majority of human solid tumors, an increased number of tumor-infiltrating FOXP3 positive TR is associated with worse outcome. However, in follicular lymphoma (FL) the impact of the number and distribution of TR on the outcome still remains controversial. In this study, we present a novel method to detect and enumerate nuclei from FOXP3 stained images of FL biopsies. The proposed method defines a new adaptive thresholding procedure, namely the optimal adaptive thresholding (OAT) method, which aims to minimize under-segmented and over-segmented nuclei for coarse segmentation. Next, we integrate a parameter free elliptical arc and line segment detector (ELSD) as additional information to refine segmentation results and to split most of the merged nuclei. Finally, we utilize a state-of-the-art super-pixel method, Simple Linear Iterative Clustering (SLIC) to split the rest of the merged nuclei. Our dataset consists of 13 region-of-interest images containing 769 negative and 88 positive nuclei. Three expert pathologists evaluated the method and reported sensitivity values in detecting negative and positive nuclei ranging from 83-100% and 90-95%, and precision values of 98-100% and 99-100%, respectively. The proposed solution can be used to investigate the impact of FOXP3 positive nuclei on the outcome and prognosis in FL.

Allo-SCT for myelofibrosis: reversing the chronic phase in the JAK inhibitor era?

At present, allo-SCT is the only curative treatment for patients with myelofibrosis (MF). Unfortunately, a significant proportion of candidate patients are considered transplant ineligible due to their poor general condition and advanced age at the time of diagnosis. The approval of the first JAK inhibitor, ruxolitinib, for patients with advanced MF in 2011 has had a qualified impact on the treatment algorithm. The drug affords substantial improvement in MF-associated symptoms and splenomegaly but no major effect on the natural history. There has, therefore, been considerable support for assessing the drug's candidacy in the peritransplant period. The drug's precise impact on clinical outcome following allo-SCT is currently not known; nor are the drug's long-term efficacy and safety known. Considering the rarity of MF and the small proportion of patients who undergo allo-SCT, well designed collaborative efforts are required. In order to address some of the principal challenges, an expert panel of laboratory and clinical experts in this field was established, and an independent workshop held during the 54th American Society of Hematology Annual Meeting in New Orleans, USA on 6 December 2013, and the European Hematology Association's Annual Meeting in Milan, Italy on 13 June 2014. This document summarizes the results of these efforts.

Acute myeloid leukemia: advances in diagnosis and classification.

Acute myeloid leukemia is an aggressive myeloid neoplasm characterized by ≥20% myeloblasts in the blood or bone marrow. Current treatment strategies for acute myeloid leukemia are based on both patient-related parameters such as age and performance status as well as the intrinsic characteristics of particular disease subtypes. Subtyping of acute myeloid leukemia requires an integration of information from the patient's clinical history (such as any prior preleukemic myeloid neoplasm or cytotoxic potentially leukemogenic therapy), the leukemia morphology, cytogenetic findings, and the mutation status of particular genes (NPM1, FLT3, and CEBPA). In recent years, a barrage of information has become available regarding gene mutations that occur in acute myeloid leukemia and their influence on prognosis. Future therapies for acute myeloid leukemia will increasingly rely on the genetic signatures of individual leukemias and will adjust therapy to the predicted disease aggressiveness as well as employ therapies targeted against particular deregulated genetic pathways. This article reviews current standards for diagnosing and classifying acute myeloid leukemia according to the 2008 WHO Classification. Data that have subsequently accumulated regarding newly characterized gene mutations are also presented. It is anticipated that future leukemia classifications will employ a combination of karyotypic features and the gene mutation pattern to stratify patients to increasingly tailored treatment plans.

Clonal X-chromosome inactivation suggests that splenic cord capillary hemangioma is a true neoplasm and not a subtype of splenic hamartoma.

