Blood Cell Identification: 2010-B Mailing: Primary Myelofibrosis · blood cells and resultant cytopenias (anemia, leukopenia or thrombocytopenia). ... (MPN) are clonal disorders of

Blood Cell Identification: 2010-B Mailing: Primary Myelofibrosis

All material is © 2010 College of American Pathologists, all rights reserved

1 - Education

Please Note: To view the Figures and Images contained within this education activity in color, access the

electronic version of the reading.

CASE HISTORY

This blood film is from a 64-year-old male with history of chronic idiopathic myelofibrosis and rising WBC.

Laboratory data include: WBC = 96.2 x 109/L; RBC = 4.73 x 1012/L; HGB = 13.8 g/dL; HCT = 48%;

MCV = 100 fL; and PLT = 138 x 109/L.

DISCUSSION

This case illustrates some of the morphologic findings seen in the blood smear of a patient with primary

myelofibrosis (PMF). Those features included abnormal platelets (giant and large forms, hypogranular

forms), circulating micromegakaryocytes, a mild basophilia, nucleated red blood cells, and anisocytosis and

poikilocytosis with increased polychromatophilic cells, and occasional teardrop-shaped cells (dacrocytes).

These morphologic features are not specific for PMF only, and should raise suspicion for a

myeloproliferative neoplasm (discussed in more detail below). In this case, the diagnosis of primary

myelofibrosis was known and the patient was presenting with a rising WBC of 96.2 x 109/L. The rising

WBC is of concern in this patient, as myeloproliferative neoplasms (MPN) such as PMF can transform into

acute leukemia. A manual differential should be performed on the blood smear and examination of the bone

marrow is necessary for an accurate diagnosis. Recognition that the peripheral blood smear is abnormal and

may represent a neoplastic process involving the myeloid lineage is the first step in making a diagnosis of

primary myelofibrosis.

Myeloid Neoplasms

Neoplastic proliferations of the myeloid series are categorized into major diagnostic subtypes based on their

characteristic clinical and pathologic features at diagnosis (Table 1. below).

Table 1. Proliferation and Differentiation in Myeloid Disorders

MDS AML MPN

Differentiation

Impaired

Impaired

Normal

Proliferation / Survival

Impaired

Preserved

Increased

All of these neoplastic myeloid proliferations are clonal disorders of hematopoietic stem cells in the bone

marrow. However, differences in the ability of the neoplastic cells to proliferate and differentiate give rise

to different clinical disorders. Myelodysplastic syndromes (MDS) are characterized by impairment of both

proliferation and survival of myeloid cells as well as impairment of normal differentiation, giving rise to the

characteristic clinical syndrome of abnormal (dysplastic or ineffective) bone marrow maturation of one or

more cell lineages (i.e., erythroid, myeloid or megakaryocytic) that causes decreased production of mature



2 - Education

blood cells and resultant cytopenias (anemia, leukopenia or thrombocytopenia). Similarly, acute myeloid

leukemia (AML) is a clonal myeloid disorder in which the neoplastic cells have near normal rates of

proliferation and survival but have lost the ability to differentiate normally, giving rise to the myeloid blast

population that is characteristic. Myeloproliferative neoplasms (MPN) are clonal disorders of hematopoietic

stem cells in which the bone marrow is typically hypercellular due to increased proliferation and survival of

the myeloid cells; however, these cells retain full maturational capacity.

Both MPN and MDS may transform to AML. MPN differ from MDS, however, in that bone marrow

dysplasia is absent, maturation is normal, and the production of cells is effective, causing elevated

peripheral blood counts in one or more lineages, (i.e., erythrocytosis, leukocytosis, and thrombocytosis).

Patients also tend to have hepatosplenomegaly (an unusual finding in MDS) because of sequestered blood

cells, extramedullary hematopoiesis, or infiltration by neoplastic cells. MPN also differ from AML in the

slower disease onset and more protracted course, measured in months to years for MPN, compared to

weeks for untreated AML.

