Multiple Myeloma 21 Epidemiology

Multiple myeloma, also termed plasma cell myeloma, is a currently incurable neoplastic disorder. MM accounts for slightly more than 10 of hematopoietic malignancies and has a slight predilection for males (male female, 3 2). The disease is rare in individuals less than 35 years of age the peak incidence is in the eighth decade of life. Approximately 14,400 new patients present annually in the United States, with approximately 50,000 total patients affected.8 The incidence of myeloma is...

PStat5

Histogram of anti-CD15 and anti-phospho-Stat5 staining in a peripheral blood mononuclear cell sample spiked with K562 cells, a BCR-ABL positive cell line that shows constitutive activation of Stat5. These results show detection of the constitutive high expression p-Stat5 in the CD15+ K562 cells and the ability to modulate that expression with imatinib mesylate (STI571) treatment. The importance of single cell measurements, as opposed to bulk measurements on cell lysates, in...

Summary

In this review, we have highlighted recent advances in chronic lymphoproliferative disorders that commonly involve the peripheral blood. As we have seen, our concepts of certain diseases are changing. Molecular genetic and immunophenotypic studies are allowing more precise characterization of CLL and defining important biologic markers that predict clinical behavior. Prolymphocytic leukemia is now more narrowly defined and its relationship to nucleolated variants of MCL is now apparent. With...

References

Oscier DG, Matutes E, Copplestone A et al. Atypical lymphocyte morphology an adverse prognostic factor for disease progression in stage A CLL independent of trisomy 12. Br J Haematol. 1997 98 934-939 2. Matutes E, Oscier D, Garcia-Marco J et al. Trisomy 12 defines a group of CLL with atypical morphology correlation between cytogenetic, clinical and laboratory features in 544 patients. Br J Haematol. 1996 92 382-388 3. Frater JL, McCarron KF, Hammel JP et al. Typical and atypical chronic...

Chronic Lymphocytic Leukemia

From a pathologist's standpoint, the diagnosis of CLL can be relatively straightforward from examination of the blood smear and immunophenotyping. Most cases demonstrate the typical morphology of mature lymphocytes with condensed, clumped nuclear chromatin, round nuclear contours, and scanty cytoplasm. Routine use of multiparameter flow cytometry confirms the morphologic impression when the typical phenotype of CD5+, CD19+, CD20+ (usually low level expression), CD23+, CD79b dim negative, FMC7...

Nmyc

The next most common IgH translocation, t(4 14), detected by either FISH or reverse transcription-polymerase chain reaction (RT-PCR), has been associated with poor responses to primary chemotherapy and a very poor prognosis.76,77 The t(4 14) is strongly associated with chromosome 13 abnormalities detected by FISH they are seen together in up to 85 of MM.78 The t(4 14) is also found in MGUS but at this point is of unclear significance in this disorder.79 The t(14 16) is the third most common IgH...

Lymphoma

The World Health Organization recognizes that genetic anomalies are one of the most reliable criteria for classification of malignant lymphomas.62 Conventional cytogenetic studies can detect many of these anomalies but this method is difficult, expensive and requires fresh tissues for analysis. Thus, many investigators today use FISH to study cells from touch preparations or paraffin-embedded lymphoid tissue.24'112'113'114 All patients with Burkitt lymphoma have a t(8 14)(q24 q32) or a variant...

Introduction

Fine needle aspiration biopsy (FNAB) is a minimally invasive technique utilized for rapid diagnosis of mass lesions through evaluation of cytological details of the aspirated material. This technique has been well accepted in Europe,1-3 and barring a few countries, FNAB has seen a renaissance worldwide over the last thirty years.4 This is especially true for lesions involving the thyroid gland, breast and salivary glands.4 However, apart from a few select medical centers,5 FNAB of lymph nodes...

Normal Plasma Cell Differentiation

In normal plasma cell differentiation, immature B-cells, that have undergone VDJ recombination, migrate from the bone marrow to lymphoid tissues (Figure 5-1). Within the lymph node, the immature B-cells either 1) mature into short lived plasma cells that remain in the local tissue, secrete IgM, and have a half life of approximately three days or, 2) enter follicular germinal centers and undergo programmed cell death unless rescued by antigen selection. Cells selected for survival undergo IgH...

Landmark Studies In Leukemia And Lymphoma

The reports by Golub on leukemia and Alizadeh on lymphoma illustrated the potential for high density gene expression arrays to contribute to the understanding of hematologic malignancies.4, 5 While two different microarray platforms were chosen in the two studies, powerful results were obtained by both methods. Golub et al demonstrated that the major subtype of acute myeloid leukemia (AML) could be distinguished from acute lymphoid leukemia (ALL) while using an oligoprobe microarray designed by...

Targeting Cd33positive Acute Myeloid Leukemia

The CD33 antigen is a transmembrane protein expressed on the leukemic cells for the majority of patients with AML.104 This antigen is not expressed on non-hematopoietic cells. These observations make it an appealing target for antibody therapy.105,106 Gemtuzumab ozogamicin is an immunoconjugate of a humanized anti-CD33 monoclonal antibody chemically linked to a semisynthetic derivative of a very potent cytotoxic antitumor antibiotic, calicheamicin.107 (Figure 3-6) A phase I dose-escalation...

Large cell lymphomas

Large cell lymphomas account for 40 of adult lymphomas148 and one third of pediatric cases.149 The majority of adult large cell lymphoma (LCL) are B cell in origin, whereas in children there is a near equal division of B lineage, T lineage and intermediate phenotypes.149 CD30 positivity is frequently seen in pediatric LCL's.149 Diffuse large B-cell lymphoma accounts for about 30-40 of adult NHL in the western hemisphere.30 The age range is wide with cases reported in children. 92, 149 Although...

