Hematopathology Case Study: The Case of Lymphocytosis

Case History

53 year old female was found to have leukocytosis upon a wellness examination. A CBC was performed and found a WBC of 34.0, HgB 13.2, and Plt of 263,000. The WBC differential consisted of 80% Lymphocytes and 12% neutrophils. The patient states that she is feeling well, no fever, chills, or night sweats. She denies any adenopathy. Flow Cytometry was recommended as well as morphologic review along with Cytogenetics and FISH (fluorescence in-situ hybridization).

Lab Identification

CBC

WBC               33.68  [103/uL] NEUT             4.17 [103/uL]      12.3 [%]
RBC                4.54  [106/uL] LYMPH          26.91 [103/uL]    79.9 [%]
HGB               13.2   [g/dL] MONO           2.04 [103/uL]      6.1  [%]
HCT               40.0   [%] EO                  0.39 [103/uL]      1.2  [%]
MCV               88.1   [fL] BASO             0.11 [103/uL]      0.2  [%]
MCH               29.1   [pg] IG                    0.06 [103/uL]      0.2  [%]
MCHC            33.0   [g/dL] NRBC             0.00 [103/uL]      0.0  [%]
RDW-CV         14.3   [%]
PLT                263   [103/uL]
MPV                9.4   [fL]

Morphologic Review

CLL1.jpg
Image 1. 40x magnification showing lymphocytosis.
CLL2.jpg
Image 2. 60x magnification showing lymphocytosis with occasional smudge cells.

Flow Cytometry: Population of Interest – Abnormal Lymphocytes

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Cytogenetics: 46,XX[20] Normal Female Karyotype

It is not unusual to observe a normal karyotype in CLL due to the limited number of abnormal cells and/or low spontaneous proliferative activity of the malignant cells. Fluorescence in situ hybridization studies may identify cytogenetic abnormalities of prognostic significance in interphase nuclei not observed in the metaphase cells analyzed. In cases of CLL molecular profiling may be performed to aid in predicting course of the disease. If clinically indicated these studies may be considered. Standard cytogenetic analysis may not detect subtle submicroscopic rearrangements and may not include metaphases from abnormal cell populations with low mitotic rates or present in low levels.

Metaphases Counted: 20 Metaphases Analyzed: 20
Metaphases Karyotyped: 2 Culture Type: 48EB, 72IL2/DSP30
Banding Technique: GTG Banding Resolution: 400

Fluorescence in-situ Hybridization: Abnormal – 13q14 deletion present

Fluorescence in situ hybridization (FISH) analysis was performed using a specific set of probes for Chronic Lymphocytic Leukemia (CLL). This study revealed a 13q14 deletion. Counts for all other probes were within the normal reference range. This finding represents an ABNORMAL result. Deletion of 13q14 is the most common deletion in CLL being reported in 10-20% of cases by conventional cytogenetics and up to 64% of cases by FISH analysis. When present as a sole abnormality this deletion is associated with a good prognosis and a median survival longer than CLL patients with a normal karyotype.

Del(6q) Not Detected
Del(11q)(ATM) Not Detected
Trisomy 12 Not Detected
Del(13q)/-13 DETECTED
t(11;14) Not Detected
Del(17p)(TP53) Not Detected

Discussion

Chronic Lymphocytic Leukemia is a neoplasm of about 5 cases per 100,000 people with a median age of around 70 years old. The neoplasm is composed of monotypic mature B-cells that typical express CD5. Other immunophenotypic characteristics of the leukemic B-cells include CD19, CD20, CD22, and CD79b with dim surface expression of one of the immunoglobulin light chains, Kappa or Lambda. These cells typically express CD200 which helps differentiate the leukemia from Mantle Cell Lymphoma/Leukemia. Patients found to have a mutated IGHV genes typically have a better prognosis than those with an unmutated genes. Expression of ZAP70, CD38, or CD49d is also associated with an adverse prognosis. Complex karyotypes also trend towards a poor outcome. Adverse predictive factors include rapid lymphocyte proliferations in the blood, typically doubling in < 12 months.

Monoclonal B-cell Lymphocytosis is typically characterized as a monoclonal
BCell count of <5 X109/L in the peripheral blood. Monoclonal B-cell Lymphocytosis with a Chronic Lymphocytic Leukemia-type phenotype is the most common which accounts for about 75% of all cases. It has been reported that virtually all Chronic Lymphocytic Leukemias are preceded by Monoclonal B-cell Lymphocytosis, although not all MBLs progress to CLL.