Myeloproliferative Neoplasms

The 2008 WHO Classification of myeloid neoplasms recognizes the following eight different clinical

subtypes of MPN based on clinical features and the specific cell line involved in increased cell proliferation:

Chronic myelogenous leukemia, BCR-ABL1 positive (CML)

Polycythemia vera (PV)

Primary myelofibrosis (PMF)

Essential thrombocythemia (ET)

Chronic neutrophilic leukemia (CNL)

Chronic eosinophilic leukemia, not otherwise specified (CEL, NOS)

Mastocytosis

Myeloproliferative neoplasm, unclassifiable (MPN, U)

MPN usually occur in middle-aged adults. The clinical and hematologic features of the various MPN show

significant overlap and this may complicate the ability to make a specific diagnosis in some cases. During

the early stages of disease, some MPN can show similar features, and patients initially diagnosed with one

type of MPN may later be recognized to have a different MPN, once the more characteristic clinical and

pathologic manifestations become evident. Some patients do not conform readily to current criteria, and

these should be labeled as MPN, unclassifiable. In addition, many of the MPN have specific molecular

abnormalities that involve tyrosine kinase genes, such as the ABL1 gene of chronic myeloid leukemia (CML)

or the Janus kinase 2 (JAK2) gene, that lead to increased activity of these enzymes and activation of cell

signaling pathways. Identification of these molecular abnormalities has greatly increased the ability to

accurately diagnose certain subtypes of MPN.

A point mutation in the JAK2 gene, a cytoplasmic protein-tyrosine kinase that is important in cell

proliferation and survival, occurs in about 95% of patients with polycythemia vera, 40% with essential



3 - Education

thrombocythemia, and 50% with primary myelofibrosis. This mutation (JAK2V617F) is rarely, if ever, present

in CML or in the normal population and is uncommon in chronic myelomonocytic leukemia (CMML),

hypereosinophilic syndrome, MDS, and systemic mastocytosis. Mutations in another gene, the

thrombopoietin receptor (MPL) have been detected in 5% of patients with primary myelofibrosis and 1% of

patients with essential thrombocythemia. Interestingly, mutations in MPL lead to aberrant activation of a

variety of intracellular signaling pathways, including those affected by JAK2 mutations. Furthermore, some

patients with MPL mutations also have simultaneous JAK2 mutations, suggesting that these two genes are

complementary in the development of disease. In addition to single gene mutations, nonspecific cytogenetic

abnormalities are present in about 30% of patients with primary myelofibrosis, 10-20% with polycythemia

vera, and 5-10% with essential thrombocythemia.

Myelofibrosis

Myelofibrosis is a common element in MPN, probably caused by release of various cytokines and growth

factors from megakaryocytes and other marrow cells that cause fibroblasts to proliferate and produce

collagen and fibronectin. In many cases, the disease evolves with stepwise increases in fibrosis, eventually

resulting in end-stage myelofibrosis. An increase in blasts may occur with or without concurrent

myelofibrosis, with the disease progressing through an “accelerated” phase (10-19% blasts in the blood or

bone marrow) to overt acute leukemia (≥ 20% blasts). The various MPN differ in the incidence of such

transformations.

Primary Myelofibrosis Also called “chronic idiopathic myelofibrosis,” “agnogenic myeloid metaplasia” or “myelofibrosis with

myeloid metaplasia,” this disorder consists of a clonal proliferation of megakaryocytes and granulocytic

precursors in the bone marrow, marrow fibrosis (probably from local release of fibrogenic growth factors by

the abnormal clone), and extramedullary hematopoiesis. The spleen and liver are the most common sites of

extramedullary hematopoiesis, but other sites, such as the dura mater, lymph nodes, lung, and pleura, may

also be involved. In these locations it may cause symptoms from organ enlargement, bleeding, fluid

formation, or compression of adjacent normal structures. Primary myelofibrosis (PMF) has a prefibrotic

phase, which is commonly asymptomatic and is characterized by a hypercellular marrow and minimal

fibrosis. Disease progression is characterized by increasing amounts of fibrosis until the marrow is nearly

obliterated by fibrotic tissue, termed the fibrotic phase (Table 2. on the following page).