Core Binding Factor Leukemias

Core binding factor leukemias represent subgroups of AML which have rearrangements involving gene encoding for the core binding factor (CBF) alpha-2 (AML1, RUNX1) and the beta subunits, and include FAB M2 AMLs with t(8 21)(q22 q22), inv(16)(p13q22), and t(16 16)(p13 q22).16-20 Such specific cytogenetic changes lead to the generation of fusion genes which include AML1-ETO and CBF-MYH11, respectively. Such patients have cytogenetic abnormalities which are included among the favorable-risk...

Bone Disease

Myeloma bone disease plays a profound role in patient morbidity. Bone marrow biopsy evaluation should document the presence of bony destruction and degree of osteoclastic activity. Bone lesions are found mainly in areas with adjacent myeloma cells and are caused by increased osteoclast activity and impaired osteoblast function. The myeloma cells either induce osteoblast apoptosis or block osteoblast differentiation. Myeloma cell binding to marrow stromal cells induces osteoclastogenic factors....

Hodgkin Lymphoma

Approximately 30 of all lymphomas fall into this category30 and have been divided in to (1) Nodular lymphocyte predominant HL (2) Classical HL with further subtypes including (a) Nodular sclerosis, (b) Mixed cellularity, (c) Lymphocyte-rich and (d) Lymphocyte-depleted in the WHO classification.30 Features common to all subtypes include preferential involvement of cervical lymph nodes, predominance in the younger age group and the presence of characteristic large tumor cells designated as Reed...

Variables In Mircoarray Studies In Lymphoma

A comparison of the different studies of microarray analysis in lymphoma will demonstrate that there are variables in the type of platform used, the number of probe targets present, the control (if any) RNA source utilized, the number of cases studied, and the type of software used to analyze the data (Table 1-1). Variables in tissue preservation, percent of tumor cells, and normalization of the data also likely exist, but will not be further detailed here as these data were variably reported....

Cml

-At diagnosis perform conventional cytogenetic studies on bone marrow.60 -To monitor patients during therapy, use an appropriate FISH assay to detect chromosome anomalies identified at diagnosis. In most patients, blood or bone marrow can be studied before and after treatment to track changes in the percentage of neoplastic over time. -A FISH assay for BCR ABL fusion is sometimes useful to rule out CML in the diagnosis of essential thrombocythemia or polycythemia vera. To compare the efficacy...

Historical Perspective

In addition to its utility as an adjunct to diagnosis, exciting new horizons for clinical cytometry are rapidly emerging, and developing, as tools in the therapeutic management of leukemia and lymphoma patients. Increasingly flow cytometric analyses are becoming key in therapeutic decisions and in therapeutic monitoring of patients. The explosion of antigen and ligand directed therapies for a range of hematopoietic malignancies, including anti-CD20,9-12 anti-CD22,13 anti-CD52,1U2,14...

Info

GEP patterns confirm MM is composed of many distinct molecular entities, with variability in expression of early and late differentiation genes. On hierarchical clustering analysis, four distinct subgroups of MM have been identified. The subgroups show an association with survival those with a GEP signature similar to MGUS have a better prognosis while those more similar to myeloma cell lines are more aggressive.65,86 Specific genes are also likely to have prognostic implications. For...

Neoplastic Plasma Cell Differentiation

Germinal center B-cells that survive antigen selection during normal plasma cell differentiation have marked intrinsic genetic instability. This leads to errors in IgH switch recombination or less frequently somatic hypermutation. Many of the errors involve the immunoglobulin (Ig) locus and likely represent the initial immortalizing event in plasma cell tumorgenesis. For example, juxtaposition of the IgH gene next to an oncogene is common, resulting in oncogene dysregulation.1 Genetically...

Small lymphocytic lymphoma

Small lymphocytic lymphoma (SLL) refers to the non-leukemic tissue counterpart of chronic lymphocytic leukemia (CLL) and, together, these account for 6.7 of NHL.30 Most patients are over 50 years of age with a male female ratio of 2 1.30 SLL CLL is often disseminated at presentation with generalized lymphadenopathy, involvement of peripheral blood, bone marrow, spleen and liver.6 Although indolent, it is incurable with the currently available therapeutic regimens.30 10-20 of cases may transform...

Selection of DNA Probes

Most FISH probes contain two or more separate DNA sequences that collectively hybridizes over 200 to 600 kilobases of target DNA. The selection of fluorescent-labeled DNA probes to accurately detect any hematological neoplasm is critical for a successful FISH study. Oncogenes that are directly involved with the neoplastic process are excellent probe targets for FISH studies. Evidence indicates that one or more oncogenes are located near sites of chromosome breakage for several of the specific...

N

DNA probes for FISH are classified by where they hybridize. Some probes hybridize to specific centromeres (A) and others to specific gene loci (B). Some probes hybridize to unique DNA sequences producing a paint-like pattern for a specific chromosome (C). With M-FISH each of the chromosomes can be painted a different color (D). Using ND-FISH, cells with monosomy have one red and one green signal (E), cells with trisomy have three red and three green signals (F), and cells with...

Mantle cell lymphoma

Lymphogranular Bodies

Mantle cell lymphoma (MCL) commonly presents with stage III or IV disease with bone marrow involvement, generalized lymphadenopathy, and hepatosplenomegaly (often a massively enlarged spleen).91-95 About one quarter of the cases have peripheral blood involvement and extra-nodal sites commonly include Waldeyer's ring and gastrointestinal tract.30 Figure 8-1. Small cell undifferentiated carcinoma of the lung. Malignant cells with high nuclear cytoplasmic ratio, coarse chromatin and prominent...

Cdk6 150

Chromosome 11q23 See Mixed lineage leukemia (MLL) gene (chromosome 11q23) Chronic lymphocytic leukemia 5, 7, 8, 10, 70, 77, 97, 98, 100,104, 109, 111, 121, 145-149, 155, 156, 159, 160, 161, 164, 169, 170, 179, 189, 190, 195, 200, 226, 235, 236, 240, 244, 252 angiogenesis in 226 atypical 146, 159 chromosomal abnormalities 147 FISH approach to 97, 98, 147 IgH mutational status 10 IGH mutational status 147, 148 ZAP-70 expression and prognosis (See also ZAP-70) 148 Chronic myeloid leukemia (CML)...

