Spleen

This case first appeared as Performance Improvement Program in Surgical Pathology (PIP) 2018, Case 33, and is primary myelofibrosis with extramedullary hematopoiesis in the spleen.

Sections show a spleen with an intact capsule. The red pulp is expanded and contains scattered megakaryocytes, clusters of erythroid cells, and clusters of myeloid cells. The erythroid cells have dark, smudged nuclei, are seen in small clusters, and are hemoglobin peroxidase positive. The myeloid cells have a more eosinophilic cytoplasm and can be difficult to see in some sections but are myeloperoxidase (MPO) and CD33 positive. The megakaryocytes express CD61. These findings are interpreted as extramedullary hematopoiesis (EMH). This evidence of splenic extramedullary hematopoiesis in a patient with a bone marrow showing reticulin fibrosis and a CALR mutation is diagnostic for primary myelofibrosis (PMF).

PMF, one of the chronic myeloproliferative neoplasms (MPN), is a clonal stem cell disorder which manifests as mature megakaryocytic and granulocytic proliferation that ultimately leads to fibrosis. In the early stages of the disease, the patient has variable anemia, thrombocytosis, and leukocytosis with minimal to absent bone marrow fibrosis. The bone marrow typically contains very pleomorphic/dysplastic megakaryocytes. However, in the later stages, the bone marrow shows marked reticulin fibrosis as well as osteosclerosis of bone, and the peripheral blood shows leukoerythroblastosis with nucleated red blood cells and precursor myeloid cells. Dacrocytes (tear drop cells) are also seen.

Molecular testing has become a requirement for myeloproliferative neoplasms and PMF shows JAK2 V617F mutation in ~60% of patients, CALR mutation in 25% of patients, and MPL mutation at codon 515 in 5% of cases. Only ~9% of cases are negative for JAK2, CALR, and MPL mutations. Survival is thought to be improved in CALR mutated cases. Studies have shown that cases with JAK2 mutation have an overall survival of 9.2 years; whereas cases with CALR mutation have an overall survival of 18 years. In contrast, cases without JAK2, CALR, and MPL mutations only have a three-year survival. Leukemic transformation is one of the poorest prognostic indicators for PMF and occurs in 4%-20% of patients. PMF has an incidence of 0.2/100,000 in the United States. It is usually diagnosed in adults with a median age diagnosis of 67 years. There is no significant sex predilection.

In this case, we have the spleen specimen to examine and only the report of the bone marrow biopsy and peripheral blood. The spleen shows extramedullary hematopoiesis, a common finding in the spleen of patients with MPN. EMH is not specific for PMF, but can be found in many diseases including myeloid neoplasms (myeloproliferative neoplasms, myelodysplastic/myeloproliferative neoplasms, acute myeloid leukemia), hemolytic anemia, and other reactive conditions such as idiopathic thrombocytopenic purpura, thrombotic thrombocytopenic purpura/hemolytic uremic syndrome, systemic infections, and bone marrow transplant.

By definition, EMH is hematopoiesis occurring in organs other than bone marrow. The hematopoietic precursors are not due to functional hematopoiesis but are rather due to trapping of progenitor cells and/or circulating maturing precursors from peripheral blood. On histology, we see erythroid cells, myeloid cells, and/or megakaryocytes. One, two, or all three hematopoietic cell lines may be present with predominant type depending on the associated condition. No dysplasia is seen in the three cell types, with the exception of myelodysplastic syndrome-associated EMH, in which dysplasia is present.

Patients with EMH typically present with splenomegaly showing diffuse involvement, but EMH can also produce a discrete mass. However, it is known that rare bone marrow elements can be found in most normal spleens, although no significant functional hematopoiesis occurs in human spleen. EMH is usually not a clinically significant finding; however, the patient may need to be treated for the underlying condition that was associated with the EMH. If the hematopoietic cells are difficult to identify, immunohistochemistry can be used to highlight each cell lineage. CD42b, factor VIII, and CD61 are positive in megakaryocytes. Myeloperoxidase, CD15, and lysozyme are positive in myeloid cells. Hemoglobin peroxidase is positive in erythroid cells. CD34 and CD117 can be helpful in assessing immature precursors.

In this case, the differential diagnosis includes other myeloproliferative neoplasms that can show similar molecular mutations such as essential thrombocythemia as well as other lesions which can have atypical cells that look similar to EMH, such as classical Hodgkin lymphoma, infectious mononucleosis, metastatic carcinoma, and T-cell/histiocyte rich large B cell lymphoma.