Table 2. Clinical and Laboratory Features in PMF

Prefibrotic Phase Fibrotic Phase Clinical Features Minimal or mild splenomegaly

and/or hepatomegaly

May be asymptomatic or have

non-specific symptoms

Moderate to marked splenomegaly and/or hepatomegaly

Symptoms from anemia, hypermetabolic state

Hematologic Abnormalities

Variable Mild to moderate anemia Mild to moderate leukocytosis

and thrombocytosis

Variable Moderate to marked anemia WBC and platelets may be low, normal

or increased Blood Smear Findings Occasional nRBCs and immature

myeloid cells No or few teardrop cells Large platelets

Leukoerythroblastosis with nRBCs, immature myeloid cells and blasts

Teardrop cells Large, abnormal platelets Circulating megakaryocytic nuclei or

micromegakaryocytes Bone Marrow Findings Hypercellular marrow

Neutrophilic and megakaryocyte proliferation

Megakaryocytic atypia Large size

Abnormally lobulated nuclei

No or minimal reticulin fibrosis

Variable marrow cellularity Megakaryocytic proliferation with

atypia Large aggregates

Large size

Abnormally lobulated nuclei

Increased reticulin/collagen fibrosis New bone formation

About 30% of patients present in the prefibrotic phase and the remainder in the fibrotic phase (Figure 1

below).

Figure 1.

The two phases of primary myelofibrosis. The majority of patients are diagnosed

during the fibrotic phase of the disease.


4 - Education



5 - Education

PMF is a rare disorder with an incidence of 0.5-1.5 cases diagnosed/100,000 population/year. The average

age at diagnosis is about 60 years, and the average life expectancy is approximately 3-7 years, for patients

diagnosed in the fibrotic stage, with 5-30% of patients eventually developing acute leukemia. About 25%

of patients are asymptomatic at diagnosis, but may have splenomegaly or abnormal blood tests. By far the

most common presenting symptom is a gradual increase in fatigue. Most of the presenting symptoms are

non-specific and typically occur because of anemia, hypermetabolism from high cell turnover,

splenomegaly, or thrombocytopenia. Hypermetabolism may cause weight loss, fever, sweats, or problems

associated with hyperuricemia, including attacks of gout or kidney stones. Anemia can lead to complaints

of fatigue, dyspnea, weakness, and palpitations. Splenomegaly can produce left upper quadrant discomfort,

a sensation of early satiety from compression of the stomach, or diarrhea from pressure on the bowels. On

physical examination, splenomegaly is nearly universal and may range from slight enlargement early in the

disease to enormous at later stages. Hepatomegaly is present in about half of patients. Thrombocytopenia

or altered platelet function may cause cutaneous hemorrhage. Extramedullary hematopoiesis affecting the

serosal surfaces can cause pleural or pericardial effusions and ascites. Portal hypertension due to hepatic

involvement or increased blood flow from an enlarged spleen may also cause ascites and esophageal

varices; the latter may cause gastrointestinal hemorrhage.

Peripheral Blood and Bone Marrow Findings in PMF

In the prefibrotic phase the characteristic blood findings are anemia, thrombocytosis, and mild to moderate

leukocytosis. The blood smear may show occasional nucleated red cells, teardrop-shaped erythrocytes

(dacrocytes), immature granulocytes, and large platelets. A bone marrow biopsy is essential for the

diagnosis of PMF. The bone marrow at this stage is hypercellular, with increased neutrophils and atypical

megakaryocytes. The megakaryocytes are characterized by a larger size, abnormal chromatin clumping,

altered nuclear-cytoplasm ratios, and abnormally lobulated nuclei. Fibrosis, as detected by special stains for

reticulin and collagen, is absent or minimal.

