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Bethesda Proposals for Classification of Non‑lymphoid
Hematopoietic Neoplasms in Mice: Supplemental Materials Scott C. Kogan, Jerrold M. Ward, Miriam R. Anver, Jules J. Berman, Cory Brayton, Robert D. Cardiff, John S. Carter, Sherri de Coronado, James R. Downing, Torgny N. Fredrickson, Diana C. Haines, Alan W. Harris, Nancy Lee Harris, Hiroshi Hiai, Elaine S. Jaffe, Ian C. M. MacLennan, Pier Paolo Pandolfi, Paul K. Pattengale, Archibald S. Perkins, R. Mark Simpson, Mark S. Tuttle, Joanne F. Wong, Herbert C. Morse III Contents
Overview and representative examples of murine non-lymphoid
hematopoietic neoplasms Practical guide to characterization and classification Glossary
Appearance of leukemia in
peripheral blood Erythroid
Granulocytic
Histiocytic
Immature forms/blasts
Mast Cells
Megakaryocytic
Monocytic
Monocytic component
Neutrophilic
Appearance
of leukemia in peripheral blood
If arising in a previously healthy animal, as defined by any combination of: A. Neutropenia B. Anemia C. Thrombocytopenia D. Presence of
immature forms/blasts in blood If arising in an animal with pre-existing abnormalities in the peripheral blood, as defined by any combination of: A. Progression of
neutropenia, anemia, and/or thrombocytopenia B. Appearance of
increasing numbers of immature forms/blasts in blood Criterion 5B1 for diagnosis of non-lymphoid leukemia (Table 2) is applicable to animals whose blood is sampled at least monthly. BiphenotypicHematopoietic cells that exhibit a mix of lymphoid and non-lymphoid characteristics Lymphoid features required for a diagnosis of biphenotypic as defined by any combination of: A. CD3+
with presence of clonal T-cell receptor gene rearrangement (VDJ or VJ not just
DJ) B. CD19+
with presence of immunoglobulin gene rearrangement (VDJ or VJ not just DJ) C. Other means of
identifying lymphoid cells that may become available‡ Non-lymphoid features required for a diagnosis of biphenotypic as defined by any combination of: A. Myeloperoxidase positive. C.
Chloroacetate esterase positive D. Ly-6G(Gr-1)+
and CD11b+ E. Other means of identifying non-lymphoid hematopoietic
cells that may become available‡ Erythroid
As defined by any combination of: A. Erythroid
morphology B. CD71+, Ly-76(Ter119)hi C. Other means of
identifying erythroid cells that may become available‡ Granulocytic
As defined by any combination of: A. Granulocytic
morphology (neutrophilic, eosinophilic, or basophilic) B. Chloroacetate
esterase positive and cells are not mast cells C. Expression of both
Ly-6G(Gr-1) and CD11b D. Other means of
identifying granulocytic cells that may become available‡ Histiocytic
As defined by any combination of: A. Histiocytic
morphology B. Non-specific
esterase positive but not erythroid C. Mac-2+ D. Ly-71(F4/80)+ Immature
forms/blasts
We propose that the immature cells of non-lymphoid neoplasms be referred to as “immature forms/blasts” and that these cells be enumerated according to the following guidelines: Size: medium-sized to large Cytoplasm: basophilic, with or without azurophilic granules Nuclear to Cytoplasmic Ratio: high Nuclei: predominantly round to oval, but modest peripheral indentation or a small central opening can be present Chromatin: fine to moderately fine Nucleoli: small but often present; can be indistinct or absent Position of Nucleus: whereas lymphoblasts and erythroblasts typically have a central nucleus, myeloblasts often have a nucleus that is more peripherally located. Notes: 1. Some of the immature cells present in leukemias can have rather condensed chromatin or be somewhat small. If these cells otherwise have features of immature forms/blasts they should be included in the differential count as such. 2. This proposal for terminology and definition of the immature cells of murine non-lymphoid hematopoietic neoplasms should be seen as one step in an ongoing process aimed at identifying features of disease that reflect underlying cellular biology and predict pathogenic behavior. Additional information may result in revisions to this recommendation. 3. “Young forms” has been used a synonym for immature forms/blasts. Mast Cells
As defined by any combination of: A. Mast cell
morphology B. Toluidine blue
strongly positive C. Chloroacetate
esterase strongly positive D. Other means of
identifying mast cells that may become available‡ Megakaryocytic
As defined by any combination of: A. Megakaryocytic
morphology B. VWF positive C. CD41+and
Glycoprotein V+ D. Other means of identifying megakaryocytic cells that may become available‡ Monocytic
As defined by any combination of: A. Mature monocyte
morphology B. Non-specific
esterase positive and not Ly-76(Ter119)hi C. CD16/32+,
Ly-71(F4/80) +, and CD45hi D. CD11b+,
Gr-1neg-lo, CD45R(B220)-, E. Immunoelectron microscopy
reveals myeloperoxidase negative 1° granules F. Other means of
identifying monocytic cells that may become available‡ Monocytic
component
As defined by any combination of: A. Monocytosis (³20% of leukocytes in peripheral blood) B. Monocytosis in peripheral
blood ³5× normal C. ³20% monocytic cells in bone marrow Neutrophilic
As defined by any combination of: A. Neutrophil
morphology (immature cells of indeterminate lineage excluded) B. Chloroacetate
esterase positive but not mast cells C. Other means of
identifying neutrophilic cells that may become available‡ Neutrophilic
component
As defined by any combination of: A. Neutrophilia (³20% of leukocytes in peripheral blood) B.
Neutrophilia in peripheral blood ³5× normal C. ³20% neutrophilic cells in bone marrow Footnote
‡Including this criterion allows additional cytochemical, immunohistochemical, flow cytometric, or genetic means of identifying these cells to be used when such means have documented lineage specificity (reflecting our recognition that the proposed criteria are not comprehensive). Because neoplastic cells may express features absent from normal cells, it is important that both neoplastic and normal murine hematopoiesis be considered when attributing lineage specificity to markers. We request investigators to submit alternative criteria to the MMHCC hematopathology subcommittee so that the lists above can be updated. Return to Contents of Supplement Overview of Non-lymphoid
Hematopoietic Neoplasms
Representative Examples
AML
without maturation (MRP8 PMLRARA
transgenic mice)
Histiocytic
Sarcoma (NFS.V+ mice with liver masses)
Myeloproliferative
disease (Lethally irradiated recipients of Nf1-/-
fetal liver cells)
Classification Table with Selected Examples MRP8
PMLRARA transgenic mice
Does the disease fulfill
criteria for non-lymphoid leukemia?
Criterion 1: Yes. Diffusely involves spleen, bone marrow Criterion 2: Yes. Mice are anemic with marked thrombocytopenia Criterion 3: Yes. Spleen is enlarged with immature myeloid cells; marrow myeloid cells are increased with decreases in erythroid and lymphoid cells. Criterion 4: Yes. 4A: immature myeloid cells are present in periportal and sinusoidal liver, lymph nodes, kidneys, lungs. Meninges and reproductive tract are infiltrated in some cases. Criterion 5: Yes. 5A and 5B2. ~90% of bone marrow cells are immature forms/blasts. Disease transplants to histocompatible unirradiated or sub-lethally irradiated recipients and is lethal to such recipients in ~5 weeks. How should the disease be
sub-classified?
>90% of marrow non-erythroid cells are immature forms/blasts with cytochemical and immunophenotypic features of immature granulocytic cells [Sudan Black B positive, Ly-6G(Gr-1)+, CD11b(Mac-1)variable, CD117(c-kit)+]. There is no evidence of monocytic differentiation. Multilineage dysplasia is absent. ·
Diagnosis:
Acute myeloid leukemia without maturation.
NFS.V+
mice with liver masses
Does the disease fulfill criteria for non-lymphoid
hematopoietic sarcoma?
Criterion 1: Yes. Arises as a solid tumor mass. Criterion 2: Yes. Bone marrow is not diffusely involved, although malignant histiocytes can be present in the marrow. Criterion 3: Yes. Cells are infiltrative in the liver and spread to other tissues. How should the disease be
sub-classified?
The cells have a histiocytic morphology and stain with histiocyte markers including Ly-71(F4/80). ·
Diagnosis:
Histiocytic sarcoma
Lethally
irradiated recipients of Nf1-/- fetal liver cells
Does the disease fulfill criteria for non-lymphoid leukemia?