Essential thrombocythemia (ET) is a MPN which also may show JAK2 (60% of cases), MPL (<5% of cases), and CALR (25% of cases) mutations, as well as EMH. ET typically shows a striking megakaryocytic proliferation with increased numbers of loosely clustered megakaryocytes, many of which are markedly enlarged and hyperlobated. The megakaryocytes in ET do not show the dysplastic features of those in PMF. In contrast to PMF, there is either no or very minimal fibrosis. The peripheral blood does not show leukoerythroblastosis or prominent dacrocytes. ET is important to distinguish from PMF because it has a much more favorable prognosis with less transformation to acute leukemia.

Acute myeloid leukemia (AML) with extramedullary hematopoiesis could also be a consideration as a diagnosis in this case. It is known that some cases of AML can have JAK2, CALR, or MPL mutations. However, no blasts are identified in the spleen and none are reported in the bone marrow; moreover, immature markers CD34 and CD117 do not show any increased immature cells.

In classical Hodgkin lymphoma, the Reed-Sternberg (RS) cells may resemble EMH megakaryocytes. However, RS cells are CD30(+), CD15(+/-), PAX5(+), MUM1(+), CD45(-), BOB.1(-), and negative for megakaryocytic markers (CD42b, CD61). Therefore, the phenotype of the large atypical cells in this case is that of megakaryocytes and not Reed Sternberg cells.

EMH can also be seen in association with metastatic carcinoma; however, the lack of cytokeratin immunostaining on the spleen effectively excludes a diagnosis of carcinoma.

1. Which of the following panels of immunohistochemical stains is most consistent with classical Hodgkin lymphoma?
   
   
   1. CD15-, PAX5-, CD30-, CD45-, BOB1-
   2. CD15-, PAX5+, CD30-, CD45+, BOB1+
   3. CD15+, PAX5+, CD30+, CD45-, BOB1-
   4. CD15+, PAX5-, CD30+, CD45-, BOB1+
   5. CD15+, PAX5-, CD30+, CD45+, BOB1-
2. Which of the following findings would favor a diagnosis of essential thrombocythemia over primary myelofibrosis?
   
   
   1. Dacrocytes
   2. Enlarged hyperlobated megakaryocytes
   3. Leukoerythroblastosis
   4. Osteosclerosis of bone
   5. Reticulin fibrosis
3. Which of the following statements is correct?
   
   
   1. CALR mutations are specific for primary myelofibrosis.
   2. CD61, MPO, and hemoglobin peroxidase can be helpful to identify extramedullary hematopoiesis.
   3. Extramedullary hematopoiesis is only found in primary myelofibrosis.
   4. Leukemic transformation is a favorable prognostic indicator in primary myelofibrosis.
   5. Molecular testing is not required for primary myelofibrosis.

1. Chabot-Richards J, Reichard K, Vasef M, Auerbach A. Diagnostic Pathology: Molecular Oncology. 1st ed. Salt Lake City, UT: Elsevier; 2016:2:86-91.
2. Foucar K. Diagnostic Pathology: Blood and Bone marrow. 2nd. Ed Salt Lake City, UT Amirsys Inc. 2016: 2:34-38.
3. Miyata T, Masuzawa M, Katsuoka K, Higashihara M. Cutaneous extramedullary hematopoiesis in a patient with idiopathic myelofibrosis. J Dermatol. 2008;35(7):456-461.
4. Haniffa MA, Wilkins BS, Blasdale C, Simpson NB. Cutaneous extramedullary hematopoiesis in chronic myeloproliferative and myelodysplastic disorders. J Am Acad Dermatol. 2006;55(2 Suppl):S28-31.
5. Vos J, Auerbach A. Diagnostic Pathology: Spleen. 1st ed. Salt Lake City, UT: Amirsys Inc; 2014:2:86-91.

Aaron Auerbach, MD MPH
Surgical Pathology Committee
Joint Pathology Center
Silver Spring, MD

1. CD15+, PAX5+, CD30+, CD45-, BOB1- (c)
2. Enlarged hyperlobated megakaryocytes (b)
3. CD61, MPO, and hemoglobin peroxidase can be helpful to identify extramedullary hematopoiesis. (b)