As PMF progresses to the fibrotic phase, hematologic findings include anemia and a leukoerythroblastic

blood smear (nucleated erythrocytes, immature myeloid elements) with numerous dacrocytes (Image 1. on

the following page).


Image 1.

Source: Image 1. George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am. 2003;17:1101-1127. The characteristic teardrop cells or dacrocytes are formed by mechanical

damages to the red cell, as demonstrated in Figure 2. below).

Figure 2.

White cell and platelet counts vary widely, but a few myeloblasts are common, and platelet abnormalities

include large size, bizarre shapes as well as circulating megakaryocyte nuclei or micromegakaryocytes. The

current case discussed with this education activity is an example of primary myelofibrosis in which many

abnormal platelets and micromegakaryocytes are seen in the blood smear. Bone marrow aspiration is

usually unsuccessful due to the increase in fibrosis, but the bone marrow biopsy demonstrates variable

cellularity, with marked reticulin or collagen fibrosis and numerous atypical megakaryocytes in large

aggregates (Image 2A, 2B on the following page).


6 - Education


Image 2A.

A low power image of a bone marrow biopsy

showing areas of cellular streaming indicative of

fibrosis (arrows) and dilated marrow sinuses

(arrowhead).

Image 2B.

Clusters of atypical megakaryocytes are present. The

megakaryocytes show atypical morphology with dark

and dense “hyperchromatic” chromatin and variation

in size.

Osteosclerosis (new bone formation) may be present and is thought to occur due to the factors secreted by

the abnormal clonal myeloid and megakaryocytic cells (Image 3A below, Image 3B on the following page).

Image 3A.

Source: George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe’s Atlas of Clinical Hematology. Lippincott Williams and Wilkins, Inc; 2007:113.

Normal adult bone marrow is made up of cellular marrow and fat interspersed among thin

bony trabeculae that appear, on gross examination, as red and white tissue respectively

(upper panel). In primary myelofibrosis, by contrast, cellular marrow is replaced by

deposition of collagen and appears uniformly white (lower panel). The bone is also seen

to be thickened (osteosclerosis).


7 - Education


Image 3B.

Source: George TI, Arber DA. Pathology of the myeloproliferative diseases. Hematol Oncol Clin North Am. 2003;17:1101-1127.

Osteosclerosis is evident as thickened bone formation which occupies more

than one-half of the bone marrow in this image. In normal bone marrow there is only scattered fine reticulin fibers that form part of the bone marrow

stroma that allows for hematopoietic cell proliferation and differentiation. An increase in the amount of

fibrotic tissue, also termed myelofibrosis, may occur secondary to a variety of stimuli including as a

response to injury, inflammation, infection or metastatic disease as well as being associated with a variety

of myeloid neoplasms, most notably primary myelofibrosis (Table 3 below).

Table 3. Causes of Bone Marrow Fibrosis

Neoplastic Conditions Non-Neoplastic Conditions

Myeloproliferative Diseases Chronic myelogenous leukemia, BCR-

ABL1 positive Primary myelofibrosis Polycythemia vera

Other Neoplasms Acute panmyelosis with myelofibrosis Myelodysplastic syndromes with fibrosis Systemic mast cell disease Acute myeloid leukemia, particularly acute

megakaryoblastic leukemia Hodgkin lymphoma Hairy cell leukemia Non-Hodgkin lymphoma Multiple myeloma Acute lymphoblastic leukemia Metastatic carcinoma

Inflammatory Reactions Infections/granulomatous reactions, such

as tuberculosis Systemic lupus erythematosus Autoimmune myelofibrosis

Miscellaneous Paget disease Vitamin D deficiency (Rickets) Renal osteodystrophy Hyperparathyroidism Hypoparathyroidism Systemic sclerosis Hereditary osteopetrosis Gray platelet syndrome


8 - Education


Because myelofibrosis is not specific to PMF, these other entities must be excluded before a definitive

diagnosis may be made. Marrow reticulin fibrosis is evaluated by staining with a silver stain, such as a

Jones or Gomori stain (Image 4 below).