References

  1. Dohner H, et al. N Engl J Med 2000; 343:1910-6.
  2. Hamblin TJ.Best Practice & Research Clinical Haematology. 2007; 20(3):455 – 68.
  3. Swerdlow SH, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H. WHO classification of tumours of haematopoietic and lymphoid tissues, fourth edition. Lyon, France: IARC; 2017
  4. Nowakowski GS, et al. Br J Hematol. 2005; 130:36 – 42.
  5. Atlas of Genetics and Cytogenetics in Oncology and Hematology http://atlasgeneticsoncology.org/

 

Troy-Krieger-small

Troy G. Krieger, MS, MLS(ASCP)CMSCYMCMQLSCMCLS(MT) graduated from Montana State University Billings with a BS in Biology, Medical Laboratory Science option. He received a NAACLS Certificate and clinical training from the University of North Dakota in Grand Forks, ND, where he also received his Master’s degree. He is a Medical Laboratory Scientist / Flow Cytometrist at Yellowstone Pathology Institute, Inc in Billings, MT and his interests include Hematology, Immunopathology, and Flow Cytometry.

Hematopathology Case Study: A 43 Year Old Man with History of Latent TB

Case History

43 year old Vietnamese speaking man with a history of treated latent TB who presented with one month of fevers, night sweats, weight loss, and acute left facial swelling with associated pain, nasal congestion and 2 nose bleeds. The patient was found to have a polypoid mass within the left interior nasal cavity.

Biopsy Left Nasal Mass

nkt-he10x
H&E 10x
nkt-he40x
H&E 40x
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CD3
nkt-cd5
CD5
nkt-cd56
CD56
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Perforin
nkt-67
KI-67
nkt-eber.jpg
EBER

Diagnosis

The biopsy shows nasal mucosa with a dense submucosal lymphoid infiltrate and large areas of necrosis. The lymphocytes are somewhat pleomorphic, medium to large in size with irregular nuclear contours, vesicular chromatin and inconspicuous nucleoli. There are scattered mitoses and apoptotic cells.

By immunohistochemistry, CD3 highlights the lymphoma cells, which comprise the majority of the lymphoid infiltrate. The lymphoma cells co-express CD56 and CD7 (dim) and are negative for CD2 and CD5. The lymphoma cells also express cytotoxic markers perforin and granzyme (major subset). CD20 highlights only rare small clusters of B-cells. The lymphoma cells are also positive for EBER (Epstein-Barr Virus encoded RNA) in situ hybridization.  The Ki67 (MIB1) proliferation index is 60% with focal areas exhibiting up to 80%.

Taken together, the morphologic and immunophenotypic findings are consistent with an extranodal NK/T cell lymphoma, nasal type.

Discussion

Extranodal NK/T cell lymphoma, nasal type is an aggressive lymphoma that is more prevalent in Asian and South American populations. It occurs most often in adults and is more common in men than women. It is generally located in the upper aerodigstive tract, with the nasal cavity being the prototypical site. Patients tend to present in a manner similar to the patient described in this case, with rhinorrhea, pain, nasal obstruction and epistaxis due to a mass lesion. 1 The term “lethal midline granuloma” was once used to describe this entity because patients can present with locally destructive mid-facial necrotizing lesions. The early non-specific symptoms can pose a diagnostic challenge, and often result in treatment delays, which makes the aggressive disease more lethal. 2

The entity is described as NK/T cell lymphoma because although most cases are of NK-cell origin, some cases are comprised of cytotoxic T-cells. Natural killer (NK) cells are non-T and non-B lymphocytes that are part of the innate immune system. They respond immediately to antigenic challenge and are able to directly kill virally infected cells without the help of antigen presenting cells. They also secrete cytokines to increase the innate immune response. NK cells are classically positive for cytoplasmic CD3 and CD56, as well as cytotoxic molecules granzyme and perforin. Of note, NK-cells lack recombination activating gene enzymes and therefore have no clonal molecular marker for gene rearrangement such as the T-cell receptor or Immunoglobulin heavy chain.  3

Microscopically, the involved sites generally have widespread mucosal destruction. There is an angioentric and angiodestructive growth pattern that results in extensive necrosis. Another important diagnostic consideration is the very strong association with EBV. EBV is present in a clonal episomal form. This means that the infection occurs prior to and likely plays a pathogenic role in the development of NK/T cell lymphomas. 3

Following diagnosis, staging and management of the disease involves quantification of circulating EBV DNA. This can be used as a laboratory marker for disease status and progression or remission of disease. PET/CT is performed for accurate staging and patients are most commonly treated with a combination of radiotherapy and the SMILE regimen, which includes dexamethasone, methotrexate, ifosfamide, L-asparaginase and etoposide. NK/T cell lymphomas are aggressive and patients tend to have a short survival and poor overall response to therapy. 3

A recent study by Kwong, et al. showed the potential use of PD1 (programmed death ligand 1) blockade drug pembrolizumab in the treatment of relapsed or refractory NK/T-cell lymphoma. As mentioned above, the lymphocytes in this entity are invariably infected with EBV. PD1 is known to be up regulated in cells infected with EBV. In the study, seven patients who had failed treatment with the SMILE regimen were treated with pembrolizumab. After a medium follow-up of 5 months, 5 patients remained in complete remission and all patients had objective responses to treatment. 4 This shows promise as a potential new treatment for patients with this uncommon, but deadly disease.