Importance of the Bone Marrow Microenvironment

The bone marrow microenvironment plays a critical role in plasma cell survival, growth and differentiation. Adherence of plasma cells to the bone marrow extracellular matrix induces the production of numerous cytokines by stromal cells that have both autocrine and paracrine functions. Interleukin-6 (IL-6) is a key growth and survival factor for myeloma that additionally mediates many of the secondary effects of the malignancy, including osteolysis, anemia and immunodeficiency. Surprisingly,...

Demonstration of Clonality

Results of FISH studies demonstrate clonality of hematological malignancies. The concept of clonal expansion is based on the premise that the formation of each neoplastic clone begins with a chromosome anomaly in a single cell. This cell then proliferates to produce two cells, then four cells and so forth until millions of abnormal cells with the same chromosome anomaly occur. FISH probes that detect the primary chromosome anomaly associated with a neoplastic process will produce an abnormal...

Bcell Chronic Lymphocytic Leukemia

The prognosis and clinical course of patients with B-CLL are highly variable. Studies with conventional cytogenetics, FISH, molecular genetics, immunophenotyping, and mutation analysis of the immunoglobulin heavy chain variable regions have all been used to study B-CLL.23,91-95,96 Each of these approaches helps to assess prognosis in this disease, but the oncogenic events that lead to the origin of B-CLL remain unknown. Based on conventional cytogenetic studies, the most common anomalies in...

Angiogenesis In Acute Lymphoblastic Leukemia

Perez-Atayde et al.2 showed that microvessel density in bone marrow biopsies obtained from children with ALL was significantly higher than normal control bone marrow biopsies. The microvessels in ALL bone marrows were tortuous and arborizing, while microvessels in normal bone marrows were straight and with no branching.2 Urine levels of bFGF were higher in ALL patients, compared to age-matched controls.2 Urine bFGF levels and bone marrow microvessel density were decreased after therapy, but...

Identification of Minimal Residual Diseases by Immunophenotyping

The identification of minimal residual disease by detecting aberrant phenotypes by multiparametric flow cytometry in patients in morphologic CR has emerged as a potentially useful strategy.51,52 Such techniques appear sensitive and quantification of the number of residual cells with the leukemic phenotype has been used to classify patients into risk categories with different risks of relapse. (Figure 3-3) High-risk patients may be candidates for alternative novel treatment strategies.

Clinical Features

A unique triad of osteolytic bone lesions, atypical bone marrow plasmacytosis, and monoclonal gammopathy characterizes myeloma. Many of the clinical and laboratory findings at diagnosis reflect or are secondary to increased circulating immunoglobulin and bone destruction (Table 5-1). The most common symptoms at presentation are bone pain (58 of cases), fatigue (32 ), and weight loss (24 ).6 Osteolytic bone lesions are a major cause of morbidity and are therefore further discussed in section...

Lymphocyte depletion HL

The number of cases diagnosed as LD HL has decreased since immunophenotyping became available. LD HL is the most rare subtype (< 1 )71 and cases classified as LD in the past are now considered to be mostly non-Hodgkin's lymphoma (especially large cell lymphomas, anaplastic large cell lymphoma), other subtypes of HL (e.g syncytial variant of NS), or occasionally non-hematopoietic neoplasms.1,71,72 The lymphocyte depletion subtype is associated with advanced stage and B symptoms in the majority...

Classifications

2.1 French-American-British Classification Although the French-American-British (FAB) classification remains a useful common language among hematopathologists, clinical investigators, and clinicians, important new insights into the biology of AML have limited its utility and provided a foundation for new classifications.1,8,9 The FAB classification is based solely on the morphology of the leukemic cells and basic cytochemical stains. Subsequent to the initial publication describing the FAB...

Ndfish

The most common FISH strategy to detect aneuploidy and chromosome deletions uses a probe of one color for a control site and a probe of another color for an interstitial target site on the same chromosome.2 For purposes of this chapter, this method is referred to as ND-FISH to indicate that this method detects numeric anomalies and or deletions. An illustration of ND-FISH is shown in Figure 4-3. The most common colors of fluors used with ND-FISH are red and green. This FISH strategy produces...

Quantitation of Minimal Residual Disease by Realtime Reverse Transcriptase Polymerase Chain Reaction PCR

Molecular techniques to amplify and detect DNA and RNA from leukemic cell s now permit the identification of minimal amounts of leukemia in blood and bone marrow.46 The presence of fusion transcripts in patients with CBF leukemias not only is important in establishing the correct diagnosis, but also facilitates the identification of those patients in apparent CR with minimal residual disease (MRD).47 Recent studies have suggested one important difference between patients with the t(8 21)(q22...

Specimen Collection

The frequently used biopsy techniques are well described in several reference texts. ' ' Non-aspiration techniques have also utilized. Superficial, palpable lymph nodes are most amenable to FNAB while deep-seated lymph nodes require radiological guidance. FNAB is best performed either by a trained cytopathologist or interventional radiologist who is aware of the clinical presentation. 1 39, 40 Facilities for on-site adequacy assessment and preliminary interpretation, reduces concerns about...

Perspective

There are now numerous reports showing increased angiogenesis and increased production of various angiogenic factors (most notably VEGF) in hematologic malignancies. Two questions remain to be clarified, and are undergoing active investigation by many groups. First, what is the basis of regulation of angiogenesis All indications are that, similar to solid tumors, angiogenesis in hematologic malignancies is regulated by a complex interplay between pro-angiogenic factors and anti-angiogenic...

Fish Strategies

DNA probes are classified by where they hybridize.1 Some FISH probes hybridize to specific centromeres (Figure 4-2A)32,33 and others to gene loci (Figure 4-2B)3'21'34'35. Some probes hybridize to unique DNA sequences over the length of the entire chromosome to produce a paint-like pattern for a single chromosome (Figure 4-2C) or to paint all chromosome pairs a different color (Figure 4-2D).36 DNA probes can be used individually or in combinations and can be labeled with different colored...