Criterion 1: Yes Involves bone marrow and spleen Criterion 2: Yes Mice can be anemic Criterion 3: Yes Spleen is enlarged with immature myeloid cells; marrow myeloid cells are increased with decreases in erythroid and lymphoid cells. Criterion 4: Yes 4A: myeloid cells are present in periportal liver and can be seen in other tissues Criterion 5: No 5A, immature forms/blasts are not increased. 5B, the disease can be evident in the peripheral blood for extended periods of time (months) prior to the mice becoming visibly ill. Does the disease fulfill criteria for myeloid dysplasia?
Criterion 1: No. Mice exhibit leukocytosis Criterion 2: No. Dyspoiesis is absent or minimal. Immature forms/blasts are well less than 20% in the bone marrow. Does the disease fulfill criteria for myeloid proliferation
(non-reactive)?
Criterion 1: Yes. Expansion in myeloid cells is non-reactive, persistent, and caused by the absence of NF1 protein. Criterion 2: Yes. 2A & 2B, there is leukocytosis in the peripheral blood and increased non-lymphoid hematopoietic cells in the bone marrow and spleen. How should the disease be
sub-classified?
The expansion in cells in evident in the blood as well as the spleen and marrow. ·
Diagnosis:
Myeloproliferative Disease
Classification
Table with Selected Examples
For each diagnosis, selected mice in which the neoplasm has been observed are listed. This list will be amended as additional animals are reviewed. Non-lymphoid
Leukemias
Myeloid leukemias Myeloid leukemia without maturation BXH-2 mice MRP8 PML-RARA transgenic mice Cathepsin
G PLZF-RARA/RARA-PLZF doubly transgenic mice Myeloid leukemia with maturation BXH-2 mice Cathepsin G PML-RARA transgenic mice MPD-like myeloid leukemia Retroviral transduction with BCR-ABL Fusion Retroviral transduction with Hoxb8 plus IL-3 Cathepsin
G PLZF-RARA transgenic mice Myelomonocytic leukemia BXH-2 mice Retroviral
transduction with AML1-MDS1-EVI1 fusion Monocytic leukemia Retroviral transduction with MLL-ELL fusion Erythroid leukemia Recipients
of Friend leukemia virus complex Megakaryocytic leukemia NFS/N
recipients of Friend-Moloney virus Biphenotypic leukemia ? Non-lymphoid
Hematopoietic Sarcomas
Granulocytic sarcoma ENU treated Runx1-ETO knock-in mice Histiocytic
sarcoma Retroviral transduction with BCR-ABL Fusion Spontaneous,
particularly in C57BL/6 and NFS.V+ mice Mast cell sarcoma Retroviral transduction with BCR-ABL Fusion Spontaneous
in NFS.V+ or DBA/2 Myeloid
Dysplasias
Myelodysplastic syndrome ? Cytopenia with increased blasts Pre-leukemic MRP8 PML-RARA/BCL2 doubly transgenic mice Myeloid
Proliferations (non-reactive)
Myeloproliferation (genetic) Pre-leukemic Cathepsin G PML-RARA transgenic mice Myeloproliferative disease Nf1+/-
mice; Recipients of Nf1-/- fetal liver cells Return to Contents of Supplement Characterization and classification of hematopoietic
neoplasms in mice: a practical guide emphasizing non-lymphoid hematopoietic
disorders. Note: this document is an overview. It does not include detailed protocols for many techniques utilized in the study of mouse hematopoiesis. Such protocols are under ongoing development and revision by the MMHCC hematopathology subcommittee. Requests for protocols can be directed to skogan@cc.ucsf.edu I. Evaluating Mice for Hematologic Illness A. Observation B. Physical examination C. Blood counts II. Characterization of Hematologic Disease in Mice A. Blood Collection B. Necropsy C. Initial Preparation of Tissues for Analysis D. Blood, Bone Marrow, and Cell Suspensions E. Decision Point: What tissues should be studied with flow cytometry? What antigens should be assessed? F. Additional Analysis 1. Cytochemical Stains 2. Histopathology 3. Histochemical stains 4. If the disease is lymphoid or may be biphenotypic 5. Assessing clonality 6. Transplantation: excluding reactive processes and assessing aggressiveness of disease III. Proposals for Differential Cell Counting in the Mouse IV. Classification of Murine Non-lymphoid Hematopoietic Neoplasms A. Is the disorder a non-lymphoid hematopoietic sarcoma? B. Is the disorder a non-lymphoid leukemia? C. Is the disorder myeloid dysplasia? D. Is the disorder a myeloid
proliferation (non-reactive)? I. Evaluating Mice for Hematologic Illness You may believe that some mice you are working with will develop a hematopoietic neoplasm. Following such mice for illness can be done by simple observation, physical examination, and blood counts. One approach is for such mice to be observed daily, physically examined once a week, and bled once a month. A.
Observation · poor grooming · hunched posture · visible weight loss · decreased activity · development of masses B.
Physical examination · looking at the ears and feet for pallor · palpation for enlarged lymph nodes [especially cervical (the front of the neck) and axillary (under the forelimbs)] · palpation for an enlarged spleen · decreased motion when the mouse is returned to the cage C.
Blood counts · blood counts and review of a stained blood smear often reveals disease Return to Characterization and Classification II. Characterization of Hematologic Disease in Mice A.
Blood Collection · It is essential to obtain blood prior to euthanasia. · Blood can be obtained from the lateral tail vein, orbital plexus, lateral saphenous vein, or by cardiac puncture. · Blood should be collected such that it is anticoagulated with EDTA. · A blood cell counter with veterinary software is important for obtaining accurate values for white blood cell count, hemoglobin, and platelet count. · Automated white blood cell differential counts are often inaccurate in mice with hematopoietic neoplasms. · It is usually necessary to establish control values from age/sex/treatment matched healthy mice. B.
Necropsy
Here are some particular aspects useful in evaluating hematopoietic neoplasms. Incise the skin and open the mouse to observe the lymph nodes and internal organs. - Note the location and color of enlarged lymph nodes. o A greenish hue can be seen in some cases of myeloid leukemia. - Flush bone marrow from the hind limbs with buffered saline and keep on ice until it is evaluated. - View inner side of the sternum. o A color other than medium red may indicate an abnormality; white marrow is often seen in leukemia or lymphoma. - Note the size and color of the liver and spleen. o In reactive conditions the liver often appears normal, whereas the liver is expanded and may change color when involved by a neoplasm. o Histiocytic sarcoma can be found as a solid tumor mass in the liver. - The spleen can be markedly enlarged in both reactive and neoplastic processes. o In reactive processes the spleen is usually its normal deep red. o In erythroid leukemia the spleen may be cherry red. o In myeloid leukemia the spleen may be browner than normal and in some cases may have a greenish hue. o Lymphoid neoplasms in the spleen are often apparent on cut section as a lacy or reticular pattern. - Observe the thymus. o The presence of a large anterior mediastinal mass is almost always due to a T cell precursor lymphoblastic leukemia/lymphoma. - View the lungs. o They may show hemorrhages due to neoplastic infiltration. - View the intestine for expansion of Payer’s patches or distension that can be associated with obstruction or infiltration. - View other organs. o Pay particular attention to the possible presence of non-hematopoietic neoplasms or areas of inflammation because these can cause reactive myeloid hyperplasia. - In addition to other tissues that may be of interest, remove the sternum, liver, spleen, thymus, kidney, lungs, selected lymph nodes (for example mesenteric, cervical, axillary/brachial, inguinal), and any solid tumor mass.
C.
Initial Preparation of Tissues for Analysis 1 Blood
2. Bone Marrow
3. Spleen
4. Other tissues
Lymph Nodes
Bisect enlarged lymph nodes and divide between fixative and preparation of a cell suspension. Thymus
If the thymus is enlarged you may wish to divide for fixation and cell suspension. Tumor Mass If there is a tumor mass in the liver or elsewhere prepare touch imprints of the mass. Bisect the tumor mass and place half into fixative and use the other half for a cell suspension (although many tumor masses cannot be easily disaggregated). Sternum The sternum must undergo at least brief decalcification. If Bouin’s is used for fixation it will decalcify as well as fix. If formalin is used, three hours in a formalin-based decalcification solution is sufficient to permit sectioning. Others Remaining tissues should be placed into fixative.
