EHE1-16 Participants Identification No. % Evaluation

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1 Cell Identification EHE1-16 Participants Identification No. % Evaluation Blast cell Educational Lymphocyte Educational Lymphocyte, reactive Educational Lymphoma cell (malignant) Educational Myeloblast with Auer rod Educational Neutrophil, promyelocyte Educational Neutrophil necrobiosis (degenerated neutrophil) Educational Immature or abnormal cell Educational The arrowed cell is a blast as correctly identified by 90.1% of participants. When blasts are observed in patients with myelodysplastic syndromes (MDS), these typically are myeloblasts which are the most immature cells of the myeloid series. While the normal myeloblast is usually a large cell with a high nuclear-to-cytoplasmic ratio, blasts seen in MDS may be smaller in size. The blasts in MDS will still have a high nuclear-to-cytoplasmic ratio and may show agranular cytoplasm which is scant in quantity. The nucleus of the MDS blast shows finely reticulated chromatin and may show distinct nucleoli. 4

2 EHE1-17 Participants Identification No. % Evaluation Macrocyte oval or round (excluding polychromatophilic red cells) Educational Ovalocyte (elliptocyte) Educational Stomatocyte Educational Parasite seen, referred for definitive identification Educational Immature or abnormal cell Educational The arrowed cell is a macroovalocyte as correctly identified by 39.0% of participants. Macrocytes are erythrocytes that are large in size with a mean corpuscular volume of greater than 100fL. Oval macrocytes may be seen in a variety of conditions and are not specific for MDS. Macroovalocytes may be seen in megaloblastic anemias (vitamin B12 or folic acid deficiency), chemotherapy, as well as MDS. 5

3 EHE1-18 Participants Identification No. % Evaluation Platelet, hypogranular Educational Platelet, normal Educational Platelet, giant Educational Immature or abnormal cell Educational The arrowed cell is a hypogranular platelet as correctly identified by 72.1% of participants. Hypogranular platelets differ from normal platelets by the absence, or marked diminishment, of purple-red granules. Two well-granulated giant platelets can be seen nearby for contrast. The size and shape of hypogranular platelets can vary, but the cytoplasm is typically a pale blue or blue-gray color. Hypogranular and dysplastic platelets may be seen in myelodysplastic disorders and in myeloproliferative neoplasms or mixed myelodysplastic/myeloproliferative neoplasms. EDTA-induced artifact can also rarely show platelet degranulation, which can mimic hypogranular platelets. 6

4 EHE1-19 Participants Identification No. % Evaluation Neutrophil with dysplastic nucleus and/or hypogranular Educational cytoplasm Neutrophil, segmented or band Educational Neutrophil with Pelger-Huët nucleus Educational (acquired or congenital) Monocyte Educational Neutrophil, metamyelocyte Educational Neutrophil, giant band Educational Lymphocyte, reactive Educational Monocyte immature (promonocyte, monoblast) Educational Immature or abnormal cell Educational The arrowed cell is a dysplastic neutrophil as correctly identified by 55.2% of participants. This dysplastic neutrophil is characteristic of a myelodysplastic syndrome. In MDS, the normal synchronous maturation of nucleus and cytoplasm is disrupted resulting in decreased to absent primary and secondary granules. This causes the cytoplasm to appear pale. Abnormal nuclear lobation and coarse chromatin are typical. Pseudo-Pelger-Hüet or hypolobated neutrophils may also be seen where the nucleus has a pince-nez appearance. 7

5 EHE1-20 Participants Identification No. % Evaluation Platelet, giant Educational Platelet, normal Educational Platelet, hypogranular Educational Babesia Educational Immature or abnormal cell Educational The arrowed cell is a normal platelet as correctly identified by 41.9% of participants. This normal platelet was chosen to contrast with the hypogranular platelet identified in EHE1-18. Normal platelets contain a normal complement of fine azurophilic granules or an aggregate of granules in the center of the platelet, and most normal-sized platelets are 1.5 to 3 µm in diameter. The arrowed cell is not a giant platelet. This is because the term giant platelet is used when the platelet is larger than the size of the average red cell in the field. As seen above, the arrowed platelet is smaller than surrounding red blood cells. 8