Splenic hamartoma is a rare tumor-like lesion composed of structurally disorganized red pulp elements. It has been hypothesized that two other splenic lesions, cord capillary hemangioma and myoid angioendothelioma, may fall within the spectrum of splenic hamartoma, simply representing morphological variants. In this study, we compared the vascular and stromal composition of cord capillary hemangioma and myoid angioendothelioma with those of classical hamartoma. In addition, we assessed the clonal vs polyclonal nature of the lesions in nine female cases by performing clonality analysis for X-chromosome inactivation at the human androgen receptor locus (HUMARA) on laser-assisted microdissected samples. In 15 of 17 cases, increased reticulin and/or collagen content was observed. The classical hamartoma cases showed a vasculature predominantly composed of CD8+ CD31+ CD34- splenic sinuses, whereas cases of cord capillary hemangioma and myoid angioendothelioma contained many CD8- CD31+ CD34+ cord capillaries, but very little CD8+ vasculature. All cases lacked expression of D2-40 and Epstein Barr virus-encoded RNA. All cases showed a proliferation index of ≤5% by Ki-67. Cases of classical hamartoma lacked significant perisinusoidal expression of collagen IV and low-affinity nerve growth factor receptor. Both markers were variably expressed in the other lesions. Increased CD163-positive histiocytes were found in four cases (three cord capillary hemangiomas and one myoid angioendothelioma). HUMARA analysis was informative in all nine tested cases, of which three cases showed a non-random X-chromosome inactivation pattern, indicating clonality. All three clonal cases were cord capillary hemangiomas. Our study has shown that in spite of considerable morphologic heterogeneity and overlapping features, classical hamartoma and cord capillary hemangioma and myoid angioendothelioma are different in terms of their vascular and stromal composition. Clonality analysis supports a true neoplastic origin for the cord capillary hemangioma. A larger study using additional immunohistochemical and molecular studies is necessary to further evaluate the biological significance of the current findings.

Bone marrow findings correlate with clinical outcome in systemic AL amyloidosis patients.

AIMS: Bone marrow sampling is a key investigation in the work-up of amyloid light chain (AL) amyloidosis, but the relationship between bone marrow findings and the varied phenotype and clinical outcome of AL amyloidosis is unclear. The aim was to determine if bone marrow pathological parameters at diagnosis were related to clinical behaviour in AL amyloidosis patients. METHODS AND RESULTS: Bone marrow findings, clinical features and outcome of 80 patients referred with a diagnosis of systemic AL amyloidosis were evaluated; six patients were subsequently excluded due to re-categorization as other forms of amyloidosis. At latest follow-up (median 66 months), 11 of the 18 patients with no identifiable bone marrow neoplastic cells (61%) versus only seven of the 56 patients with neoplastic plasma cells or non-Hodgkin's lymphoma (13%) were alive (P = 0.0046). However, neither the quantity of the neoplastic cells nor the serum light chain levels were correlated with amyloid burden or patient survival. CONCLUSIONS: Identification of a neoplastic population in the bone marrow of AL amyloidosis patients by histology and immunohistochemistry correlates with poor outcome; however, the neoplastic cell burden is not prognostically significant, suggesting that additional factors are important in determining disease behaviour in AL amyloidosis.

Optimal processing of bone marrow trephine biopsy: the Hammersmith Protocol.

Specimens of bone marrow trephine biopsy (BMT) are transported and fixed in acetic acid-zinc-formalin fixative, decalcified in 10% formic acid-5% formaldehyde and processed with other specimens to paraffin-wax embedding. Sections, 1-microm-thick, are cut by experienced histotechnologists and used for haematoxylin and eosin, Giemsa, reticulin silver and other histological stains. Further, all immunohistochemical procedures used in the laboratory, including double immunostaining, can be used on these sections with no or minimal modifications. About 10,000 BMT specimens have been analysed using this procedure since 1997 and diseases involving the bone marrow have been classified successfully. More recently, standardised polymerase chain reaction-based analysis and mRNA in situ hybridisation studies have been conducted. Excellent morphology with good antigen, DNA and RNA preservation is offered by the Hammersmith Protocol.

Quantitation of bacteria in bone marrow from patients with typhoid fever: relationship between counts and clinical features.