 

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Mallya V, Singh A, Pahwa M. Lethal midline granuloma. Indian Dermatology Online Journal. 2013;4(1):37-39. doi:10.4103/2229-5178.105469.
  3. Tse, E, Kwong, Yok-Lam. The diagnosis and management of NK/T-cell lymphomas. Journal of Hematology and Oncology. 2017:10:85. Doi: 10.1186/s13045-017-0452-9.
  4. Yok-Lam, K, Thomas, S.Y. Chan, Daryl Tan, et. al. PD1 blockade with pembrolizumab is highly effective in relapsed or refractory NK/T-cell lymphoma failing L-asparaignase. Blood. 2017:129:2437-2422. Doi: 10/1182/blood-2016-12-758641.

 

Marcus, Chelsea_099-Edit

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: A 48 Year Old Woman with Left Upper Quadrant Pain

Case History

A 48-year-old female presents with a one-month history of left upper quadrant pain. Laboratory investigation reveals pancytopenia. Radiology work-up demonstrates splenomegaly. CT scan confirms splenomegaly at 22 cm. There is no lymphadenopathy appreciated in the abdomen. A bone marrow biopsy is performed.

Figure HSTCL
Image 1. H&E and CD3 stains at varying magnification.

Bone Marrow Findings

The bone marrow core biopsy reveals a normocellular marrow space (approximately 50% cellular marrow) with progressive trilineage hematopoiesis. Clusters of small, slightly irregular, mature-appearing lymphocytes are seen within the sinusoids. The marrow aspirate smears reveal mild erythroid hyperplasia without morphologic evidence of dysplasia. There is no increase in blasts. Lymphocytes comprise 18% of a 500-cell differential count on the marrow aspirate smears.

The sinusoidally distributed lymphocytes demonstrate immunopositivity (flow and/or IHC) for CD2, CD3, CD7, CD16, CD56, and γδ. These neoplastic lymphocytes are negative for granzyme B, CD4, CD5, CD8, CD57, and αβ.  PCR for T-cell receptor clonality was positive. Cytogenetics revealed a normal female karyotype. FISH for 5p/5q and 7p11/7q31 was normal.

Diagnosis

Taken together, the patient’s clinical presentation along with the presence of an abnormal gamma-delta population of T cells in a sinusoidal distribution with PCR evidence of T-cell clonality is diagnostic of a T-cell lymphoma. The pattern of distribution, granzyme B negativity, lack of concurrent adenopathy favor a diagnosis of Hepatosplenic T-cell lymphoma.

Discussion

Hepatosplenic T-cell lymphoma (HSTCL) is an uncommon entity that represents <1% of all non-Hodgkin lymphomas and 1%-2% of all T/natural killer cell lymphomas. It most commonly affects young adult men, with a median age of 35 years. This high-grade malignancy is most often characterized by γδ T-cells. The most consistent symptoms among patients are fever, splenomegaly, hepatomegaly, bone marrow involvement, peripheral blood cytopenia, and less commonly, adenopathy. Hepatosplenic T-cell lymphoma has a poor prognosis with median survival rates varying from a few months to 16 months in different studies.

Immune suppression (such as solid- organ transplant, or immune dysregulation secondary to malignancy or infection) is thought to play a role in the lymphomagenesis in around 20% of cases. Inflammatory bowel disease and the use of immunosuppressive agents (e.g., antitumor necrosis- α agents) and antimetabolite therapy (e.g., 6TG, 6MP) had also been associated with development of HSTCL.

HSTCL initially infiltrates the cords and sinusoids of the splenic red pulp. The white pulp is often atrophic or absent. Eventually, the neoplastic T cells diffusely replace the spleen. The lymphoma cells often involve the liver and bone marrow sinusoids. At the time of diagnosis, the bone marrow is almost always involved and commonly hypercellular. The neoplastic cells are mostly intermediate in size, with pale agranular cytoplasm and round nuclei with condensed chromatin and inconspicuous nucleoli.