6 Interpretive Questions - EHE Common features of myelodysplastic syndrome at diagnosis do not include: Response No. % (A) Anemia - - (B) Thrombocytopenia (C) Neutropenia (D) Monocytosis (E) Thrombocytosis Intended Response: Monocytosis The diagnosis of myelodysplastic syndrome specifically requires the exclusion of patients with an absolute monocyte count greater than 1 x 10 9 /L. Patients with features of a myelodysplastic syndrome who also have a persistent monocytosis are better classified as myelodysplastic/myeloproliferative neoplasms (MDS/MPN). Of course, a bone marrow examination must be performed as the diagnosis of MDS/MPN, such as chronic myelomonocytic leukemia, requires blood and marrow blast percentages be less than 20% to exclude an acute myelogenous leukemia (AML). Thrombocytosis was chosen by many laboratories as the response. Normal or elevated platelet counts may be seen in a specific subtype of myelodysplastic syndrome, MDS with del(5q). 2. MDS with isolated deletion of chromosomes 5q (long arm of chromosome 5) is typically associated with an elevated or normal platlet count. Response No. % (A) True (B) False Intended Response: True MDS with del(5q) is associated with an elevated platelet count in up to one-half of patients; normal platelet counts may also be seen, but thrombocytopenia is very uncommon. This contrasts with most patients with other subtypes of MDS who present with thrombocytopenia. MDS with del(5q) show deletion of chromosome 5q as the sole cytogenetic abnormality. These patients are primarily elderly women who present with anemia with or without other cytopenias, and with thrombocytosis or a normal platelet count. Less than 1% blasts are seen in the peripheral blood and less than 5% blasts are seen in the bone marrow. In the bone marrow, characteristic small hypolobated megakaryocytes are characteristic. 9

7 Interpretive Questions - EHE1-21 (con t.) 3. Which of the following can mimic myelodysplasia? Response No. % (A) Poor stain quality - - (B) Drug effect (C) G-CSF (Granulocyte colony stimulating factor) (D) Inherited Pelger-Hüet anomaly (E) All of the above Intended Response: All of the above Myelodysplasia in blood smears is typically characterized by neutrophils with hypogranular cytoplasm and hypolobated or occasionally hyperlobated nuclei. Morphologic mimics of myelodysplasia in peripheral blood are numerous which makes the diagnosis of myelodysplastic syndrome tricky. Poor stain quality can give the impression of so-called hypogranular neutrophils, but examination of the blood smear should show areas of the slide with well-granulated neutrophils and the neutrophils will lack nuclear abnormalities. A variety of different drugs can cause drug effect in granulocytes including hypolobated neutrophils with coarse chromatin in combination with well-granulated cytoplasm. This combination should prompt one to consider which medications the patient may be on. The growth factor, G-CSF, can show a variety of changes in the peripheral blood including neutrophilia, left-shift in granulocytes including blasts, toxic granulation and Döhle bodies in neutrophils, and neutrophils showing a range in granularity of cytoplasm. The inherited Pelger-Hüet anomaly shows hypolobation of granulocytes with coarse chromatin accompanied by normal cytoplasmic granulation. 10

8 Discussion on Myelodysplastic Syndrome (MDS) This peripheral blood smear is from a 61-year-old male with worsening pancytopenia. Laboratory data include: WBC = 6.2 x 10 9 /L; HGB = 8.5 g/dl; MCV = 86.0fL; RDW = 14.4%. An automated platelet count could not be given due to interference by giant platelets. Myelodysplastic syndromes (MDS), or preleukemias, represent a group of clonal neoplasms that involve the peripheral blood and bone marrow. These diseases are characterized by cellular bone marrows with ineffective maturation resulting in cytopenias. These diseases typically progress to acute myeloid leukemia, with the rate of progression depending on the subtype of MDS. The diagnosis of a myelodysplastic syndrome requires integration of data from the clinical history, drug history, complete blood cell count, peripheral blood smear, bone marrow aspirate smears and biopsy, iron stain, and cytogenetic studies. From the peripheral blood and bone marrow aspirate smears, the percentage of blasts is needed; a 200 leukocyte differential should be performed on the peripheral blood smear, while a 500 nucleated cell differential is performed on the bone marrow aspirate. The cellular types affected and degree of dysplasia is also necessary for classification of MDS and can be determined from evaluation of the peripheral blood smear and bone marrow specimen. Morphologic features of MDS seen in the peripheral blood may be seen in red blood cells, white blood cells, and/or platelets. Interpretation of myelodysplasia in a peripheral blood smear depends on a well-prepared and well-stained preparation using a fresh blood specimen made with less than 2 hour exposure to anticoagulant. Dyserythropoiesis (dysplasia of erythrocytes) typically presents with a hypoproductive anemia reflecting an inadequate reticulocytosis with minimal polychromatophilic red cells (Table 1). Table 1. Finding Associated with Dyserythropoiesis in Peripheral Blood Anemia Normocytic and normochromic erythrocytes Macrocytic erythrocytes Dimorphic red cells Basophilic stippling Poikilocytosis including oval macrocytes, dacrocytes, and fragmented cells Minimal polychromatophilic red blood cells Nucleated red blood cells 11