D.
Blood, Bone Marrow, and Cell Suspensions 1. Blood
o You may wish to consider supplementing differential counts based on blood smear morphology with flow cytometric immunophenotyping (see below) or cytochemical stains (see below). o For cytochemical stains of blood it may be useful to deplete the blood of dead cells and red blood cells with a density cushion such as Sigma Histopaque 1119. The post-Histopaque sample can then be used to prepare four cytospins. 2. Bone Marrow · Observe the morphology of cells in stained bone marrow smears or cytospins. · It may be useful to deplete the bone marrow of dead cells and red blood cells with a density cushion such as Sigma Histopaque 1119. o The post-Histopaque sample may be useful for flow cytometric immunophenotyping and for cytochemical stains (four cytospins can be prepared). 3. Suspensions of Spleen, Lymph Nodes, Thymus, Tumor Mass · It may be useful to deplete these preparations of dead cells and red blood cells with a density cushion such as Sigma Histopaque 1119. o The post-Histopaque sample may be useful for flow cytometric immunophenotyping or preparation of stained cytospins (two to four cytospins can be prepared). E.
Decision Point: What tissues should be studied with flow cytometry? What antigens should be assessed? At this time you should perform flow cytometry on a cell suspension from a tissue that is heavily involved by the disease. · For lymphoid neoplasms, this may be spleen, thymus or lymph node. · For most non-lymphoid neoplasms, this may be bone marrow or spleen. · If the disease was predominantly a solid tumor mass that could be easily disaggregated then these cells could be used for flow. (If the disease was predominantly a solid tumor mass that could not be disaggregated you could be dealing with a non-hematopoietic tumor or with a non-lymphoid hematopoietic sarcoma, the diagnosis of which can generally be made in paraffin sections.) · In some cases, for example early in your work with a type of mouse, you may wish to perform flow cytometry on more than one of your cell suspensions. · If there is the possibility that you are dealing with a myeloid leukemia you will wish to include blood in your plans for flow immunophenotyping. Recommendations for antibodies to be used for flow cytometric immunophenotyping is an area of ongoing discussion by the MMHCC hematopathology subcommittee. Below are our current recommendations. Extensive three color antibody panel useful in evaluating
most hematopoietic neoplasms:
Minimal three color antibody panel useful in evaluating
non-lymphoid hematopoietic neoplasms:
Three color antibody panel useful for evaluating the
blood of mice that may have myeloid leukemia:
F.
Additional Analysis 1. Cytochemical Stains If the disease appears to be a disease of immature non-lymphoid cells, granulocytes, and/or monocytes you may want to stain cytospins of red-cell depleted blood, bone marrow and spleen for: 1) Sudan Black B, which variably stains neutrophilic, eosinophilic, and monocytic cells, 2) Chloroacetate Esterase, which strongly stains mast cells, and variably stains neutrophilic and occasionally other cell types, and 3) Non-specific Esterase, which variably stains monocytic and occasionally other cell types. o You will want to perform these stains promptly, because the quality of the stains decreases quickly when slides are stored. 4) Romanowsky Stain for correlating cytologic appearance with staining characteristics. 2. Histopathology · Tissues should be embedded in paraffin, sectioned, stained with hematoxylin and eosin, and the histopathology of the tissues reviewed. · To enable a better comparison of information, standardized techniques for the preparation of histological slides have been established for all organs of rats (refer to http://www.ita.fhg.de/reni/trimming/TR_F.HTM). Many of these recommendations have been successfully applied to mice. 3. Histochemical stains
The following immunohistochemical stains can be important in classification of mouse non-lymphoid hematopoietic neoplasms:
4. If the disease is lymphoid or may be biphenotypic
5. Assessing clonality
6. Transplantation: excluding
reactive processes and assessing aggressiveness of disease Transplantation can be useful in excluding reactive processes and in assessing the aggressiveness of a lesion. · 1 x 106 cells can be injected into the tail vein of histocompatible unirradiated or sub-lethally irradiated mice. · At least three such mice should be injected. · The time from injection until the mice become ill enough that they should be euthanized should be noted. More generally, if transplants are done, be certain to record the following information: - Type of recipient o Immunodeficient, Nude o Immunodeficient, Other o Histocompatible, lethally irradiated o Histocompatible, sub-lethally irradiated o Histocompatible, unirradiated - Route of injection - Number of cells injected - Whether recipients become ill - Character of disease in recipients (localized vs. disseminated) - Time from transplantation to o Illness o Euthanasia/death Return to Characterization and Classification III.
Proposals for Differential Cell Counting in the Mouse
Differential cell counting of Romanowsky-stained (eg. Wright’s Giemsa, May-Grünwald-Giemsa) blood smears, bone marrow smears or cytospins, and spleen touch preps or cytospins has an important role in the classification of non-lymphoid hematopoietic neoplasms of mice. 1. Immature forms/blasts
We propose that the immature cells of non-lymphoid neoplasms be referred to as “immature forms/blasts” and that these cells be enumerated according to the following guidelines: Size: medium to large Cytoplasm: basophilic, with or without azurophilic granules Nuclear to Cytoplasmic Ratio: high Nuclei: predominantly round to oval, but modest peripheral indentation or a small central opening can be present Chromatin: fine to moderately fine Nucleoli: small but often present; can be indistinct or absent Position of Nucleus: whereas lymphoblasts and erythroblasts typically have a central nucleus, myeloblasts often have a nucleus that is more peripherally located. Notes: 1. Some of the immature cells present in leukemias can have rather condensed chromatin or be somewhat small. If these cells otherwise have features of immature forms/blasts they should be included in the differential count as such. 2. This proposal for terminology and definition of the immature cells of murine non-lymphoid hematopoietic neoplasms should be seen as one step in an ongoing process aimed at identifying features of disease that reflect underlying cellular biology and predict pathogenic behavior. Additional information may result in revisions to this recommendation. 3. “Young forms” has been used as a synonym for “immature forms/blasts.” 2. Mature Forms, Neutrophilic
Cells with pale blue or neutrophilic cytoplasm, With ring form nuclei with diameter of center of ring >50% of diameter of nucleus or fully developed segmentation With coarsely distributed chromatin. [Note, indented rather than ring form nuclei can be seen in murine neutrophils in some circumstances. When such cells are present they should be counted as “Mature Forms, Neutrophilic” when the nuclear indentation exceeds >50% of the diameter of the nucleus] 3. Mature Forms, Monocytic
Largest leukocytes With round, oval or bean-shaped nucleus Often with nongranular slightly basophilic cytoplasm, with or without vacuoles. Forms that cannot be distinguished from Immature Forms/Blasts should be counted as Immature Forms/Blasts. Some forms cannot be distinguished morphologically from large lymphocytes 4. Intermediate Forms (including neutrophilic and monocytic
cells)
All other cells of the neutrophilic or monocytic series, that is, cells showing partial maturation of cytoplasm and nucleus in various combinations. 5. Eosinophilic Cells
6. Lymphocytes
7. Nucleated Red Blood Cells
8. Other Cells
Includes megakaryocytes, plasma cells, reticuloendothelial cells, mast cells, stromal cells, endothelial cells, and unidentified cells. In some settings, it is desirable to separately enumerate sub-types of Other Cells. Note regarding ring-shaped nuclei. In contrast to humans, normal blood cells of mice can have ring-shaped nuclei. Ring forms are not limited to neutrophilic and eosinophilic granulocytes. Ring-shaped nuclei can also be seen in monocytic lineage cells and in cells that are relatively undifferentiated (Biermann H, et al. Murine leukocytes with ring-shaped nuclei include granulocytes, monocytes, and their precursors. J of Leuk Biol. 1999;65:217-231). Return to Characterization and Classification IV.
Classification of Murine Non-lymphoid Hematopoietic Neoplasms This section is meant as a “user’s guide” to the Bethesda proposals for the classification of murine non-lymphoid hematopoietic neoplasms. The proposals themselves and “Questions and Answers about the Bethesda Proposals” contain additional useful information. As discussed in the proposals and in the Q & A, it is important to consider the possibility that an expansion of hematopoietic cells represents a reactive process. What follows is only applicable to lesions that are not reactive. A.