Enteric fever is the only bacterial infection of humans for which bone marrow examination is routinely recommended. A prospective study of the concentrations of bacteria in the bone marrow and their relationship to clinical features was conducted with 120 Vietnamese patients with suspected enteric fever, of whom 89 had confirmed typhoid fever. Ninety-three percent of the Salmonella enterica serovar Typhi samples isolated were resistant to ampicillin, chloramphenicol, and co-trimoxazole. For 81 patients with uncomplicated typhoid and satisfactory bone marrow aspirates, the number of serovar Typhi CFU in bone marrow aspirates was a median value of 9 (interquartile range [IQR], 1 to 85; range, 0.1 to 1,580) compared to 0.3 (IQR, 0.1 to 10; range, 0.1 to 399) CFU/ml in simultaneously sampled blood. The ratio of individual blood counts to bone marrow counts was 10 (IQR, 2.3 to 97.5). The number of bacteria in blood but not bone marrow was correlated inversely with the duration of preceding fever. Thus, with increasing duration of illness the ratio of bone marrow-to-blood bacterial concentrations increased; the median ratio was 4.8 (IQR, 1 to 27.5) during the first week compared with 158 (IQR, 60 to 397) during the third week. After lysing the host cells, the median ratio of viable bone marrow to blood increased, reflecting the higher concentration of intracellular serovar Typhi in the bone marrow. Effective antibiotic pretreatment had a significantly greater effect in reducing blood counts compared to bone marrow counts (P < 0.001). Thus, bacteria in the bone marrow of typhoid patients are less affected by antibiotic treatment than bacteria in the blood. The numbers of bacteria in bone marrow correlated negatively with the white blood cell (R = -0.3, P = 0.006) and platelet counts (R = -0.32, P = 0.01) and positively with fever clearance time after treatment (R = 0.4, P < 0.001). The bacterial load in bone marrow therefore may reflect the clinical course of the infection, and high levels may suppress neutrophil proliferation.

Acute erythremic myelosis (true erythroleukaemia): a variant of AML FAB-M6.

AIMS: Classic erythroleukaemia (acute myeloid leukaemia M6, or M6 AML) is defined as an excess of myeloblasts in an erythroid predominant background. Leukaemia variants in which the primitive blast cells are demonstrably erythroid are extremely rare and poorly characterised. Variably referred to as "true erythroleukaemia" or "acute erythremic myelosis", they are often included within the M6 AML category even though they do not meet strict criteria for this type of AML. METHODS: Two cases of acute erythroid neoplasia are presented with clinical, morphological, immunophenotypic, and cytogenetic analysis. RESULTS: Both patients presented with profound anaemia, one in a setting of long standing myelodysplasia. Bone marrow examination revealed a predominant population of highly dysplastic erythroid cells in both cases. In one case, the liver was infiltrated by neoplastic erythroid cells. Both patients died within four months of diagnosis. CONCLUSIONS: This report illustrates that cases of acute leukaemia occur in which the dominant neoplastic cell is a primitive erythroid cell without an accompanying increase in myeloblasts. This does not preclude the neoplastic clone originating in a multipotent haemopoietic stem cell, as suggested by cases arising in patients with myelodysplasia. Acute erythremic myelosis should be recognised as a distinct variant of M6 AML.

Thyroid hormones regulate hypertrophic chondrocyte differentiation and expression of parathyroid hormone-related peptide and its receptor during endochondral bone formation.

Hypothyroidism in children causes developmental abnormalities in bone and growth arrest, while thyrotoxicosis accelerates growth rate and advances bone age. To determine the effects of thyroid hormones on endochondral bone formation, we examined epiphyseal growth plates in control, hypothyroid, thyrotoxic, and hypothyroid-thyroxine (hypo-T4)-treated rats. Hypothyroid growth plates were grossly disorganized, contained an abnormal matrix rich in heparan sulfate, and hypertrophic chondrocyte differentiation failed to progress. These effects correlated with the absence of collagen X expression and increased parathyroid hormone-related protein (PTHrP) messenger RNA (mRNA) expression. In thyrotoxic growth plates, histology essentially was normal but PTHrP receptor (PTHrP-R) mRNA was undetectable. PTHrP is a potent inhibitor of hypertrophic chondrocyte differentiation that acts in a negative feedback loop with the secreted factor Indian hedgehog (Ihh) to regulate endochondral bone formation. Thyroid hormone receptor alpha1(TRalpha1), TRalpha2, and TRbeta1 proteins were localized to reserve zone progenitor cells and proliferating chondrocytes in euthyroid rat cartilage; regions in which PTHrP and PTHrP-R expression were affected by thyroid status. Thus, dysregulated Ihh/PTHrP feedback loop activity may be a key mechanism that underlies growth disorders in childhood thyroid disease.