Tcell lymphoma

T-cell lymphomas comprise a heterogeneous group of neoplasms and have been divided into immature (precursor T-lymphoblastic leukemia lymphoma) and mature phenotypes (mature T-cell and NK-cell neoplasms) in the recent WHO classification.30 These neoplasms account for approximately 10 of NHL in North America but are found with increased frequency in Asian countries.5 T-cell lymphoma is a difficult diagnosis by FNAB.67 A specific sub-classification of the mature subtypes is not possible based on...

Brief Overview Of Tumor Angiogenesis

The hypothesis that tumor growth is angiogenesis-dependent was first proposed in 1971.3 Many lines of evidence indicate that growth of tumors beyond a diameter of 2-3 mm requires formation of new blood vessels.4-6 These blood vessels supply oxygen and nutrients to the growing tumor, and also promote metastasis.5 Specific anti-angiogenesis agents such as endostatin7-9 and anti-VEGF antibodies10 suppress tumors in animal models, providing strong support for the hypothesis that tumors are...

Automated Versus Manual Blood Analysis

Major clinical laboratories perform CBCs and leukocyte differential counts by automated methods. Modern automated hematology analyzers offer rapid, high throughput analysis, low cost-per-test, high precision (due to both the high number of cells analyzed and excellent between-run reproducibility), and superb accuracy. Current automated systems are limited by an inability to quantify certain cell types (such as blasts and immature granulocyte forms), and they also require manual validation from...

Angiogenesis In Chronic Lymphocytic Leukemiacll

We have shown that there is increased microvessel density in bone marrow biopsies from patients with CLL, compared to normal control biopsies.81 There was a positive correlation between microvessel density and Rai clinical stage.81 We also showed that urine levels of bFGF were significantly higher in CLL patients as compared to healthy controls.81 Urine levels of VEGF were also shown to be higher in CLL patients, but the difference was not statistically significant.81 Molica et al.82 also found...

Pu1

In contrast to classic RS cells, L& H cells do not express CD15, CD30, or fascin, but they do express B-cell markers CD20, CD79a, and CD75 as well as CD45 32-36. In addition, L& H cells express BCL-6 (attesting to their germinal center origin), EMA in approximately 50 of cases, and they also synthesize J chains (a feature of B-cells).1,34-37 They are also positive for OCT 2, BOB.1, and PU.1, whereas classic RS cells are almost always negative.38, 39 Light chain restriction in RS cells has...

Lymphocyterich Classical Hodgkin Lymphoma 271 Clinical features

LRC HL shares many of the clinical features of NLP HL that are distinct from other forms of CHL. It comprises about five percent of all HL.34,41 There is a male predominance (approximately 70 ), and the median age at diagnosis is greater than that of other types of classical HL as well as that of NLP HL. Like patients with NLP HL, patients with NLRC HL present with low stage disease (stages I II) and usually lack B symptoms, bulky disease, and mediastinal involvement. In contrast to NLP HL,...

Myeloproliferative Disorders Other Than CML

At least 27 chromosome anomalies have been associated with MpD.60,6i,62,63,64 Although not diagnostic, a relatively strong association exists between del(13)(q12q14) with myelofibrosis65,66 , t(5 12)(q33 p13) with eosinophilia67, and del(20)(q11q13), +8, +9 with polycythemia vera68. These and other chromosome anomalies are readily detected by conventional cytogenetic studies, but good FISH probes are available to detect most of them as well. An algorithm for cytogenetic and FISH testing in MPD...

Burkitt lymphoma Burkittlike lymphoma

Previously referred to as small non-cleaved lymphoma,115 these are highly aggressive lymphomas that may present in a leukemic phase116 (L3 type of acute lymphoblastic leukemia) or as an extra-nodal mass.117, 118 The endemic form, representing the commonest childhood malignancy in equatorial Africa,30 presents as a jaw or orbital mass,117-119 or an extra-nodal mass with involvement of the bowel, omentum, ovaries, kidneys or breast. 117, 119, 120 Climactic factors may correspond to endemicity in...

Genetics Of Splenic Lymphoma With Villous Lymphocytes Slvl

SLVL (splenic marginal zone lymphoma, SMZL) is a rare entity characterized by a proliferation of post-germinal center B cells present initially in the spleen and bone marrow, generally with a lack of lymph node involvement at presentation. The clinical course is generally indolent. The cytogenetic and molecular biologic findings of SLVL SMZL have been recently reviewed.36 The most commonly encountered genetic lesions in this lymphoma include loss of chromosome 7q31-q32 (up to 40 of cases) and...

Cytomics

The sequencing of the human genome and historic advancements in information technology have brought about the age of omics. Functional genomics (transcriptomics), and proteomics allow us to analyze disease patterns and physiologic states not simply by the analysis of a few select characteristics, but by broad patterns that emerge in the simultaneous analysis of thousands of expressed genes or proteins. Within each individual cell, gene expression is regulated by a complex network of mechanisms...

Microscope Analysis

FISH studies require the use of a high quality fluorescence microscope equipped with a 100-watt mercury lamp and appropriate filter sets. To observe FISH signals produced by a set of probes, one of which is red and the other which is green, it is best to use a combination of a single DAPI filter and a dual-pass SpectrumGreen and SpectrumOrange filter set. With this setup, the nucleus appears blue from the DAPI stain, and red and green FISH signals can be observed within the nucleus the...

Diagnosis

The bone marrow examination is essential for the diagnosis of MM, even when immunologic or radiographic studies are strongly supportive of this disease. Studies of bone marrow also provide prognostic information and a basis from which to measure therapeutic response. Adequate sampling, including obtaining both aspirate smears and a trephine biopsy, is critical as focal lesions may be widely spaced and irregularly distributed. In approximately 9 of myeloma, the bone marrow cannot be aspirated...