Cytogenetic studies in HSTCL most commonly show isochromosome 7q and trisomy 8. Molecular analysis of HSTCL characteristically shows expression of a γδ T-cell type and flow cytometric analysis typically reveals a CD2+, CD3+, CD7+/−, CD4−, CD5−, and CD8− phenotype with positivity for natural killer cell-associated markers CD11b, CD16, and CD56.

Activating mutations in PI3KCD and STAT signaling genes have also been described in HSTCL, providing potential molecular target therapies for this aggressive lymphoma.

The differential diagnosis of HSTCL includes other types of T-cell lymphoma and leukemia, and non-neoplastic such as immune thrombocytopenia or acute hepatitis. In most instances, the distinctive presentation of spleen, liver and bone marrow involvement, the immunophenotype and T-cell monoclonality distinguishes HSTCL from other entities.

The outcomes of the patients using standard chemotherapy regimens are dismal, and allogeneic SCT appears to be a reasonable approach to achieve the best possible patient outcome.

References

  1. Yabe M, Miranda RN, Medeiros LJ. Hepatosplenic T-cell Lymphoma: a review of clinicopathologic features, pathogenesis, and prognostic factors. Hum Pathol. 2018 Apr; 74:5-16.
  2. McThenia SS, Rawwas J, Oliveira JL, Khan SP, Rodriguez V. Hepatosplenic γδ T-cell lymphoma of two adolescents: Case report and retrospective literature review in children, adolescents, and young adults. Pediatr Transplant. 2018 Aug;22(5): e13213.

 

levent photo

-Levent Trabzonlu, MD is a postdoctoral researcher in the department of pathology at Johns Hopkins University in Baltimore, MD. Follow Dr. Trabzonlu on twitter @aflevent

Mirza-small

-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: A 55 Year Old Woman with Fatigue, Nausea, and Vomiting

Case History

55 year old woman with no significant past medical history presented with two weeks of increasing fatigue, nausea and vomiting. She was subsequently found to have a leukocytosis (WBC = 31.1) and marked splenomegaly (19 cm).

Peripheral Blood

mcl-peri-1

mcl-peri-2

 

Bone Marrow Biopsy 

mcl-bm-20x
aspirate 20X
mcl-bm-40
aspirate 40X
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core biopsy 10X
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core biopsy 40X

 

mcl-core-cd20
core biopsy CD20
mcl-core-bcl1
core biopsy BCL1 (CCND1)

 

Flow Cytometry

mcl-flow

 

Cytogenetics

mcl-cyto-kary
Karyotype
mcl-cyto-IGH
IGH/CCND1 gene rearrangement
mcl-cyto-deletion
deletion of TP53
mcl-cyto-MYC
MYC amplification

 

Diagnosis

The peripheral blood shows a population of atypical cells that at first may look like blasts. However, the variable size, round to markedly irregular nuclear contours, large prominent nucleoli and mild to moderate amounts of cytoplasm favor lymphoma cells.

The bone marrow aspirate shows the vast majority of the cellularity is composed of a pleomorphic population of lymphoma cells that are varied in size from small to large with mild to moderate cytoplasm, round to irregular nuclear contours and prominent nucleoli. Occasional maturing erythroid and myeloid precursors are present.

The core biopsy shows a marrow with a cellularity of approximately 70%. There is an interstitial infiltrate of atypical mononuclear cells with frequent scattered mitoses occupying 70% of the overall cellularity. By immunohistochemistry performed on the core biopsy, B-cell marker CD20 highlights the majority of the infiltrating lymphocytes, which co-express BCL1 (CCND1).

Flow cytometry revealed a population of CD19 and CD20 positive kappa (bright) restricted B-cells that were also positive for CD23 in a subset. They did not express any other characteristic antigens including CD5, CD10 and CD11c. Importantly, as there was initial concern for acute myeloid or lymphoblastic leukemia, no abnormal events were identified in the CD45 dim “blast” gate, with CD34 positive blasts showing normal maturation.

Cytogenetics revealed a complex abnormal karyotype. The most important finding was a translocation involving the long arms of chromosome 11 and 14 resulting in the IGH/CCND1 translocation that is characteristic of mantle cell lymphoma. Interestingly, the FISH probe showed that there were 4 IGH/CCND1 fusions indicating an extra copy of the derivative chromosome 14. Additional FISH probes showed a deletion of the TP53 gene on 17p13 and greater than 10 copies of the MYC gene on chromosome 8, consistent with MYC amplification.

Overall, the findings are consistent with a pleomorphic variant of mantle cell lymphoma with leukemic peripheral blood involvement. The cytogenetic findings portend an unfavorable prognosis.