9 Discussion on Myelodysplastic Syndrome (MDS) While typically a macrocytic anemia is seen with oval macrocytes (Figure 1), other patients with MDS may show a normocytic or microcytic anemia. Figure 1. Oval macrocytes can be seen in this blood smear from a patient with MDS. 12

10 Discussion on Myelodysplastic Syndrome (MDS) A dimorphic red cell picture is more commonly seen in a specific subtype of MDS, refractory anemia with ring sideroblasts, with a population of hypochromic microcytes and normocytic normocytes (Figure 2). Rare nucleated red cells with cytoplasmic siderotic granules can also be seen; this can be confirmed by an iron stain on the peripheral blood smear. Dysplastic features in nucleated red blood cells need to be interpreted with caution (ie, blebbing, satellite nuclei), as a brisk turnover of red cells in the bone marrow can show similar features with dysplastic nucleated RBCs. Marked anisocytosis and poikilocytosis can also be seen including bizarre microcytes, teardrop-shaped cells (dacrocytes), oval macrocytes, and coarse basophilic stippling. However, none of these changes are specific for MDS. Figure 2. A dimorphic population of red cells is present from this patient with MDS, refractory anemia with ring sideroblasts. (Reprinted with permission from Pereira I, George TI, and Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Lippincott Williams & Wilkins, Fig 9.3.) 13

11 Discussion on Myelodysplastic Syndrome (MDS) Dysgranulopoiesis (dysplasia in granulocytes) is characterized by abnormalities in the cytoplasm and nucleus of granulocytes (Table 2). Table 2. Finding Associated with Dysgranulopoiesis in Peripheral Blood Hypogranularity Nuclear hypolobation, often with clumped nuclear chromatin Pseudo-Pelger-Hüet nucleus Ring nucleus Hypersegmentation Abnormal granules Auer rods Small or unusually large size of granulocytes The peripheral blood is the preferred place to look for findings of dysgranulopoiesis as the bone marrow of MDS patients may show limited granulocytic maturation. Common findings of dysgranulopoiesis include hypogranular neutrophils that are small in size with clumped nuclear chromatin that fail to segment fully (Figure 3). Figure 3. This neutrophil is dysplastic with hypogranular cytoplasm and Döhle bodies. (Reprinted with permission from Pereira I, George TI, and Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Lippincott Williams & Wilkins, Fig 9.4B.) 14

12 Discussion on Myelodysplastic Syndrome (MDS) These neutrophils may be bilobed in appearance (pseudo-pelger-hüet cell) or hypersegmented (Figure 4). Figure 4. This pseudo Pelger-Hüet neutrophil is from a patient with MDS. Note the chromatin which is coarser than normal. (Modified with permission from Pereira I, George TI, and Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Lippincott Williams & Wilkins, Fig 9.4D.) 15

13 Discussion on Myelodysplastic Syndrome (MDS) A careful drug history is necessary to distinguish MDS-related dysgranulopoiesis from true inherited Pelger-Hüet anomaly or drug effect. Poor staining can also mimic hypogranular neutrophils. Abnormal granules (pseudo Chediak-Highashi granules) can be seen in dysplastic granulocytes, as can Auer rods. Immature granulocytes, including blasts, can also be seen in MDS patients; some immature granulocytes will be difficult to classify given the amount of dysplasia present and the lack of granulation. The blasts seen in patients with MDS are myeloblasts, but will show morphologic features ranging from blasts with scant agranular cytoplasm, to blasts with Auer rods, to blasts with abundant basophilic cytoplasm (Figure 5). Figure 5. A dysplastic neutrophil is present at left with hypogranular cytoplasm and hypercondensed nuclear chromatin adjacent to a small blast. The blast contains a scant amount of basophilic cytoplasm and partially condensed chromatin. Blasts seen in MDS may be smaller than blasts seen in normal individuals, and MDS-type blasts may contain slightly more condensed chromatin than seen in typical myeloblasts. (Modified with permission from Pereira I, George TI, and Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Lippincott Williams & Wilkins, Fig 9.7.) If more than 20% blasts are present on a peripheral blood smear, then a diagnosis of acute leukemia can be made; however, if less than 20% blasts are present then examination of the bone marrow must be performed before a final diagnosis can be made. Exact enumeration of blasts on a 200 leukocyte differential from a blood smear is necessary as the subclassification of MDS is based on the percentage of marrow and blood blasts. Enumeration of monocytes is also important as patients with MDS are required to have an absolute monocyte count less than 1 x 10 9 /L. Patients with features of MDS who also have persistently elevated numbers of monocytes greater or equal to 1 x 10 9 /L in the peripheral blood and less than 20% blasts in the marrow are best classified as mixed myelodysplastic/ myeloproliferative neoplasms (ie, chronic myelomonocytic leukemia). 16