Is the disorder a non-lymphoid hematopoietic sarcoma? 1. Was there a solid tumor mass? 2. What does the bone marrow look like? Is it diffusely involved by cells similar to those in the primary solid tumor mass or is hematopoiesis focally preserved 3. Is the tumor mass invasive or is it present at more than one site? If there is a solid tumor mass, hematopoiesis is focally preserved in the bone marrow, and the tumor is invasive or present at more than one site the diagnosis may be “non-lymphoid hematopoietic sarcoma.” Immunohistochemical stains for cytokeratins and smooth muscle actin can be important in excluding non-hematopoietic tumors. Spindle cell carcinomas (EMT tumors) and histiocytic sarcomas may exhibit similar histology. If non-hematopoietic tumors have been excluded, you should assess which type of hematopoietic sarcoma is present: granulocytic, histiocytic, or mast cell. Granulocytic Sarcoma
Lesion is composed predominantly of granulocytic and/or
relatively undifferentiated non‑lymphoid hematopoietic cells. Granulocytic
as defined by any combination of: A.
Granulocytic morphology (neutrophilic, eosinophilic, or basophilic) B.
Chloroacetate esterase positive and cells are not mast cells C.
Expression of both Ly-6G(Gr-1) and CD11b D. Other means of
identifying granulocytic cells that may be available
Histiocytic Sarcoma
Lesion is composed
predominantly of histiocytic cells Histiocytic as defined by any combination of: A. Histiocytic morphology B. Non-specific esterase positive but not erythroid C. Mac-2+ by immunohistochemistry D. Ly-71(F4/80)+ by immunohistochemistry Mast Cell Sarcoma
1. Lesion has characteristic cytology: uniform, closely packed cells with round nuclei and clear abundant cytoplasm with or without granules. 2. Cells exhibit features of mast cells Mast cells as defined by any combination of: A. Mast cell morphology B. Toluidine blue strongly positive C. Chloroacetate esterase strongly positive D. Other means of identifying mast cells that may
be available B.
Is the disorder a non-lymphoid leukemia? 1. In addition to histopathologic examination you will
need to: · Compare the blood counts of the mouse to appropriate normal controls · Count the percentage of white blood cells in the blood, spleen, and bone marrow that are immature forms/blasts Immature forms/blasts defined as: Size: medium-sized to large Cytoplasm: basophilic, with
or without azurophilic granules Nuclear to Cytoplasmic
Ratio: high Nuclei: predominantly round
to oval, but modest peripheral indentation or a small central opening can be
present Chromatin: fine to
moderately fine Nucleoli: small but often
present; can be indistinct or absent Position of Nucleus:
whereas lymphoblasts and erythroblasts typically have a central nucleus,
myeloblasts often have a nucleus that is more peripherally located. Note: Some of the immature
cells present in leukemias can have rather condensed chromatin or be somewhat
small. If these cells otherwise have
features of immature forms/blasts they should be included in the differential
count as such. · Note whether you performed serial blood counts (at least monthly) on the mouse before it became moribund. · Note whether you performed transplantations, and how long after transplantation the mice were moribund. 2. Does the disorder meet the following five criteria? 1. Diffusely involves hematopoietic tissue, generally both the spleen and bone marrow. 2. Presence of anemia, neutropenia, and/or thrombocytopenia. 3. Non-lymphoid hematopoietic cells are increased in spleen (generally in both bone marrow and spleen). 4. Neoplastic
cells are disseminated as defined by any combination of: 5. Disorder
exhibits additional aspect of malignancy in appearance or behavior as defined
by any combination of: § “Appearance of leukemia” 3. If the disease is a non-lymphoid leukemia, you may
wish to apply additional descriptors to the leukemia. · If the disease meets criteria 5A and (5B1 or 5B2), it can be called “acute” · If the disease is secondary to therapy (for example alkylators or epipodophyllotoxins) it may be called “therapy related” · Is there dyserythropoiesis, dysgranulopoiesis or dysmegakaryocytopoiesis? If there is dysplasia in two or three of these lineages, the disease can be called “with multilineage dysplasia.” At
this time, recommendations regarding the morphologic features that should be
considered dyspoiesis in mice are speculative.
Characteristics described as dyspoiesis in cats and dogs as well as
those described in the FAB proposals for classification of human MDS may be
applicable to mice (Bennett JM, et al. Br J Haematol. 1982;51:189-199. Raskin RE. Vet Clin N Amer-Small Anim.
1996;26:1023-1042.). o
For erythroid cells,
dyspoiesis may include maturation of the cytoplasm preceding maturation of the
nucleus (megaloblastic change), nuclear fragmentation, irregular nuclear
contours, abnormal mitotic figures, ringed sideroblasts, and multiple nuclei. o
For megakaryocytes,
dyspoiesis may include cells with multiple separated nuclei, small cells with
one or more small oval nuclei in mature cytoplasm (micromegakaryoctyes), large
cells with unlobated nuclei, and large cells with bizarre hypersegmentation. o
For neutrophilic cells,
dyspoiesis may include abnormal cytoplasmic maturation, manifested as pale
basophilic cytoplasm in mature neutrophils, and abnormal nuclear maturation as
evidenced by the presence of open chromatin in mature cells. In addition, we have noted that the presence
of neutrophils with lobated as opposed to ring-shaped nuclei may represent
dysplasia. 4. You may wish to sub-classify the leukemia. Bi-phenotypic Leukemia
· Are ³90% of cells in hematopoietic tissue (bone marrow, or spleen excluding lymphocytes) immature forms/blasts? · Are the cells biphenotypic? In bi-phenotypic leukemias the immature forms/blasts
exhibit a mix of lymphoid and non-lymphoid characteristics Lymphoid features required for a diagnosis of bi-phenotypic as defined by any combination of: A.
CD3+ with presence of clonal T-cell receptor gene rearrangement (VDJ
or VJ not just DJ) B.
CD19+ with presence of immunoglobulin gene rearrangement (VDJ or VJ
not just DJ) C.
Other means of identifying lymphoid cells that may be available Non-lymphoid features required for a diagnosis of bi-phenotypic as defined by any combination of: A. Myeloperoxidase positive. C. Chloroacetate esterase positive D.
Ly-6G(Gr-1)+ and CD11b+ E. Other means of identifying non-lymphoid hematopoietic cells that may
be available v If so the diagnosis is biphenotypic leukemia. Erythroid Leukemia
· Are ³50% of cells in hematopoietic tissue nucleated erythroid cells? Erythroid
as defined by any combination of: A.
Erythroid morphology B.
CD71+, Ly-76(Ter119)hi C.
Other means of identifying erythroid cells that may be available v If so the diagnosis is erythroid leukemia. Megakaryocytic Leukemia
· Are ³50% of cells in hematopoietic tissue megakaryocytic cells? Megakaryocytic
as defined by any combination of: A.
Megakaryocytic morphology B.
VWF positive C.
CD41+and Glycoprotein V+ D. Other means of identifying megakaryocytic cells that may become available† v If so the diagnosis is megakaryocytic leukemia. Myeloid Leukemia
· If the disorder is a leukemia of granulocytic and/or monocytic cells and their precursors the diagnosis is myeloid leukemia. · You may wish to sub-classify the myeloid leukemia. MPD-like leukemia
· How many of the cells in the hematopoietic tissue are immature forms/blasts? v If <20% the diagnosis is MPD-like leukemia. Monocytic leukemia, Myelomonocytic leukemia · Have you demonstrated the presence of a monocytic component to the leukemia? Monocytic component means any combination of: A. Monocytosis (³20% of leukocytes in peripheral blood) B. Monocytosis in peripheral blood ³5× normal C. ³20% monocytic cells in bone marrow Monocytes/monocytic cells can be defined by any
combination of: A. Mature monocyte morphology B. Non-specific esterase positive and not
Ly-76(Ter119)hi C. CD16/32+, Ly-71(F4/80) +,
and CD45hi D. CD11b+, Gr-1neg-lo,
CD45R(B220)-, E. Immunoelectron microscopy reveals
myeloperoxidase negative 1° granules F. Other means of identifying monocytic cells that
may become available‡ · Have you demonstrated the presence of a neutrophilic component to the leukemia? Neutrophilic
component means any combination of: A. Neutrophil
morphology (immature cells of indeterminate lineage excluded) B. Chloroacetate
esterase positive but not mast cells C. Other means of
identifying neutrophilic cells that may become available† Neutrophils/neutrophilic
cells can be defined by any combination of : A. Neutrophilia (³20% of leukocytes in
peripheral blood) B. Neutrophilia in
peripheral blood ³5× normal C. ³20% neutrophilic cells
in bone marrow v If ³20% of the cells in the hematopoietic tissue are immature forms/blasts, a monocytic component is present, and a neutrophilic component is absent the diagnosis is monocytic leukemia. v If ³20% of the cells in the hematopoietic tissue are immature forms/blasts, a monocytic component is present, and a neutrophilic component is present the diagnosis is myelomonocytic leukemia. Myeloid leukemia without maturation, Myeloid leukemia with maturation· When nucleated erythroid cells, lymphocytes, plasma cells, macrophages and mast cells are excluded are ³90% of cells in hematopoietic tissue immature forms/blasts? v If ³20% of the cells in the hematopoietic tissue are immature forms/blasts, a monocytic component is absent, and ³90% of non-lymphoid, non-erythroid cells are immature forms/blasts the diagnosis is myeloid leukemia without maturation. v If ³20% of the cells in the hematopoietic tissue are immature forms/blasts, a monocytic component is absent, and <90% of non-lymphoid, non-erythroid cells are immature forms/blasts the diagnosis is myeloid leukemia with maturation. 5. Making comparisons with human leukemias You may wish to state that the leukemia has (i) features of a particular human leukemia or (ii) features that are associated with a particular genetic abnormality in humans. For example, acute myeloid leukemia with maturation, with features of human AML with t(8;21)(q22;q22). C.