Recurrences in nodal T-cell lymphoma. Changes in histologic appearance and immunophenotype over the course of disease.

We examined the patterns of relapse or persistence in 37 cases of nodal peripheral T-cell lymphoma (PTCL) to address the morphologic and immunophenotypic findings. Relapses were documented in lymph node (25 cases) and/or a variety of extranodal sites at a mean of 21 months after presentation; several cases recurred as late as 13 years. Persistent bone marrow involvement was a feature of angioimmunoblastic lymphoma (AIL) and histiocyte-rich and small-cell tumors. Relapses in anaplastic tumors often involved unusual extranodal sites. The majority of relapsed PTCLs retained a similar histologic appearance, pattern of nodal involvement, and immunophenotype. Histologic progression, as assessed by increased numbers of large cells, was seen in 3 cases of AIL, in 1 case with an initial small cell morphologic appearance, and in 2 cases of PTCL with an initial mixed small and large cell appearance. Immunostains for T-cell activation markers showed increased immunoreactive cells in 5 of the 6 cases, whereas increased numbers of p53-positive tumor cells were noted in 3 of the 6 cases. The discrete large cell transformation occasionally seen in B-cell lymphoma and extranodal T-cell lymphoma was not observed in these cases.

Normal and chronic phase CML hematopoietic cells repopulate NOD/SCID bone marrow with different kinetics and cell lineage representation.

INTRODUCTION: Chronic myelogenous leukemia is characterized by a clonal expansion of abnormal hematopoietic cells, which eventually replaces normal hematopoiesis. We wanted to test the hypothesis that the growth kinetics of CML and normal hematopoietic cells are different. MATERIALS AND METHODS: We compared the growth kinetics and the phenotype of engraftment of chronic phase CML and normal human CD34(+) precursor cells in the bone marrow of immune deficient mice. RESULTS: High levels of engraftment of normal precursors occurred early and consisted of myeloid, erythroid, megakaryocytic, and lymphoid elements. This level and pattern of engraftment were maintained at later assessments. The level of CML cell engraftment was initially much lower, but it increased progressively at late time-points with no indication of a plateau in growth. Early engraftment of CML cells consisted almost entirely of myeloid and mast cells but soon after only mast cells were detectable. Conversely mast cells were infrequent in mice engrafted with normal progenitors. CONCLUSION: We conclude that in contrast to normal cell engraftment, engraftment of CML cells in NOD/SCID mice is characterized by a slow but progressive myeloid infiltration, which eventually consists almost entirely of mast cells.

Allo-major histocompatibility complex-restricted cytotoxic T lymphocytes engraft in bone marrow transplant recipients without causing graft-versus-host disease.

Previous experiments in humans and mice have shown that allogeneic donors can serve as a source of cytotoxic T lymphocytes (CTL) specific for proteins, such as cyclin-D1 and mdm-2, expressed at elevated levels in tumor cells. In vitro, allo-major histocompatibility complex (MHC)-restricted CTL against these proteins selectively killed allogeneic tumor cells, including lymphoma, but not normal control cells. This suggested that these CTL may be useful for adoptive tumor immunotherapy, provided that they (1) survive in MHC-disparate hosts, (2) maintain their killing specificity, and (3) do not attack normal host tissues. Here, we used cloned allo-restricted CTL isolated from BALB/c mice (H-2(d)) that killed H-2(b)-derived tumor cells expressing elevated levels of the mdm-2 target protein. When these CTL were injected into bone marrow transplanted (BMT) C57BL/6 (H-2(b)) recipients, they consistently engrafted and were detectable in lymphoid tissues and in the bone marrow (BM). Long-term survival was most efficient in spleen and lymph nodes, where CTL were found up to 14 weeks after injection. The administration of CTL did not cause graft-versus-host disease (GVHD) normally associated with injection of allogeneic T cells. These data show that allo-restricted CTL clones are promising reagents for antigen-specific immunotherapy in BMT hosts, because they engraft and retain their specific killing activity without causing GVHD.

High expression of CD23 in the proliferation centers of chronic lymphocytic leukemia in lymph nodes and spleen.