Discussion

Mantle cell lymphoma is generally characterized as an aggressive lymphoma of mature B-cells. It accounts for approximately 3-10% of non-Hodgkin lymphomas and tends to occur in older men. Lymph nodes are the most commonly involved site; however the bone marrow and peripheral blood are frequently involved as well. There are multiple morphologic variants of mantle cell lymphoma. The two aggressive variants include blastoid and pleomorphic. The blastoid variant has cells that resemble lymphoblasts with dispersed chromatin and large prominent nucleoli. The pleomorphic variant is characterized by a spectrum of cells, with many large cells with irregular nuclear contours, pale cytoplasm and variably prominent nucleoli. These two variants are clinically significant because they portend a worse prognosis. 1

The patient’s cytogenetic findings also portend a poor prognosis. IGH/CCND1 is a translocation between the immunoglobulin heavy chain on chromosome 14 and cyclin D1 on chromosome 11. This translocation leads to the overexpression of cyclin D1. However, Cyclin D1 is a “weak” oncogene and is not sufficient by itself to lead to the development of lymphoma. There are numerous secondary chromosomal aberrations and mutations that must occur to result in the presentation of mantle cell lymphoma. A paper by Beà et al. performed whole genome and/or whole exome sequencing on 29 cases of mantle cell lymphoma. They detected around 3,700 somatic mutations per tumor. ATM, CCND1 and TP53, which have previously been described as drivers in mantle cell lymphoma, were found frequently mutated. TP53 mutations were found in 28% of the lymphomas.2

MYC is a potent proto-oncogene located on chromosome 8. It mainly functions as a transcription factor and its activation leads to increased DNA replication, protein synthesis and alterations in cell metabolism among many other changes. The ultimate effect is increased cell proliferation and tumorigenesis. MYC amplification or translocation was shown to occur more often in blastoid/pleomorphic mantle cell lymphoma variants. This finding was associated with a shortened overall survival and progression-free survival. 3

References

  1. Swerdlow, Steven H. WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues. Revised 4th ed., International Agency for Research on Cancer, 2017.
  2. Beà S, Valdés-Mas R, Navarro A, et al. Landscape of somatic mutations and clonal evolution in mantle cell lymphoma. Proceedings of the National Academy of Sciences of the United States of America. 2013;110(45):18250-18255. doi:10.1073/pnas.1314608110.
  3. Choe, JY, Yun, JY, Na, HY, et al. MYC overexpression correlates with MYC amplification or translocation, and is associated with poor prognosis in mantle cell lymphoma. 2016 Feb;68(3):442-9. doi: 10.1111/his.12760. Epub 2015 Jul 28.

 

Marcus, Chelsea_099-Edit

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: An 85 Year Old Man with Pancytopenia

Case History

An 85 year old man presented with pancytopenia and weakness. His labs include WBC of 3.2, HgB of 9.9 and platelets of 137.

Bone Marrow Biopsy

hairycellbm10x
Bone Marrow Aspirate, 10x
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Bone Marrow Aspirate, 40x
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Core Biopsy, 10x
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Core Biopsy, 40x

Flow Cytometry

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hairycellplasmacell

hairycellplasmacellgate

Diagnosis

The bone marrow aspirate shows multiple cellular spicules with a prominent population of lymphoid cells with oval to reniform nuclei, dispersed chromatin and abundant pale cytoplasm. Scattered plasma cells are also present.

The core biopsy shows an infiltrating population of atypical lymphocytes with moderate amounts of pale eosinophilic cytoplasm and mature chromatin that stain positive for CD20. Frequent mononuclear cells consistent with plasma cells are also seen scattered throughout the bone marrow and stain positive for CD138.

Flow cytometry revealed that 80% of the lymphoid gate represented a kappa light chain restricted population that co-expressed B-cell markers CD19, CD20 and CD22 along with classic hairy cell leukemia specific markers CD11c, CD25 and CD103. A second population of kappa restricted cells fell in the plasma cell gate. The cells co-expressed CD138, CD56 and were largely negative for CD19 and CD20.

Overall, there is a hypercellular bone marrow with a prominent mononuclear lymphoid infiltrate consistent with hairy cell leukemia and a concurrent population of plasma cells consistent with plasma cell neoplasm.