Image 4.

Source: George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe’s Atlas of Clinical Hematology. Lippincott Williams and Wilkins, Inc; 2007:127. Reticulin staining of bone marrow. A normal bone marrow is shown at top left, with increasing

reticulin fibrosis shown in primary myelofibrosis.

The silver deposits on reticulin fibers. Late in the disease there may be formation of collagen fibrosis, which

is typically evaluated using a trichrome stain such as Masson’s trichrome, which stains collagen blue.

Fibrosis is scored using a semi-quantitative scoring system.

Diagnostic Criteria for PMF

The 2008 WHO Classification has established specific criteria for the diagnosis of PMF that utilize clinical,

pathologic and molecular data. They are as follows:

Diagnosis of PMF requires satisfying each of the 3 major and 2 of the minor criteria.

Major Criteria 1. Presence of megakaryocytic proliferation and atypia, usually accompanied with either reticulin or

collagen fibrosis

or

In the absence of fibrosis, the megakaryocytic proliferation must be accompanied by an increase in

marrow cellularity characterized by granulocytic proliferation and often decreased erythroid

elements

2. Not meeting the WHO criteria for PV, CML, MDS, or other myeloid neoplasms


9 - Education



10 - Education

3. Demonstration of JAK2 V617F or other clonal marker such as MPL W515K/L

or

In the absence of a clonal marker, no evidence that the bone marrow fibrosis or other changes are

secondary to infection, autoimmune disorder or other chronic inflammatory condition, hairy cell

leukemia, or other lymphoid neoplasm, metastatic malignancy, or toxic (chronic) myelopathies

Minor Criteria 1. Leukoerythroblastosis

2. Increase in serum lactate dehydrogenase

3. Anemia

4. Splenomegaly

Major criteria for diagnosis include identification of appropriate marrow findings of myelofibrosis,

megakaryocytic proliferation and/or hypercellularity, exclusion of other clonal myeloid neoplasms and

identification of a molecular marker such as JAK2 (50% of PMF patients) or MPL (5% of PMF patients).

Minor criteria include characteristic blood findings of leukoerythroblastosis (teardrop-shaped erythrocytes,

immature granulocytes, and nucleated red cells), elevated serum lactate dehydrogenase (LDH), anemia or

splenomegaly. Thus, work-up of a patient with suspected PMF should include a complete blood count with

morphologic examination of the blood smear, bone marrow biopsy with appropriate staining to demonstrate

reticulin and collagen fibrosis, blood analysis for LDH and appropriate molecular testing to detect JAK2 or

MPL mutations and to exclude other disorders, such as BCR-ABL1 testing to exclude CML. By using these

diagnostic criteria and by carefully excluding other causes of marrow fibrosis, a definitive diagnosis of PMF

may be made.

PMF is usually a progressive disorder with gradual transition between the prefibrotic and fibrotic stages

(Image 5. on the following page). This leads to the variable clinical and hematologic findings described

above. A small proportion of patients, estimated at 5-30%, may have increasing numbers of blasts in the

blood and marrow and eventually transform to AML.


Image 5.

fibrosis

Source: George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe’s Atlas of Clinical Hematology. Lippincott Williams and Wilkins, Inc; 2007:130.

The image at left shows the prefibrotic phase of primary myelofibrosis. The marrow

appears hypercellular with increased megakaryocytes, some of which are present in

clusters. The image at right shows cellular streaming typical of myelofibrosis and

represents the fibrotic phase of primary myelofibrosis.

PMF has a median survival of 5 years when diagnosed in the fibrotic stage, with most patients dying of

progressive bone marrow failure or infection. Older patients, patients with severe anemia, and patients with

increased myeloid immaturity or hypermetabolic symptoms appear to do more poorly. Treatment is limited.