Variant Chromosome Anomalies

In clinical practice, it is important to detect common chromosome anomalies and their variant forms. This is best done by conventional cytogenetic studies, but FISH can also accomplish this goal because variant chromosomes possess the same underlying molecular abnormality as the more common anomaly. Most structural anomalies of chromosomes originate during replication or repair of DNA when chromosomes are uncoiled and different chromosomes are overlapped or in close association with one...

Summation

Over the last twenty years, cytometric evaluation of hematopoietic malignancies has evolved from an adjunctive test which was secondary in the diagnostic work-up, typically following by several days the primary diagnostic work-up, to one which although still adjunctive to morphology is now a key aspect of the primary diagnostic package. The maturing of the technology in this setting is satisfying however, more exciting are the glimpses of the next evolution of this technology in a clinical...

Whole Chromosome Paints and Multi Color FISH

For cells in metaphase, whole chromosome paints can be used to identify most major structural anomalies of a few chromosomes at a time (Figure 4-2C).39 A variant of whole chromosome paints is called M-FISH (multi-color FISH) because it paints each of the 24 types of human chromosomes a different color (Figure 4-2D). This method is particularly useful to characterize chromosomal anomalies in complex karyotypes that are difficult to define by conventional cytogenetic studies.36 However, these...

Future Potential Roles

Perhaps even more exciting is the potential role that cytometric analyses may play in assessing and monitoring of the new generation of therapies such as imatinib mesylate (STI571, Gleevec)50'51 that more specifically target abnormal or dysregulated proteins in the malignant or abnormal cells in a patient. Driven by the Human Genome and Proteomics initiatives, the pace at which genes and molecules associated with specific diseases are being identified has greatly accelerated as has the...

Advances In Lymphoma

Lymphoma Subtype

What are some of the conclusions that can be derived from microarray analysis in lymphoma First, morphologic subtypes of lymphoma do cluster together in microarray analysis. Second, large numbers of tumor cases are needed for proper analysis and subsequent validation of the data. Third, cell lineage subgroups have been biologically validated by other assays, (e.g., identification of t(14 18) translocations in only the germinal center B-cell subgroup of diffuse large cell lymphoma).9, 14 Fourth,...

Preliminary Evaluation Of Fnab Smears

A preliminary assessment of the nature of the aspirate at the time of FNAB is critical to guide procurement of additional material for ancillary studies. Details evaluated at low power include cellularity, architecture, necrosis and presence of lymphoglandular bodies (cytoplasmic fragments), especially prominent in ML (Table 8-1). High power cytomorphologic features include assessment of cell size, nuclear size, chromatin pattern and other, especially background features. (Table 8-2) Markedly...

Optimizing Current Laboratory Hematology Practice

Clinical practitioners currently enjoy the services of highly advanced clinical hematology laboratories that provide timely and accurate results. Despite rapid advances over the last four decades, the current practice of laboratory hematology is still based upon a sometimes awkward mixture of scientifically-derived principles, and lore passed down through tradition but not formally or scientifically validated. As a result, many laboratories maintain suboptimal practices, justified by their...

Fish Procedures 31 Specimen Collection

It is important that specimens referred for FISH studies include malignant cells. Bone marrow aspirates are best for patients with MPD, MDS and acute leukemia.3,21 If the disease involves circulating cells, then a peripheral blood specimen can be collected. In some cases, FISH methods can be used to study blood in preference over bone marrow, especially for CML22 and B-CLL23. Lymph node tissues can be studied using paraffin-embedded tissue, touch preparations or frozen cells.24 In certain...

Limitations

Sampling issues, either because of inadequate material or a non-representative sample, may be sources of error. The former may occur because of extensive necrosis, hemorrhage, fibrosis or dilution with blood.21 The latter involves the inability to definitively ascertain whether the sample is representative of the lesion being sampled.8 Composite lymphoma,195 concomitant lymphoma and carcinoma,196 focal large cell transformation,8 and partial nodal involvement12...

Prognostic Factors

The most commonly used prognostic indicators estimate tumor burden (i.e. or intrinsic characteristic of the plasma cell clone (i.e. proliferative rate, genotype) (Table 5-6). Prognostic factors utilized more commonly in the research setting, such as gene expression profiles (GEP), are making inroads into clinical practice and are beginning to influence treatment decisions. 2.6.1 Prognostic Relevance of Bone Marrow Morphology Although increased tumor burden is associated with decreased patient...

Plasma Cell Leukemia

This rare aggressive variant of MM may occur de novo (primary PCL) or as a terminal event in refractory relapsed MM (secondary PCL).115 Patients with primary PCL, in contrast to MM, have a higher incidence of clinical stage III disease, extramedullary involvement, and immunoglobulin light chain (Bence Jones) disease. Criteria for the diagnosis of PCL are either a peripheral blood absolute plasma cell count > 2 x 109 L or plasma cells > 20 of white blood cells.116 Circulating plasma cells in...

Mixed Cellularity HL

Approximately 20-25 of cases of HL are of the MC type.1 In the Lukes Butler and Rye schemes, MC HL included not only cases conforming to the classical description of this entity (classic RS cells in a diffuse or vaguely nodular mixed inflammatory background in the absence of a nodular sclerosing fibrosis), but it also served as a wastebasket for cases that did not readily fit into any of the other subtypes. Cases that cannot be subclassified in the REAL WHO scheme are should be diagnosed as...

Conclusion

FNAB has an important role to play in the diagnosis of potential hematopoietic neoplasms. Although surgical biopsy may be more accepted than FNAB in many institutions, it is not always the best approach in every patient. Lack of superficial lymph nodes, extremely ill patients and the need for a rapid diagnosis (i.e. highly proliferative tumors like BL) make the availability of a good FNAB service crucial. To achieve the greatest accuracy in diagnosis, FNAB should combine skilled aspiration and...

Fish Probes And Fish Nomenclature

The utility and accuracy of FISH probes to detect chromosome anomalies have been established for many hematological malignancies (Table 4-1). However, the United States Food and Drug Administration has approved only a few commercial FISH probes for this purpose. While many DNA probes can be purchased from commercial companies, home-brew probes can also be prepared. Commercial probes are usually preferred to avoid the rigorous quality control steps needed for making home-brew probes.