Discussion

Hairy cell leukemia is a rare lymphoid neoplasm that accounts for only 2% of lymphoid leukemias. Patients tend to be in their 50s-60s with a 4:1 male predominance. The tumor is generally found in the bone marrow and spleen with rare circulating cells in the peripheral blood. Patients are generally cytopenic at presentation and symptoms include weakness and fatigue. Splenomegaly is common and hepatomegaly can also be seen.. 1

Hairy cell leukemia involves the clonal expansion of B-cells with a unique immunophenotypic profile. They are bright for CD19, CD20, CD22 and CD200, negative or dim for CD5, CD23 and CD10 and positive for CD11c, CD103, CD123 and CD25. Hairy cell leukemia must be distinguished from two provisional entities, hairy cell leukemia-variant and splenic diffuse red pulp lymphoma. These two entities do not have the classic morphology or staining profile of hairy cell leukemia.2

BRAF V600E mutations are detected in more than 80% of cases of classic hairy cell leukemia. The mutation is considered to be a driver mutation, but additional mutations are usually present that lead to disease progression. Hairy cell leukemia-variant is usually negative for BRAF mutations and has a more aggressive clinical course.3

Patients with hairy cell leukemia are given purine analogues as first line treatment and generally do well. However, patients who do not respond or who undergo relapse have few options. Increasingly, BRAF V600E inhibitors are being used for patients with hairy cell leukemia. Multiple studies have now confirmed the efficacy of vemurafenib and dabrafenib, however patients can be quick to relapse once off the drugs. Combination approaches should be considered for the most effective treatment. 4

References

  1. Swerdlow SH, Campo E, Harris NL, et al. WHO Classification of Tumours of Haematopoetic and Lymphoid Tissues (Revised 4th edition). IARC: Lyon 2017.
  2. Troussard X, Cornet E. Hairy cell leukemia 2018: Update on diagnosis, risk‐stratification, and treatment. American Journal of Hematology. 2017;92(12):1382-1390. doi:10.1002/ajh.24936.
  3. Maitre E, Bertrand P, Maingonnat C, et al. New generation sequencing of targeted genes in the classical and the variant form of hairy cell leukemia highlights mutations in epigenetic regulation genes. Oncotarget. 2018;9(48):28866-28876. doi:10.18632/oncotarget.25601.
  4. Roider T, Falini B, Dietrich S. Recent advances in understanding and managing hairy cell leukemia. F1000Research. 2018;7:F1000 Faculty Rev-509. doi:10.12688/f1000research.13265.1.

 

Marcus, Chelsea_099-Edit

Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.

Hematopathology Case Study: A 67 Year Old Female with a Sore Throat

History 

A 67 year old female presents with a two-month history of sore throat. She endorses dysphagia and left-sided otalgia but denies voice changes, shortness of breath, hemoptysis, weight loss, fever or night sweats. She has smoked 1 pack/day for 41 years and occasionally drinks alcohol. Her past medical history is notable for systemic lupus erythematosus for which she takes Plaquenil.

Physical examination slightly elevated systolic blood pressure. She is afebrile. Pertinent neck exam findings include mild tonsillar asymmetry (left slightly larger than right), and a firm mass at left base of tongue, and a 3 cm lymph node in the neck (left level III). A biopsy sample was taken from the tongue mass. 

Biopsy

EBV-1

H&E stained sections reveal sheets of large lymphocytes. The lymphoid cells are medium to large in size with irregular nuclear contours and prominent nuclei. Areas of necrosis are prominent. No specific areas of epithelial ulceration are noted. Immunophenotypic characterization of the larger cells reveals positivity for CD20, CD30, CD79a, PAX5, MUM1, Epstein Barr virus encoded RNA (EBER) and a variable Ki-67 proliferation index, which is up to 60-70% in the larger cells, but around 20-30% overall. Only rare cells are positive for BCL-2 and BCL-6. The lymphoma cells are negative for keratin AE1/AE3, CD10, CD4, CD8, CD21, CD23, CD7, CD5, Cyclin D1, CD68, CD56, and CD43. The background T cells express CD5 and CD7 and are a mixture of CD4 and CD8 with CD4 predominance.

We considered the diagnosis of EBV-positive mucocutaneous ulcer (a more indolent entity); however, the lack of history of an ulcer/ulceration and the presence of a mass-lesion (with additional adenopathy) does not support this diagnosis.

The findings are most consistent with EBV-positive DLBCL, NOS (WHO 2017), previously known as EBV positive DLBCL of the elderly (WHO 2008). 

Discussion 

Epstein Barr Virus, a member of the Herpesviridae family is mostly known for causing Infectious Mononucleosis. However, the ubiquitous virus which is present in about 90% of adults but often asymptomatic1, has a predilection for epithelial cells including B-cells.2 Incorporation of the viral genome and viral takeover of the cells proliferative machinery underlies the pathogenesis of any EBV-related disease/malignancy. It has been associated with a gastric carcinoma, fulminant hepatitis, undifferentiated nasopharyngeal carcinoma, and B cell, T cell and NK cell lymphomas3, including EBV+ diffuse large B-cell lymphoma, not otherwise specified (DLBCL-NOS).