Is the disorder myeloid dysplasia? 1. Considerations in diagnosing myeloid dysplasia If you have gotten this far, you have excluded non-lymphoid hematopoietic sarcoma and non-lymphoid leukemia. The following questions relate to the diagnosis of myeloid dysplasia. · Is there dyserythropoiesis, dysgranulopoiesis or dysmegakaryocytopoiesis? · Although the disease does not meet criteria for non-lymphoid leukemia, are ³20% of cells in hematopoietic tissue immature forms/blasts? · How do peripheral blood counts compare to appropriate control mice? Is there anemia, thrombocytopenia, or neutropenia? Is there erythrocytosis, thrombocytosis, or leukocytosis? Disorders more similar to human myelodysplastic syndromes than to human chronic myeloproliferative diseases or human myelodysplastic/myeloproliferative diseases should in mice be called myeloid dysplasias. Conversely, disorders more similar to human chronic myeloproliferative diseases or human myelodysplastic/myeloproliferative diseases than to human myelodysplastic syndromes should in mice be called myeloid proliferations (non-reactive). 2. Defining Criteria If the disorder is not a non-lymphoid leukemia and meets the criteria below, then the diagnosis is myeloid dysplasia.
3. You may wish to sub-classify the myeloid dysplasia. · The cell types that are dysplastic should be stated. · The cell types that are decreased in the peripheral blood should be stated. v If a disease is characterized by increased immature forms/blasts without morphologic dyspoiesis it may be designated as a “cytopenia with increased blasts.” v If a disease is characterized by morphologic dyspoiesis it may be designated as a “myelodysplastic syndrome.” v If a disease has features of a particular human myelodysplastic syndrome it may be designated as a “myelodysplastic syndrome with features of a named human MDS.” D.
Is the disorder a myeloid proliferation (non-reactive)? 1. Considerations in diagnosing myeloid proliferation
(non-reactive). If you have gotten this far, you have excluded non-lymphoid hematopoietic sarcoma, non-lymphoid leukemia, and myeloid dysplasia. Nevertheless, you believe the process to be non-reactive, persistent, and genetically determined. If this setting, the following questions relate to the diagnosis of myeloid proliferation (non-reactive). · How do the peripheral blood counts compare with appropriate control mice? Is there erythrocytosis, thrombocytosis, or leukocytosis? · Are non-lymphoid hematopoietic cells increased in spleen or bone marrow, compared to appropriate control mice? 2. Defining Criteria If the disorder is neither a non-lymphoid leukemia nor a myeloid dysplasia and meets the criterion below, then the diagnosis is myeloid proliferation (non-reactive). Mice exhibit increased non-lymphoid hematopoietic cells as
evidenced by any combination of: 3. You may with to sub-classify the myeloid
proliferation. · The cell types that are increased should be stated. · If dysplastic forms are present this should be stated. v If a disorder is limited to increased non-lymphoid hematopoietic cells in spleen and/or bone marrow without increased counts in the peripheral blood it may be designated as a “myeloproliferation (genetic).” v If a disease is characterized by increased blood counts along with increased non-lymphoid hematopoietic cells in spleen and/or bone marrow it may be designated as a “myeloproliferative disease.” v If a disease has features of a particular human chronic myeloproliferative disease or myelodysplastic/myeloproliferative disease it may be designated as a “myeloproliferative disease with features of a named human MPD or MD/MPD.” Return to Characterization and Classification Return to Contents of Supplement Questions and answers about the Bethesda proposals
for the classification of murine non-lymphoid hematopoeitic neoplasms. Questions raised during
the development of these proposals are presented below. These questions and answers are organized as
follows: General Should not a pathologist be able to make diagnoses based solely
on tissue sections? Definition of Neoplasm What should guidelines be for defining a hematopoietic disorder as a neoplasm? Definition of Blasts Classification Has acute leukemia been eliminated as a diagnostic entity? Are all leukemias transplantable? What criteria distinguish
non-lymphoid leukemia from myeloid dysplasia? What criteria distinguish non-lymphoid leukemia from myeloid proliferation (non-reactive)? What criteria distinguish myeloid dysplasia from myeloid
proliferation (non-reactive)? How is monocytic leukemia distinguished from histiocytic sarcoma with dissemination into the blood? Can dyspoiesis be observed in diseases other than myeloid
dysplasias? Sub-classification Is definitive sub-classification necessary? Why is “promyelocytic
leukemia” not listed as one of the types of myeloid leukemia? Why are leukemias of eosinophilic and basophilic cells not included in the proposals? Comparison to Existing Human
and Rodent Classifications How do the proposals
correspond with the WHO classification of human hematopoietic neoplasms? How do the proposals correspond with the international
classification of rodent tumors? Assorted Is not leukocytosis a consistent finding in murine leukemias? Why are in vitro studies not included in these proposals? General Why
not wait until we understand more about normal and malignant murine hematology
before drafting proposals? The lack of current guidelines has been an impediment to clear communication and has generated some unneeded disagreements. The proposals will certainly need to be revised in light of additional data, but they represent an important step toward consensus nomenclature. It is hoped that the use of electronic communication will facilitate the adoption and dissemination of such revisions. Return to Questions and Answers Should
not a pathologist be able to make diagnoses based solely on tissue sections? It would certainly be useful if all relevant information about an animal could be obtained by post-mortem examination. However, it is felt that integration of clinical/biological, morphologic, cytochemical, immunophenotypic, and genetic information should serve as the basis for a classification system of particular value for studies of genetically engineered mice. Return to Questions and Answers Can
a diagnosis of non-lymphoid leukemia be made from histologic specimens only
when blood and bone marrow cytology are not available? In some cases a presumptive diagnosis of non-lymphoid leukemia can be made by histologic section: when an animal has a disease (i) comprised predominantly of immature non-lymphoid hematopoietic cells and (ii) these immature cells dominate hematopoietic tissues, extensively infiltrate the liver, and have spread to other tissues such as the lungs, kidneys, and lymph nodes. There are leukemias in mice that don't have these features and therefore histologic analysis is insufficient to diagnose many cases of leukemia. Histology is also generally insufficient to distinguish among non-lymphoid leukemias with differentiation, myeloid dysplasias, myeloid proliferations, and reactive processes. Return to Questions and Answers Why
is splenic pathology given more weight in this murine classification than it is
in human classification? The spleen remains a hematopoietic organ throughout life in mice. Return to Questions and Answers Definition of Neoplasm What should guidelines be
for defining a hematopoietic disorder as a neoplasm?