Il2

Distribution of the number of cells versus log p-Stat5 staining intensity. Peripheral blood mononuclear cells isolated by ficoll-hypaque separation were either stimulated with IL-2, IL-4, or not stimulated as shown. Figure 10-3 illustrates the ability to detect the high levels of p-Stat5 in the spiked K562 cells based on their expression of CD15 and high level p-Stat5, and to modulate that expression in vitro with imatinib mesylate treatment. In general, coupling the measurement of...

Follicular lymphoma

Follicular lymphoma (FL) comprises approximately 35 of adult NHL in the United States.30 It is much less common in Europe, Asia and developing countries.106 Patients are over 50 years of age with widespread, but asymptomatic disease commonly involving lymph nodes, spleen and bone marrow. FL's recapitulate the lymphoid constituents of a normal follicle with presence of both centrocytes (small lymphoid cells with irregular, cleaved nuclei, inconspicuous nucleoli and scarce cytoplasm) and...

Interpretation of Results

Most FISH studies are informative about the chromosomal locus that they are designed to detect, but are not helpful to identify other chromosome anomalies. Thus, a normal FISH result does not rule out other chromosomal or genetic abnormalities that might be associated with a neoplastic clone. The standard FISH definition of a clone requires the observation of more nuclei with neoplastic FISH signal patterns than is observed in normal individuals. This requires establishing strict scoring...

Carla S Wilson

Advances in the Diagnosis and Classification of Chronic Lymphoproliferative Disorders 145 Laboratory Hematology Practice Present and Future 167 Gerald M. Davis, Kay Lynne Lantis, William G. Finn Role of Fine Needle Aspiration In Lymphoma 181 Angiogenesis in Leukemia and Lymphoma 221 Clinical Flow Cytometry A Transition in Utilization 239 Charles L. Goolsby, Mary Paniagua, Laura Marszalek Hodgkin Lymphoma Clinical, Morphologic, Immunophenotypic, and Bertram Schnitzer and Riccardo Valdez

Angiogenesis In Myeloproliferative Disorders

Increased blood flow in myeloproliferative disorders was noted over 30 years ago.57 Subsequent studies have documented increased bone marrow vascularity or blood flow in myelofibrosis,58-65 chronic myelogenous leukemia (CML),22, 58, 63, 66, 67 essential thrombocythemia (64) and polycythemia vera.64 The mediators of angiogenesis in myeloproliferative disorders appear to be VEGF and bFGF. In myelofibrosis, megakaryocytic cells have been shown to express increased levels of bFGF,65, 68, 69 and...

Lcp

MRNA Microarray Analysis in Lymphoma and Leukemia 1 Timothy C. Greiner Myelodysplastic Myeloproliferative Diseases 13 Relevance of Pathologic Classifications and Diagnosis of Acute Myeloid Leukemia to Clinical Trials and Clinical Practice 45 Molecular Cytogenetic Studies for Hematological Malignancies 69 Gordon W. Dewald, Stephanie R. Brockman, Sarah F. Paternoster

Prolymphocytic Leukemia Pll And Its Relationship To Mantle Cell Lymphoma

MCL is a B-cell lymphoma with a characteristic translocation t(11 14)(q13 q32) resulting in the juxtaposition of the BCL1 CCND1 gene on chromosome 11q13 with the IGH gene on chromosome 14q32. This produces a constitutively upregulated BCL1 CCND1 -encoded cyclin D1 protein, an important cell cycle regulator. Like CLL, the malignant cells characteristically aberrantly express the T-cell associated antigen CD5, but unlike most cases of CLL, the malignant population is CD23 negative. The cells of...

Chromosomal Basis Of Malignancy2 21 Chromosome Anomalies5

The results of FISH can be used to detect neoplastic clones with either numeric or structural anomalies of chromosomes. The term polyploid refers to chromosome complements that are multiples of 23 the haploid number of chromosomes for humans. Diploid refers to 46 chromosomes, triploid to 69 chromosomes, and tetraploid to 92 chromosomes. In neoplastic disorders, most polyploid clones are associated with advanced stages of disease and are derived from fusion of neoplastic cells or...

Fish In Clinical Practice

Most treatment protocols for hematological malignancies require quantitative procedures to assess tumor burden and response to therapy. This can be done by using various genetic tests including quantitative cytogenetic studies, FISH and molecular genetic techniques. No single genetic testing procedure fulfills all the needs of clinical care for patients with hematological malignancies. Thus, it is important to use combinations of testing methods that are both accurate and cost-effective for...

Emerging Therapies

Emerging Therapies for Multiple Myeloma Immunomodulating derivative or ImiDs (Revimid, CC-5013) Proteasome inhibitors (Bortezomib, VELCADE , PS-341) Bcl-2 antisense oligonucleotide (oblimersen sodium, Genasense ) Arsenic trioxide (AS2O3) Bisphosphonates (zoledronate, pamidronate, etc.) Osteoprotegerin (AMGN-0007) Alemtuzumab (Campath 1 H) Monoclonal antibody to IL-6 Skeletal targeted radiotherapy (166 Holmium-DOTMP) Induces either apoptosis of MM cells or G1 growth arrest, blocks...

Immunophenotype

Immunophenotyping in paraffin sections is exceedingly useful in the diagnosis and subclassification of HL. Because no single marker is highly sensitive nor specific for RS cells, a panel of antibodies must be employed to characterize these cells including CD15, CD30, CD45, CD20, and CD3. In problematic cases, EMA, bcl-6, fascin, EBV-latent membrane protein (LMP), and additional B- and T-cell antibodies may be added to the panel.1 While the immunophenotype of RS cells in all types of classical...

Laboratory Automation Systems

Total laboratory automation (TLA) systems are composed of instruments and work cells along a robotic stretch of hardware used for specimen management and transportation both within and between different clinical laboratories.27,28 Centrifugation (when necessary), checking specimen suitability (hemolysis, clotting), decapping, aliquoting, barcode application, and routing are just some of the tasks performed by a TLA. Many TLA systems are available with a process control software component. They...