EBV-positive diffuse large B-cell lymphoma, not otherwise specified (EBV+ DLBCL-NOS) was formerly known as EBV-positive diffuse large B-cell lymphoma (DLBCL) of the elderly. The WHO classification substituted “not otherwise specified” in place of “for the elderly” to reflect two things: 1) EBV is associated with other specific neoplastic Large B-Cell diseases such as lymphomatoid granulomatosis, and 2) EBV+DLBCL can affect younger individuals as well as the elderly. 2

EBV+DLBCL-NOS patients may occur in nodal or extranodal sites, with up to 40% presenting with extranodal sites at least in the early stages. Patients may be asymptomatic with or without B symptoms but usually, patients present with rapidly enlarging tumors at single or multinodal sites, as well as at extranodal sites. 4

The patient’s presentation with sore throat and the finding of neck mass with EBV-positive large B-cells associated with ulcer-like necrosis raises a differential diagnosis that ranges from reactive to malignant. Table 1 shows a comparison between three differential diagnoses: EBV+DLBCL-NOS; EBV-positive mucocutaneous ulcer; and infectious mononucleosis.

EVB-t1
Table 1. Comparison of 3 EBV-positive differentials in the head and neck

Unfortunately, there is currently no uniformly agreed standard of treatment for EBV+DLBCL which has a worse prognosis than EBV negative DLBCL.2 The standard treatment for DLBCL (rituximab, cyclophosphamide, doxorubicin, vincristine, and prednisone- R-CHOP) is used but it responds poorly to treatment, with a median survival of 2 years.

Therefore, early detection by clinical suspicion and testing all DLBCL patients for EBV is very important.2 

 References

  1. Tsuchiya S. Diagnosis of Epstein–Barr virus-associated diseases. Critical Reviews in Oncology and Hematology. 2002;44(3):227-238. https://www.sciencedirect.com/science/article/pii/S1040842802001142. doi: 10.1016/S1040-8428(02)00114-2.
  2. Murthy SL, Hitchcock MA, Endicott-Yazdani T, Watson JT, Krause JR. Epstein-barr virus–positive diffuse large B-cell lymphoma. Proceedings (Baylor University.Medical Center). 2017;30(4):443-444. http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5595389/.
  3. Okano, Motohiko, MD, PhD|Gross, Thomas G., MD, PhD. Acute or chronic life-threatening diseases associated with epstein-barr virus infection. American Journal of the Medical Sciences, The. 2012;343(6):483-489. https://www.clinicalkey.es/playcontent/1-s2.0-S0002962915309435. doi: 10.1097/MAJ.0b013e318236e02d.
  4. Swerdlow S, Campo E, Harris NL, Jaffe ES, Pileri SA, Stein H, Thiele J, Arber D, Hasserjian R, Le Beau M. WHO classification of tumours of haematopoietic and lymphoid tissues. 2017.
  5. Dunmire SK, Hogquist KA, Balfour HH. Infectious Mononucleosis. Current topics in microbiology and immunology. 2015;390:211-240. doi:10.1007/978-3-319-22822-8_9.

 

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-Adesola Akinyemi, M.D., MPH, recently earned his MPH-Health Policy and Management from New York Medical College. He plans on pursuing residency training in pathology. His interests include cytopathology, neuropathology, and health outcomes improvement through systems thinking and design.

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-Kamran M. Mirza, MD PhD is an Assistant Professor of Pathology and Medical Director of Molecular Pathology at Loyola University Medical Center. He was a top 5 honoree in ASCP’s Forty Under 40 2017. Follow Dr. Mirza on twitter @kmirza.

Hematopathology Case Study: Two Cases with Surprising Hematopoetic Elements

Case 1 History

Sixty-one year old man with new diagnosis of Bud-Chiari syndrome and extensive peripheral, splenic and hepatic venous thrombosis with increasing fatigue, abdominal discomfort and abnormal liver function tests. A liver biopsy was performed and a hypercoagulability work-up, including JAK2 mutation analysis was initiated.

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Liver core biopsy 2X
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Liver core biopsy 10X

Diagnosis

The liver biopsy showed extensive hemorrhage, hepatocellular necrosis and collapse with mild portal and lobular mixed inflammation. Occasional megakaryocytes and nucleated red blood cell precursors were noted. The case was sent to hematopathology for further review.