Non-lymphoid hematopoietic neoplasms share three characteristics: they are non-reactive, persistent, and are genetically determined. Non-neoplastic disorders (such as deficiencies in cellular functions) may also have these three features, and therefore for a disorder to be considered a non-lymphoid hematopoietic neoplasm, it must also fulfill criteria for one of the four categories of non-lymphoid hematopoietic neoplasms. Return to Questions and Answers Why is monoclonality
not considered as one of the defining characteristics of non-lymphoid
hematopoietic neoplasms? In humans, neoplasms arise out of a background of normal cells (or a background of cells that have an inherited genetic change that creates a propensity to neoplastic transformation). Hence, monoclonality is a feature of human neoplasms. When genetic alterations are introduced into mice, we do not know in advance whether these changes will be fully sufficient to cause disease, or whether additional events will be necessary. Some of the genetic events associated with non-lymphoid hematopoietic neoplasms in humans can by themselves induce a polyclonal expansion of non-lymphoid hematopoietic cells in mice. Furthermore, the introduction into mice of combinations of genetic changes can create aggressive illness with all the features of acute non-lymphoid leukemia, but such disease may not be monoclonal. Nonetheless, demonstrating that a process is monoclonal greatly strengthens a contention that a disease is non-reactive. Return to Questions and Answers Definition of Blasts Why
is the term “immature forms/blasts” recommended rather than simply
“blasts?” What issues underlie the
recommendations for enumerating these cells? A number of issues contribute to potential difficulties in enumerating the immature cells of non-lymphoid neoplasms used for diagnosis and classification. Among these issues are: a. desire to enumerate these immature cells in cytologic preparations of hematopoietic tissue stained with a Romanowsky stain (eg. Wright’s Giemsa, May-Grünwald-Giemsa) b. desire that such counts have limited inter-observer variability c. desire to use terminology that facilitates comparisons between humans and mice d. desire to limit confusion that can arise when a term has different definitions in humans and mice e. observation of differences in appearance of cells depending on the cytologic preparation (eg. smear vs. cytospin); for example, preparation influences the appearance of chromatin and nucleoli f. observation of differences in presence and number of azurophilic (primary) granules depending on the particular stain used g. observation that the appearance of immature cells in neoplasms can differ from their normal counterparts h. observation that ring-form nuclei are seen not only in neutrophilic lineage cells but can also be seen in monocytic lineage cells and in cells that are relatively undifferentiated (Biermann H, et al. Murine leukocytes with ring-shaped nuclei include granulocytes, monocytes, and their precursors. J Leuk Biol. 1999;65:217-231) i. recognition of the fact that in some human leukemias promonocytes or promyelocytes are considered part of the immature cell (blast) population for the purpose of diagnosing acute leukemia j. difficulty in establishing consensus criteria for distinguishing blasts, promyelocytes and promonocytes in mice The proposal to use the term “immature forms/blasts” and the definition of this term is an attempt to balance these many issues. Return to Questions and Answers What term is recommended for describing cells between “immature forms/blasts” and mature neutrophils and monocytes? It is recommended that such cells of the neutrophilic and monocytic series be termed “intermediate forms.” Recommendations for differential cell counting of murine bone marrow cells are available in the accompanying on-line practical guide to classification. Return to Questions and Answers Classification Has
acute leukemia been eliminated as a diagnostic entity? No. If a disease is a leukemia, and if it is characterized by (A) ³20% non-lymphoid immature forms/blasts in blood, spleen, or bone marrow and by (B1) rapidly fatal to primary animal or (B2) transplantable to normal or sub-lethally irradiated histocompatible recipients and rapidly fatal to such transplant recipients, then it can be designated as an “acute” leukemia. Return to Questions and Answers Are
all leukemias transplantable? Not necessarily. Although most leukemias are transplantable, demonstrating transplantability is not required for the diagnosis of non-lymphoid leukemia. In order to expand our understanding of the significance of transplantability, it is recommended that investigators include descriptions of the cells transplanted (origin, preparation, number), route of injection, type of recipient (for example nude/other immunodeficient strain/histocompatible, unirradiated/histocompatible, sub-lethally irradiated/histocompatible, lethally irradiated), and character of disease in recipient (localized/disseminated, time from transplantation until illness). In describing mouse hematopoietic neoplasms, investigators should indicate the method and results of transplantation experiments. Return to Questions and Answers Should
not leukemias and sarcomas of similar cell types be considered as two
presentations of the same disease? Although there is overlap, the presentation and appearance of these diseases are generally distinct in the mouse. Furthermore, our understanding in mice of the evolution of these diseases over time is limited. It therefore appears appropriate to consider hematopoietic sarcomas and leukemias as separate categories of disease. Return to Questions and Answers What
criteria distinguish non-lymphoid leukemia from myeloid dysplasia? Non-lymphoid leukemias and myeloid dysplasias are characterized by cytopenias. In addition, both may exhibit increased young forms and dyspoiesis may be observed in both. A diagnosis of leukemia takes precedence and should be made when the disorder shows evidence of (i) dissemination (criterion #4 for leukemia) and (ii) either ³20% non-lymphoid young forms or clinical aggressiveness (criterion #5 for leukemia). Return to Questions and Answers What criteria distinguish non-lymphoid leukemia from myeloid proliferation (non-reactive)? Non-lymphoid leukemias and myeloid proliferations (non-reactive) are both characterized by increased numbers of non-lymphoid hematopoietic cells. A diagnosis of leukemia takes precedence and should be made when all the criteria for this diagnosis are met. In many cases, a diagnosis of myeloid proliferation (non-reactive) will be made because a disorder does not meet the strict criteria for leukemia. Return to Questions and Answers What
criteria distinguish myeloid dysplasia from myeloid proliferation
(non-reactive)? Myeloid dysplasias and myeloid proliferations (non-reactive) reflect ends of a spectrum of non-leukemic disorders. A diagnosis of myeloid dysplasia takes precedence and should be made when there is (i) evidence for a defect in maturation of non-lymphoid hematopoietic cells (criterion #2 for myeloid dysplasia) and (ii) if one or more cytopenias are present (criterion #1 for myeloid dysplasia). Classification of disorders that have a mix of cytopenias and increased non-lymphoid elements in the peripheral blood is problematic. Disorders characterized by defective maturation and neutropenia should be classified as myeloid dysplasia even in the presence of thrombocytosis (or erythrocytosis). (The basis for this recommendation is the finding that in some cases of human MDS neutropenia is accompanied by thrombocytosis.) On the other hand, if anemia or thrombocytopenia is accompanied by an increase in the peripheral blood in a different lineage, such a disorder should be classified as a myeloid proliferation (non-reactive). In many cases, a diagnosis of myeloid proliferation (non-reactive) will be made because a disorder does not meet the strict criteria for myeloid dysplasia. As noted in the proposals, the intention is that myeloid dysplasia be used for disorders most closely related to human MDS, whereas myeloid proliferation (non-reactive) is intended to be used for disorders most closely related to either human MPD or human MD/MPD. Return to Questions and Answers What
criteria distinguish myeloid proliferation (non-reactive) from non-neoplastic
myeloid hyperplasia? It can be difficult to distinguish leukemoid reactions and extramedullary hematopoiesis from myeloid proliferations (non-reactive). Evidence for a process being a myeloid proliferation (non-reactive) may include: the absence of infectious or inflammatory lesions, the absence of non-hematopoietic tumors, adequate nutrition, the absence of exposure to drugs or toxins, the abnormalities being persistent and progressive (by serial physical examination or examination of the blood), presence of clonal hematopoiesis, and the disorder being transplantable. The possibility that an increase in non-lymphoid hematopoietic cells may be due to defects in blood cell effector function, autoimmune blood cell destruction, or dysregulation of the lymphoid system (potentially causing abnormal cytokine production) should be considered. Return to Questions and Answers Although it can be difficult to
distinguish myeloid proliferation (non-reactive) from non-neoplastic myeloid
hyperplasia, how can myeloid leukemias be distinguished from non-neoplastic
myeloid hyperplasia? Dunn’s comprehensive discussion of abnormalities of the hematopoietic system of mice included a table comparing myeloid leukemia with reactive myeloid hyperplasia (Dunn, T. J Natl Cancer Inst. 1954;14:1281-1433). An updated version of this table is presented below.
These guidelines can help distinguish aggressive non-lymphoid hematopoietic neoplasms from reactive lesions. However, indolent neoplasms can have many features in common with reactive proliferations. Return to Questions and Answers What are causes of non-lymphoid hematopoietic neoplasms in mice? What are causes of non-neoplastic myeloid hyperplasia? Hematopoietic neoplasms
in mice can arise spontaneously or be induced viruses, radiation, toxins, or by
engineered genetic changes.