Hematopathology In Oncology

Rosen, M.D., Series Editor Muggia, F.M. (ed) Concepts, Mechanisms, and New Targets for Chemotherapy. 1995. ISBN 0-7923-3525-2. Klastersky, J. (ed) Infectious Complications of Cancer. 1995. ISBN 0-7923-3598-8. Kurzrock, R., Talpaz, M. (eds) Cytokines Interleukins and Their Receptors. 1995. ISBN 0-7923-3636-4. Sugarbaker, P. (ed) Peritoneal Carcinomatosis Drugs and Diseases. 1995. ISBN 0-7923-3726-3. Sugarbaker, P. (ed) Peritoneal Carcinomatosis Principles of Management. 1995. ISBN...

Myelodysplastic Syndromes

The MDS are a clinically heterogeneous group of hematological disorders.71 The most common chromosome anomalies in MDS involve chromosomes 5, 7, 8, 11, 13, 17, 20, 21 and X.72,73 Cytogenetic studies are important for these disorders as the results can provide both diagnostic and prognostic information.20 Briefly, patients with MDS who have -Y alone, 5q- alone, 20q- alone or normal metaphases have a favorable prognosis. Patients with a complex karyotype (defined as > 3 anomalies) or anomalies...

Naming FISH Probes

FISH probes are given names in various ways according to information known about the DNA fragments.28 All approved human gene symbols can be found in the Genew database.29 Arbitrary DNA fragments and loci are assigned gene symbols by the Genome Database. The symbols are made up of four parts. For example, the probe D13S319 indicates D for DNA, 13 for chromosome 13, S for a unique DNA sequence and 319 for a sequential number to make the probe designation unique. FISH probes that hybridize to...

Chronic Myeloid Leukemia

Given the available successful treatments with imatinib mesylate, arsenic trioxide, interferon and or bone marrow transplantation, CML is a particularly important disease to identify by genetic testing. About 95 of patients with CML have a t(9 22)(q34 q11.2) or a variant of this translocation. A similar translocation occurs in 6 of children with ALL, 17 of adults with ALL and 1 of patients with AML-M1. The International System for Cytogenetic Nomenclature5 designates the abnormal chromosome 22...

Validation Of Fish Probes

Validation of any new FISH assay is necessary to ensure safe and effective testing for the specific intended clinical purpose. The validation of FISH assays is a systematic multi-step process designed to assess probe performance in normal and abnormal specimens. This process also provides important experience with many cases to assure consistent scoring criteria and accurate interpretation of results.40-42 The procedures to validate FISH assays vary significantly depending upon the specific...

Lymphoplasmacytic lymphoma Waldenstrom macroglobulinemia

Lymphoplasmacytic lymphoma (LPL) is a rare entity accounting for approximately 2-3 of all NHL.6 This is a disease of older adults (median age 63 years) with a slight male predominance.101 It is a low grade B cell lymphoma with transformation to large cell lymphoma being identified in 510 of cases6. Frequently patients present with features of lymphoma (lymphadenopathy, hepatosplenomegaly and bone marrow involvement) and a monoclonal serum IgM paraprotein. They may exhibit signs of , bleeding,...

Current Therapies

Patients treated with conventional chemotherapy have a median survival of approximately 3 years.6 Therapeutic failure in MM is multifactorial with the most significant problem being drug resistance. Current chemotherapy regimens attempt to use combinations of multiple drugs affecting different, non-overlapping, and non-cross resistant pathways.94 Approximately 10 years ago, Barlogie and colleagues in Arkansas began tandem autologous stem cell transplantation (ASCT) randomized trials have...

Further Directions

While the techniques of laser microdissection and PCR have enabled significant successes towards unraveling the mysteries of Hodgkin lymphoma, even newer technologies such as cDNA and tissue microarray analyses show great promise in further divulging the pathogenesis of this intriguing disease through comprehensive gene and protein expression profiling. cDNA arrays are powerful tools useful for the identification of differentially expressed genes in malignant tumors. Similarly, tissue...

Home Brew Probes

Home-brew probes can be made from bacterial artificial chromosome (BAC) or yeast artificial chromosome (YAC) clone(s) containing the DNA covering the genome sequence of interest. The appropriate BACs and or YACs can be determined and purchased using Internet resources such as www.ncbi.nih.gov and www.sanger.ac.uk . Polymerase chain reaction (PCR) primers are designed for the gene of interest and or for markers in the genome target. Bacteria containing appropriate BACs are cultured and PCR is...

Mycosis Fungoides And Sezary Syndrome

SS is generally regarded as the leukemic form of epidermotropic cutaneous T-cell lymphoma (MF). Clinically, SS is characterized by erythroderma, palmoplantar keratoderma, partial alopecia, lymphadenopathy, and pruritis. Atypical lymphocytes are seen in the blood and take the form of lymphocytes with grooved, cerebriform nuclei of large (typical Sezary cells,) or small (so-called Lutzner cells) size. Although originally described in 1961 by Tasell and Winkelmann,49 the exact definition of SS is...

D

Nodular lymphocyte-rich classical Hodgkin lymphoma. A) CD20 positive nodules composed of expanded mantle zones. B) Higher magnification of CD20 positive expanded mantle zone containing an atrophic, eccentrically-placed germinal center. C) H& E section of an atrophic germinal center within the mantle zone. D) CD3 positive lymphocytes ring a Reed-Sternberg cell. Figure 11-2. Nodular lymphocyte-rich classical Hodgkin lymphoma. A) CD20 positive nodules composed of expanded mantle...

Laboratory Procedures

In the cytogenetic laboratory, various methods can be used to process specimens for FISH analysis. Bone marrow and blood specimens are usually processed using hypotonic solution (0.075 M KCl) for 15 minutes and fixed with 3 1 methanol-glacial acetic acid. These cells can be stored indefinitely at -70 C until FISH studies are needed. The laboratory method to perform FISH is summarized in Figure 4-1 and described in detail in various books1 and publications24,25. Briefly, fixed cells are dropped...