Chronic lymphocytic leukemia (CLL) is a B-cell neoplasm composed of a heterogeneous mixture of cells, including small lymphocytes, prolymphocytes, and large transformed cells; these last cells appear to represent the proliferating compartment. CLL cells express, in addition to B cell markers, the transmembrane receptor CD23. CD23 functions as the receptor for IgE and also appears to play a role in controlling the growth and proliferation of lymphocytes. Its level of expression among the different cells in CLL has not been examined. In this study, we show that CD23 expression is much higher in the large transformed CLL cells than in the small lymphoid population. This may provide an explanation for the observed correlation between a circulating CD23 cleavage product (soluble CD23) and prognosis in CLL. In addition, we have shown that proliferation in splenic CLL occurs preferentially in the white pulp zones, even in cases in which both the white and red pulp are extensively infiltrated.

TEL/PDGFbetaR induces hematologic malignancies in mice that respond to a specific tyrosine kinase inhibitor.

The TEL/PDGFbetaR fusion protein is expressed as the consequence of a recurring t(5;12) translocation associated with chronic myelomonocytic leukemia (CMML). Unlike other activated protein tyrosine kinases associated with hematopoietic malignancies, TEL/PDGFbetaR is invariably associated with a myeloid leukemia phenotype in humans. To test the transforming properties of TEL/PDGFbetaR in vivo, and to analyze the basis for myeloid lineage specificity in humans, we constructed transgenic mice with TEL/PDGFbetaR expression driven by a lymphoid-specific immunoglobulin enhancer-promoter cassette. These mice developed lymphoblastic lymphomas of both T and B lineage, demonstrating that TEL/PDGFbetaR is a transforming protein in vivo, and that the transforming ability of this fusion is not inherently restricted to the myeloid lineage. Treatment of TEL/PDGFbetaR transgenic animals with a protein tyrosine kinase inhibitor with in vitro activity against PDGFbetaR (CGP57148) resulted in suppression of disease and a prolongation of survival. A therapeutic benefit was apparent both in animals treated before the development of overt clonal disease and in animals transplanted with clonal tumor cells. These results suggest that small-molecule tyrosine kinase inhibitors may be effective treatment for activated tyrosine kinase-mediated malignancies both early in the course of disease and after the development of additional transforming mutations.

The kinetics and extent of engraftment of chronic myelogenous leukemia cells in non-obese diabetic/severe combined immunodeficiency mice reflect the phase of the donor's disease: an in vivo model of chronic myelogenous leukemia biology.

In vitro studies have provided little consensus on the kinetic abnormality underlying the myeloid expansion of chronic myelogenous leukemia (CML). Transplantation of human CML cells into non-obese diabetic mice with severe immunodeficiency disease (NOD/SCID mice) may therefore be a useful model. A CML cell line (BV173) and peripheral blood cells collected from CML patients in chronic phase (CP), accelerated phase (AP), or blastic phase (BP) were injected into preirradiated NOD/SCID mice. Animals were killed at serial intervals; cell suspensions and/or tissue sections from different organs were studied by immunohistochemistry and/or flow cytometry using antihuman CD45 monoclonal antibodies (MoAbs), and by fluorescence in situ hybridization (FISH) for the BCR-ABL fusion gene. One hour after injection, cells were sequestered in the lungs and liver, but 2 weeks later they were no longer detectable in either site. Similar short-term kinetics were observed using 51Cr-labeled cells. The first signs of engraftment for BV173, AP, and BP cells were detected in the bone marrow (BM) at 4 weeks. At 8 weeks the median percentages of human cells in murine marrow were 4% (range, 1 to 9) for CP, 11% (range, 5 to 36) for AP, 38.5% (range, 18 to 79) for BP, and 54% (range, 31 to 69) for BV173. CP cells progressively infiltrated BM (21%) and spleen (6%) by 18 to 20 weeks; no animals injected with the cell line or BP cells survived beyond 12 weeks. The rate of increase in human cell numbers was higher for BP (7.3%/week) as compared with CP (0.9%/week) and AP (0. 5%/week). FISH analysis with BCR and ABL probes showed that some of the human cells engrafting after injection of CP cells lacked a BCR-ABL gene and were presumably normal. We conclude that CML cells proliferate in NOD/SCID mice with kinetics that recapitulate the phase of the donor's disease, thus providing an in vivo model of CML biology.