Non-neoplastic myeloid hyperplasia can be caused by nutritional
deficiencies, infections, tumors of non-hematopoietic cells, ulcerations, other
inflammatory stimuli, toxins, autoimmune blood cell destruction, and
dysregulation of the lymphoid system. Return to Questions and Answers How is monocytic leukemia distinguished from histiocytic sarcoma with dissemination into the blood? Histiocytic sarcoma can disseminate into the blood. If on histopathologic examination the bone marrow shows areas of intact hematopoiesis whereas solid lesions composed of histiocytic cells are found elsewhere (eg. uterus, liver) the diagnosis of “histiocytic sarcoma” should be made. If however, the disease meets all criteria for leukemia including diffuse marrow involvement, the diagnosis of “monocytic leukemia” should be made. Some cases may be particularly difficult to distinguish as sarcoma or leukemia and should be noted as such. Return to Questions and Answers Can
dyspoiesis be observed in diseases other than myeloid dysplasias? Yes. Morphologic dyspoiesis can be seen in any of the neoplasms. If observed in leukemia, then the designation “with multilineage dysplasia” may be appropriate. Diseases classified as myeloid proliferations (non-reactive) may have dyspoiesis, and such diseases might therefore correspond to the myelodysplastic/myeloproliferative diseases of the human WHO classification. Return to Questions and Answers Sub-classification Is definitive sub-classification necessary? It is important to note that these proposals are intended as guidelines to be used when appropriate. The group that drafted these guidelines has neither authority nor intent to obligate studies that, while permitting classification, may or may not be scientifically valuable. On the other hand, it is suggested that the terms defined in the proposals not be used except for diseases that fulfill the proposed criteria. For this reason, investigators should sub-classify non-lymphoid hematopoietic neoplasms only to the extent that available data permit. Return to Questions and Answers Why
do the definitions of sub-types of myeloid leukemia in mice differ in small
ways from those used in humans? The proposals attempt to build on the human classification to discern the lineage of the neoplastic populations as well as to accommodate differences between mice and humans. Return to Questions and Answers Why
is “promyelocytic leukemia” not listed as one of the types of myeloid leukemia? There are murine leukemias with an apparently distinctive feature (large numbers of primary granules) suggesting that the leukemic cells are a murine equivalent of the abnormal promyelocytes present in human acute promyelocytic leukemia. This feature can be seen in gross pathology as green lymph nodes (due to high expression of myeloperoxidase), in Romanowsky stained cytologic preparations as large numbers of azurophilic granules, and in Sudan Black B stained cytologic preparations. It has been suggested that the term “promyelocytic leukemia” should be applied to leukemias that meet criteria for “myeloid leukemia without maturation” but whose immature forms/blasts show strong and fairly uniform cytoplasmic staining with Sudan Black B. However, adopting this provisional suggestion depends on study of Sudan Black B staining in a variety of murine non-lymphoid leukemias to assess whether strong staining is, in fact, able to reproducibly distinguish a sub-type of myeloid leukemia. Return to Questions and Answers Why are splenic lymphoid cells included in differential counts for the purpose of diagnosing non-lymphoid leukemia, but excluded from differential counts for the purpose of sub-classifying non-lymphoid leukemia? Splenic lymphoid cells are included for the purpose of diagnosing non-lymphoid leukemia to set a standard threshold for diagnosis in blood, marrow and spleen. Splenic lymphoid cells are excluded for the purpose of sub-classifying non-lymphoid leukemia to allow sub-classification to be based on the mix of neoplastic cells, rather than on the non-neoplastic lymphoid “bystanders.” This is in keeping with the FAB recommendations regarding excluding lymphoid and erythroid cells when sub-classifying myeloid leukemias. Return to Questions and Answers Can “myeloid leukemia without
maturation” be distinguished from “myeloid leukemia with maturation” when
cytology of spleen cells but not of marrow cells is available? The criterion recommended to diagnose “without maturation” as opposed to “with maturation” is the presence of ³90% immature forms/blasts when nucleated erythroid cells, lymphocytes, plasma cells, macrophages and mast cells are excluded. This diagnostic criterion can be applied to splenic cytology. It is generally possible, however, that maturing neutrophilic cells in a leukemic spleen may represent residual normal hematopoietic tissue. Therefore, some leukemias in which the leukemic clone itself shows very little maturation might be diagnosed as “myeloid leukemia with maturation” when splenic cytology is utilized. Return to Questions and Answers Why are leukemias of eosinophilic
and basophilic cells not included in the proposals? The committee did not observe such leukemias in the materials available for consideration. It would be important to identify and analyze such leukemias prior to their incorporation into future recommendations. Return to Questions and Answers When sub-classifying non-lymphoid leukemias as erythroid or megakaryocytic, are young forms of erythroid and megakaryocytic lineages counted toward the ³50% criterion? Yes. Cells identified as erythroid or megakaryocytic by marker expression are included even when they are not recognized by morphology. [Note: Ly-76 (TER119) is not lineage specific to erythroid cells, with modest expression being seen in some myeloid leukemias] Return to Questions and Answers When sub-classifying non-lymphoid leukemia, may flow cytometry be used to assess the percentage of cells of various lineages? Yes, results of morphology, cytochemical stains, flow immunophenotyping, and immunohistochemistry can be used for lineage assessment. In the future, analyses of gene expression are likely to also be valuable for lineage assessment. Return to Questions and Answers How can non-lymphoid leukemias be sub-classified when no cytologic samples are available, ie. when paraffin embedded tissues must be utilized? Sub-classification is difficult in the absence of cytology samples of involved hematopoietic tissues. With the use of histology and immunohistochemistry it if generally possible to sub-classify disorders as myeloid leukemia, erythroid leukemia, or megakaryocytic leukemia. Myeloid leukemias can be further described, if not sub-classified, as poorly differentiated, moderately differentiated, or well differentiated. Return to Questions and Answers Why
is one of the sub-categories of myeloid proliferation (non-reactive) called
myeloproliferation (genetic)? When
should this sub-category be diagnosed? Increases, sometimes modest, in splenic non-lymphoid hematopoietic cells have been seen in many genetically engineered mice with genetic abnormalities closely associated with human non-lymphoid neoplasms including leukemias. The possibility of considering such subtle expansions as entities outside of the classification of non-lymphoid hematopoietic neoplasms was considered, but rejected for two reasons. First, these expansions can give rise to leukemia. Second, these expansions are caused by genetic abnormalities known to be associated with human disease. If a disorder in genetically engineered mice is limited to increased non-lymphoid hematopoietic cells in spleen and/or bone marrow without increased counts in the peripheral blood it may be designated as a “myeloproliferation (genetic).” Investigators must nevertheless remain alert to the possibility of reactive causes of any observed increases in myeloid cells. Return to Questions and Answers The sub-classification for myeloproliferative diseases seems
underdeveloped relative to that used in humans. Should not the classification include such entities as chronic
myelogenous leukemia, polycythemia vera, essential thrombocythemia, and chronic
idiopathic myelofibrosis? The term myeloproliferative disease has been applied very broadly to diseases in mice, so broadly that the term currently lacks any diagnostic precision. Some of the diseases previously considered to be myeloproliferative diseases would be classified under the Bethesda proposals as MPD-like myeloid leukemia, whereas others would be considered as myeloproliferative diseases or myeloproliferations (genetic). Further sub-classification of myeloproliferative diseases in the mouse awaits additional experience. If an investigator desires, a murine disease with features of a particular human myeloproliferative disease or myelodysplastic/myeloproliferative disease may be designated as a “myeloproliferative disease with features of a named human MPD or MD/MPD.” Return to Questions and Answers Comparison to Existing Human and Rodent Classifications How
do the proposals correspond with the WHO classification of human hematopoietic
neoplasms? Human classification as described in Jaffe ES, Harris NL, Stein H, Vardiman J eds. Pathology and genetics of tumours of haematopoietic and lymphoid tissues. WHO classification of tumours. Lyon: IARC Press; 2001.
Return to Questions and Answers How
do the proposals correspond with the international classification of rodent
tumors? International classification as described in Frith CH, Ward JM, Harleman JH, et al. Hematopoietic System. In: Mohr U, ed. International classification of rodent tumors: the mouse. Heidelberg: Springer-Verlag; 2001:417-451.