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Liver core biopsy 40X
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Liver core biopsy 40X
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JAK2 Mutations Analysis

Hematopathology Diagnosis

Sections show liver parenchyma with changes of the patient’s known history of venous outflow obstruction, as well as extramedullary hematopoiesis, including scattered megakaryocytes (arrows) and erythroid precursors (circle). In the setting of a positive JAK2 V617F mutation, this constellation of findings is consistent with a myeloproliferative neoplasm.

Case 2 History

Fifty-nine year old man with a history of hypertension and alcohol abuse with posterior mediastinal lymphadenopathy. Recent bone marrow biopsy showed mildly hypercellular bone marrow with megakaryocytic and myeloid hyperplasia, and increased stromal reticulin with concern for primary myelofibrosis. A lymph node biopsy was performed.

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Lymph node biopsy 10X
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Lymph node biopsy 40X
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Myeloperoxidase
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CD71
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CD61
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CD34
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CD3
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CD20

Diagnosis

The lymph node biopsy shows fragments composed of adipocytes and maturing trilineage hematopoiesis. Multiple small to medium sized lymphoid aggregates are also seen, composed of small and mature appearing lymphocytes. The lymphocytes are a mixture of CD3 positive T cells and CD20 positive B cells with focal B cell predominance. Myeloperoxidase highlights myeloid precursors, which comprise 70-80% of the cellularity. CD71 highlights erythroid precursors, which comprise 20-30% of the cellularity. CD61 highlights megakaryocytes. CD34 highlights vessels and only rare CD34-positive cells are seen. Taken together, the findings are consistent with extramedullary hematopoiesis.

Discussion

Extramedullary hematopoiesis (EMH) is defined as hematopoiesis that occurs outside of the bone marrow. It can occur in both normal and pathologic states and has been seen in several hematologic disorders including chronic myeloproliferative neoplasms. Myeloproliferative neoplasms (MPN) are a group of clonal hematopoetic stem cell disorders that include polycythemia vera (PV), essential thrombocythemia (ET) and primary myelofibrosis (PMF).1 JAK2V617F mutation is the most frequent mutation associated with MPNs, found in roughly 96% of patients with PV and 65% of patients with ET and PMF. This mutation leads to constitutive activation of the JAK/STAT pathway and is a driver of myeloproliferation.2  The patient in case 1 was found to have a JAK2 mutation during the work-up for hypercoagulability. This suggests that he may have an underlying MPN, however JAK2 mutations have been found in patients with venous thrombosis, but without overt evidence of MPNs.3 The patient in case 2 had a bone marrow biopsy with features concerning for primary myelofibrosis. In PMF, there is generally a proliferation of myeloid cells in addition to marrow fibrosis. Increasing fibrosis can eventually result in pancytopenia as the fibrosis takes over the marrow space in addition to altering the bone marrow environment so that it is unable to support normal hematopoiesis. Ultimately, this can lead to extramedullary hematopoesis. EMH most commonly occurs in the spleen and liver, but has been described in many other sites including the mediastinum and lymph nodes. In addition to being a driver of proliferation, it is thought that JAK2 mutations make hematopoetic stem and progenitor cells more sensitive to growth factors and can cause the cells to mobilize to the liver and spleen.4  Patients with EMH can have symptoms related to the site of involvement. Depending on the extent of involvement and location, EMH may require treatment with low dose radiation. While EMH is a rare finding, it should prompt an investigation for an underlying MPN.

References

  1. Imai K, Aoi T, Kitai H,et al. A case of perirenal extramedullary hematopoiesis in a patient with primary myelofibrosis. CEN Case Reports. 2017;6(2):194-199. doi:10.1007/s13730-017-0274-1.
  2. Kim CH. Homeostatic and pathogenic extramedullary hematopoiesis. Journal of blood medicine. 2010;1:13-19.3https://www.ncbi.nlm.nih.gov/pubmed/17263783.
  3. De Stefano, V, Fiorini, A, Rossi, E, et al. Incidence of JAK2 V617F mutation among patients with splanchnic or cerebral venous thrombosis and without overt chronic myeloproliferative disorders. Journal of Thrombosis and Haemostasis. 2007;5(4):708-14. https://www.ncbi.nlm.nih.gov/pubmed/17263783.
  4. Passamonti F, Maffioli M, Caramazza D, et al. Myeloproliferative neoplasms: From JAK2 mutations discovery to JAK2 inhibitor therapies. Oncotarget. 2011;2(6):485-490.

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Chelsea Marcus, MD is a third year resident in anatomic and clinical pathology at Beth Israel Deaconess Medical Center in Boston, MA and will be starting her fellowship in Hematopathology at BIDMC in July. She has a particular interest in High-grade B-Cell lymphomas and the genetic alterations of these lymphomas.