Nodular Sclerosis HL

NS HL is the most common subtype of classical HL in developed nations, accounting for 60-80 of cases.1 It is also the most common type of HL found in adolescents and young adults, and the median age at diagnosis is 28 years. It is the only subtype in which there is no male predominance, with an almost equal male to female ratio.14, l5 A high percentage of patients present with mediastinal involvement (80 ), and massive mediastinal disease is an adverse risk factor.56 The characteristic...

X

Bone marrow biopsy from patient with CMML. Frequently, the bone marrow biopsy specimen appears to show a very prominent granulocytic proliferation, and monocytes are frequently not easily appreciated. Monocytes often show folded or irregularly shaped Splenic enlargement is usually due to leukemic infiltration that is particularly prominent in the red pulp. Lymphadenopathy is seen in a minority of patients, but lymph nodes should always be biopsied, as this may indicate...

Reed Sternberg Cell

The classic Reed-Sternberg (RS) cell or a variant RS cell (L& H cell, lacunar cell) must be present for a diagnosis of HL to be made. Not only must this cell be present, but it must be found in a cellular milieu of one of the five histologic subtypes of HL, because cells morphologically indistinguishable from RS cells may also be found in benign lymphoid proliferations as well as in non-Hodgkin's lymphomas. There are three types of RS cells 1) classic, 2) lacunar cell, and 3) lymphocytic and...

Angiogenesis In Lymphoma

In an insightful article from 1975, Wolf et al98 described angiogenesis in a cutaneous lymphoma (classified as malignant lymphoma, undifferentiated, non-Burkitt type). They showed that lymphoma fragments stimulated angiogenesis in a hamster cheek pouch model, even when separated by a Millipore membrane.98 This observation led the authors to conclude that a diffusable tumour angiogenic factor may play a vital role in tumour survival and portends a therapeutic potential.98 Experiments using a...

Tcell Prolymphocytic Leukemia Tpll And Tcell Chronic Lymphocytic Leukemia Tcll

Most of the T cell non-Hodgkin lymphomas are relatively rare compared to their B cell counterparts in the Western world, and for this reason, have been less exhaustively studied. Among these uncommon disorders are the entities referred to variously as T-PLL and T-CLL. T-PLL is a rare peripheral T-cell leukemia lymphoma with the following characteristics an absolute lymphocytosis typically with concomitant hepatosplenomegaly, anemia, and thrombocytopenia. The incidence of lymphadenopathy and...

Red Blood Cell Morphology

Abnormalities of red blood cells account for a high percentage of manual blood smear reviews in the clinical hematology laboratory,12 but properly interpreting red cell abnormalities is a deceptively difficult task. Many clinical-morphologic correlates are well-established through decades of accumulated anecdotal experience and in vitro analysis15 and are likely appropriate for clinical practice, but there are actually very few objective or scientific data indicating the sensitivity,...

E21g3

Dystrophin rel prot 2 3UTR of unk prot Uncharacterized Prot kinase C gamma 5-Hydroxytrypta 2B Rec H731 Transducin-line enhancement Uncharacterized Prot kinace C beta 1 Oviductal glycoprot Zinc fing prot C2H2-150 The identification of the predictor genes was based on the analysis of lymphoma cases treated during the past decade, largely prior to the advent of Rituximab chemotherapy. It is unknown whether the same gene subsets will be predictive of response to chemotherapy and survival in...

Classifications of Hodgkins disease

In contrast to non-Hodgkin's lymphomas (NHL), which have undergone many changes in classification, the organizational scheme for Hodgkin's disease has remained remarkably stable for almost 40 years. The first useful classification of Hodgkin's disease was published in 1947 by Jackson and Parker in their book Hodgkin's Disease and Allied Disorders.13 HL was divided into three histologic types paragranuloma, granuloma, and sarcoma (Table 11-1). However, this division was not helpful in predicting...

Monoclonal Immunoglobulin Deposition Diseases

Primary amyloidosis (AL) and light chain deposition disease (LCDD) are disorders caused by monoclonal plasma cell populations that over-synthesize free immunoglobulin light chains, which are deposited in tissues. Patients with these disorders most frequently present with symptoms of organ dysfunction due to the immunoglobulin deposition, along with weakness, fatigue, and weight loss. Kidney and liver involvement is common, while cardiac involvement has a particularly bad prognosis.106 A...

Marginal Zone Lymphoma

Heterogeneous Lymph Node

Marginal zone lymphoma (MZL) represents a subgroup of NHL comprised of low-grade B cell lymphomas occurring in nodal (monocytoid B-cell lymphoma), splenic (splenic marginal zone lymphoma) and extra nodal (low grade lymphomas of mucosa associated lymphoid tissue, MALT) locations.24 Although most commonly a disease of adults,161 recent reports document its occurrence in younger individuals.162 It is an indolent lymphoma with a tendency to remain localized for a prolonged period of time.163,164...

Nodular Lymphocyte Predominant Hodgkin Lymphoma

In the Lukes Butler classification, NLP HL was divided into two morphologic subtypes nodular and diffuse.14,15 In the Rye scheme, it was simply called NLP HL. In the REAL WHO classification, NLP HL is defined as having at least a partially nodular growth pattern, although diffuse areas may be present in a minority of cases.1,19 Table 11-1 Although there is still controversy about whether a purely diffuse LP HL exists, this entity is not recognized as a distinct category in the WHO scheme. The...

Acute Lymphoblastic Leukemia

Most published series of patients with ALL indicate that more than 85 have an abnormal clone by conventional cytogenetic studies. At least 36 chromosome anomalies are common in ALL. The most common karyotype among children with ALL is hyperdiploidy. This karyotype is associated with a good prognosis in the absence of any structural anomalies and when associated with trisomies of chromosomes 4, 10 and 17.90 The most common chromosome translocations in pediatric ALL include t 9 22 q34 q11.2 , t...