Return to Questions and Answers Assorted Are not there additional types of neoplasms of non-lymphoid
hematopoietic cells (e.g. non-malignant tumors of mast cells; processes
comparable to chronic idiopathic myelofibrosis)? Although the proposals cover most diseases already described in mice as well as some diseases that have not yet been described, they are incomplete. A category of benign soft tissue tumors composed of hematopoietic cells may need to be added. It is unclear if the suggested definition of myeloproliferative disease would include mice with a neoplasm comparable to myelofibrosis. Additional studies followed by revision of the proposals is necessary. It is hoped that the electronic means piloted to develop these proposals will serve as an effective tool for updating the recommendations. Return to Questions and Answers Friend
virus complex causes a number of different disorders in mice. Why is there only one entry in the
classification that is a neoplasm of erythroid cells, “erythroid
leukemia?” How should proliferations of
erythroid cells that do not meet the definition of erythroid leukemia be
classified? The features of other disorders caused by Friend virus are variable. Depending on the particular character of disease in an animal these disorders would be considered as non-neoplastic disorders that can give rise to erythroid leukemia or as myeloid proliferations (non-reactive). It is anticipated that future proposals for sub-classification will include a category in which erythroid predominant myeloproliferative diseases could be placed. Return to Questions and Answers Is
not leukocytosis a consistent finding in murine leukemias? Although most murine leukemias are characterized by leukocytosis, some leukemias have normal or even decreased peripheral white blood cell counts. Return to Questions and Answers Should
not markers used for defining lineage be noted as positive or negative rather
than by some difficult to reproduce quantitative measure (eg. strongly positive, Antigenhi)? Differences in level of expression can have an important role in defining lineage. A certain level of familiarity with how different populations stain is essential if certain markers are used to determine lineage. We plan to provide examples of cytologic, histologic, and flow cytometric markers at the MMHCC web-site in order to assist the community with application of the proposed criteria. Return to Questions and Answers Why
are in vitro studies not included in
these proposals? There was no agreement on how studies of the in vitro characteristics of the neoplastic cells could be used in classification. Return to Questions and Answers Return to Contents of Supplement Bibliography
General References Regarding Mouse Hematopathology Morphology of Mouse Blood Cells including Differential Cell Counting General References Regarding Mouse HematopathologyDunn,
T. Normal and Pathologic Anatomy of the
Reticular Tissue in Laboratory Mice with a Classification and Discussion of
Neoplasms. J. Natl. Cancer Inst.
14:1281-1433, 1954. Schermer,
S. The Blood Morphology of Laboratory
Animals. Philadelphia: Davis; 1967. Pattengale, PK and Taylor, CR. Experimental Models of
Lymphoproliferative Disease: the mouse as a model for human non-Hodgkin’s
lymphomas and related leukemias.
American Journal of Pathology 113:237-265, 1983. Perkins,
AS. The Pathology of Murine Myelogenous Leukemias. Current Topics in
Microbiology and Immunology 149: 3-21, 1989. Frith CH,
Ward JM, Fredrickson T, Harleman JH. Neoplastic lesions of the hematopoietic
system. In: Mohr U, Dungworth DL, Capen CC, Carlton WW, Sundberg JP, Ward JM,
eds. Pathobiology of the Aging Mouse. Washington, DC: ILSI Press; 1996:219-235 Ward, JM et
al. Thymus, Spleen and Lymph Nodes. In: Pathology of the Mouse. Maronpot, RR;
Boorman, GA; Gaul, BW. (eds), Vienna,
IL: Cache River Press, 1999. Taddesse-Heath,
L. and Morse, HD. Lymphomas in
Genetically Engineered Mice. In: Pathology of Genetically Engineered
Mice. Ward, JM; Mahler, J; Maronpot, R;
Sundberg, JP (eds.), Ames, Iowa: Iowa State University Press, 2000. Fredrickson,
TN and Harris, AW. Atlas of Mouse Hematopathology. Harwood Academic Publishers, 2000. Frith CH,
Ward JM, Harleman JH, Stromberg PC, Halm S, Inoue T, Wright JA. Hematopoietic
System. In: Mohr U, ed. International classification of rodent tumors: the
mouse. Heidelberg: Springer-Verlag; 2001:417-451. Mouse Models of Human Cancer: http://emice.nci.nih.gov/ Lab Protocols Columbia University Life Sciences: http://icg.cpmc.columbia.edu/cattoretti/Protocol/Main.html Pathology and Biology of Genetically Engineered Mice: http://www.ncifcrf.gov/vetpath/nihtg.html The Virtual Mouse Necropsy: http://geocities.com/virtualbiology/necropsy.html Morphology of Mouse Blood Cells including Differential Cell Counting Endicott,
KM; Gump, H. Hemograms and Myelograms
of Healthy Female Mice of C-57 Brown and CFW Strains. Blood Special Issue #1:60-63, 1947. Brecher, G;
Endicott, K; Gump, H; Brawner, HP.
Effects of X-ray on Lymphoid and Hemopoietic Tissues of Albino
Mice. Blood 3:1259-1274, 1948 Petri, S.
Morphologishe and Numerische Untersuchungen Ueber Knochenmarkzellen Bei
Normalen Weissen Laboratoriummaueusen
(Morphological and Numerical Studies of Bone Marrow Cells of Normal
White Laboratory Mice). ACTA
Pathologica et Micro. Scandinavica 11:1-43, 1934. The Petri article contains
detailed description and excellent illustrations. However, the Petri classification is not particularly practical,
including 32 categories of cells. Biermann, H;
Pietz, B; Dreier, R; Schmid, KW; Sorg, C; Sunderkötter, C. Murine Leukocytes with Ring-Shaped Nuclei
Include Granulocytes, Monocytes, and Their Precursors. J. Leukocyte Biology 65:217-230, 1999. Fredrickson,
TN and Harris, AW. Atlas of Mouse Hematopathology. Harwood Academic Publishers, 2000. Lagasse, E; Weissman, IL. Flow cytometric identification of murine
neutrophils and monocytes. J Immunological Methods, 1996 Oct 16, 197(1-2):139-150. Lily, L. et al. Mouse Cell Surface
Antigens: Nomenclature and Immunophenotyping. J. Immunology. 160: 3861-3868,
1998. Cattoretti, G and Fei, Q. Application of
the Antigen Retrieval Technique in Experimental Pathology: From Human to Mouse.
In: Antigen Retrieval Techniques, Shi, SR; Gu, J; Taylor, CR (eds.) Eaton
Publishing, Natick, MA, 2000. Return to Contents of Supplement Materials Considered I. Among the samples that were directly examined were materials from the following mice: Transgenics: Cathepsin G PML-RARA Cathepsin G PLZF-RARA Cathepsin G PLZF-RARA/RARA-PLZF Cathepsin G NPM-RARA MRP8 PML-RARA MRP8 PML-RARA/BCL2 Recipients of bone marrow transduced with retroviruses
that express: AML1-MDS1-EVI1 MLL-ELL BCR-ABL IL-3 & Hoxb8 Strain with endogenous retrovirus: BXH-2 Exogenous retroviruses: Cas2 Spl retrovirus in NFS Recombinant Friend-Moloney virus in NFS/N Friend leukemia virus complex or Friend helper virus alone Strains with radiation induced disease: SJL Strains with spontaneous disease: NFS.V+ C57BL/6 Knock-outs/knock-ins: Runx1-ETO knock-in Nf1 knock-out II. Selected additional materials considered from published literature included: Transgenics: Em BCL2 (irradiated) MRP8 BCL2/Faslpr/lpr Recipients of bone marrow transduced with retroviruses
that express: c-myc v-myc v-fms c-Ha-ras N-rasG12D gp55 (from Friend SFFV) E2A-Pbx1 Hoxa10 Hoxa9 Hoxa9&Meis1a Hoxa9&E2A-Pbx1 Hoxb3 HRX-ENL MLL-CBP TEL-JAK2 TEL-PDGFbR TEL-TRKC Activated GM-CSF/IL-3/IL-5 receptor beta common
subunit IL-6 GM-CSF PDGFB Exogenous retroviruses: Malignant histiocytosis sarcoma virus Strains with radiation induced disease: SJL/J Knock-outs/knock-ins: Cbfb-MYH11 knock-in Mll-AF9 knock-in Mll-lacZ knock-in Icsbp knock-out junB knock-out
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