Bone marrow transplants are curative, but the older age of most of the patients limits the use of this

approach. Chemotherapy, such as hydroxyurea, may be used to control splenomegaly, leukocytosis or

granulocytosis, and supportive transfusions or erythropoietin may be used to manage anemia. Splenectomy

may be considered in some patients with severe discomfort or hypersplenism. Specific JAK2 inhibitors are

under investigation, but no such targeted therapies are currently available.


11 - Education



12 - Education

References

1. Anastasi J, Vardiman JW. Primary myelofibrosis. In: Kjeldsberg CR, Perkins SL, eds. Practical Diagnosis

of Hematologic Disorders. 5th ed. Chicago, IL: American Society for Clinical Pathology Press;

2010:467-486.

2. George TI. Chronic myeloproliferative syndromes. In: Tkachuk D, Hirschmann JV, eds. Wintrobe’s Atlas

of Clinical Hematology. Philadelphia, PA: Lippincott Williams and Wilkins, Inc; 2007:105-136.

3. George TI. Pathology of the myeloproliferative diseases. In: Greer JP, Foerster J, Rodgers GM,

Paraskevas F, Glader B, Arber DA, Means RT, eds. Wintrobe’s Clinical Hematology. 12th ed.

Philadelphia, PA: Lippincott Williams and Wilkins, Inc; 2008:1988-2005.

4. Glassy EF, ed. Color Atlas of Hematology: An Illustrated Field Guide Based on Proficiency Testing.

Northfield, IL: College of American Pathologists; 1998.

5. Thiele J, Kvanicka HM, Tefferi A, Barosi G, Vardiman JW. Primary myelofibrosis. In: Swerdlow SH,

Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Vardiman JW, eds. WHO Classification of

Tumours of Haematopoietic and Lymphoid Tissues. Sterling, VA: Stylus Publishing, LLC; 2008:44-47.



13 - Education

Education Activity Authors

Tracy I. George, MD, FCAP: Tracy George. MD, is Director of Hematology for Stanford University

Medical Center Clinical Laboratories, which serves Stanford Hospital and Clinics and Lucile Salter Packard

Children’s Hospital. She is an assistant professor of Pathology at the Stanford University School of

Medicine in Stanford, CA. Dr. George has written over 70 papers, book chapters, books, educational

activities and abstracts in the areas of hematology, hematopathology and surgical pathology. She teaches

medical students, residents, and fellows, participates in clinical service work in hematopathology, and

performs translational research in the areas of myeloproliferative neoplasms and laboratory hematology. Dr.

George is currently Chair of the Hematology and Clinical Microscopy Resource Committee and a member of

the Council on Scientific Affairs for the College of American Pathologists (CAP).

Kyle T. Bradley, MD, MS, FCAP: Kyle T. Bradley, MD, is a Fellow in Anatomic Pathology at Vanderbilt

University Medical Center in Nashville, TN. He is board certified in anatomic pathology, clinical pathology,

and hematology by the American Board of Pathology. Dr. Bradley has authored several original articles,

book chapters, and abstracts in the fields of hematopathology and anatomic pathology and is a member of

the Hematology and Clinical Microscopy Resource Committee for the College of American Pathologists

(CAP).

Sherrie L. Perkins, MD, PhD, FCAP: Sherrie L. Perkins, MD, PhD, is a professor of Pathology at the

University of Utah Health Sciences Center and the Chief Medical Officer for ARUP Laboratories in Salt Lake

City, UT. She is the Director of Hematopathology for ARUP Laboratories and has responsibilities in

teaching, resident training, clinical service and research. Dr. Perkins has written over 140 peer-reviewed

papers and 70 book chapters in the areas of hematology and hematopathology. Dr. Perkins is currently a

member of the College of American Pathologists (CAP) Hematology and Clinical Microscopy Resource

Committee.

Documents

Blood Cell Identification: 2010-B Mailing: Primary Myelofibrosis · blood cells and resultant cytopenias (anemia, leukopenia or thrombocytopenia). ... (MPN) are clonal disorders of