14 Discussion on Myelodysplastic Syndrome (MDS) Dysmegakaryopoiesis (megakaryocyte dysplasia) is typically based on examination of the bone marrow aspirate and biopsy specimen. However, the platelets of dysplastic megakaryocytes also reflect dysplasia and may be hypogranular, giant, and/or bizarre (Table 3, Figure 6). Table 3. Finding Associated with Dysmegakaryopoiesis in Peripheral Blood Giant platelets Bizarre platelets Hypogranular platelets Micromegakaryocytes Figure 6. In the upper left of the image a giant hypogranular platelet is present adjacent to a normal well-granulated platelet in this patient with MDS. (Reprinted with permission from Pereira I, George TI, and Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Lippincott Williams & Wilkins, Fig 9.6A.) Most patients with MDS have low platelet counts, but elevated platelet counts are typically associated with MDS associated with a specific chromosomal abnormality, that of an isolated deletion in the long arm of chromosome 5. 17

15 Discussion on Myelodysplastic Syndrome (MDS) Recommended Reading: 1. Pereira I, George TI, Arber DA. Atlas of Peripheral Blood. The Primary Diagnostic Tool. Wolters Kluwer, Lippincott Williams and Wilkins, Inc., Philadelphia, PA (2012). 2. Wang E, Boswell E, Siddiqi L, et al. Pseudo-Pelger-Hüet anomaly induced by medications: a clinocpathologic study in comparison with myelodysplastic syndrome-related pseudo-pelger-hüet anomaly. Am J Clin Pathol 2011; 135: Zhou J, Orazi A, Czader MB. Myelodysplastic syndromes. Semin Diagn Pathol 2011;28: Tracy I. George, MD, FCAP, Chair Anna K. Wong, MD, FCAP Hematology and Clinical Microscopy Resource Committee 18

16 Cell Identification Participants Identification No. % Evaluation EHE1-23 Lymphoma cell (malignant) Educational Lymphocyte, reactive Educational Blast cell Educational Lymphocyte Educational Lymphocyte, large granular Educational Plasma cell with inclusion (Dutcher body, Russell body) Educational Immature or abnormal cell Educational The identified cells are prolymphocytes, which may be seen in some cases of chronic lymphocytic leukemia (CLL), and are also seen in B and T-cell prolymphocytic leukemia (PLL). Prolymphocytes are malignant lymphoid cells that are not seen in benign or reactive conditions. For the purpose of proficiency testing, prolymphocytes are best classified as lymphoma cells. These were correctly identified by 50.3% of participants. Prolymphocytes are readily distinguishable from both normal lymphocytes as well as the lymphoid cells of CLL. Prolymphocytes are notably larger, roughly twice the size of a small lymphocyte, with a greater amount of cytoplasm, and appear more immature than the small lymphocytes of CLL. The nuclear chromatin is distinctly more open than that of mature lymphocytes, but is not as immature as that of lymphoblasts. As seen in these examples, prolymphocytes almost always have a single prominent nucleolus. Infrequently, there may be more than one nucleolus. The cytoplasm is moderately basophilic and usually agranular, although a few prolymphocytes may contain scant azurophilic granules. It is important to recognize and identify when prolymphocytes are present because they may have diagnostic and prognostic significance. Prolymphocytes typically comprise less than 2% of peripheral blood lymphocytes in CLL, and by definition represent more than 55% of lymphocytes in de novo PLL, an entity quite distinct from CLL. In the current case, numerous prolymphocytes are present, and the patient has a known history of CLL; thus, this represents prolymphocytic transformation of CLL, which is a very rare occurrence. 21

17 EHE1-24 Participants Identification No. % Evaluation Basket cell/smudge cell Educational Immature or abnormal cell Educational The arrowed object is a basket or smudge cell, as correctly identified by 97.7% of participants. Smudge cells are an artifact resulting from cells, usually but not always lymphocytes, that are somewhat more fragile and are ruptured in the process of making the blood film. This results in either an irregular mass of chromatin or chromatin strands reminiscent of a basket. Cytoplasm is usually absent. Rare smudge cells may be seen in almost any blood film, but are more common in disorders that lead to increased lymphocyte fragility, such as chronic lymphocytic leukemia or infectious mononucleosis. In the current case, numerous smudge cells may be seen throughout the virtual blood film. 22

18 EHE1-25 Participants Identification No. % Evaluation Lymphocyte Educational Lymphoma cell (malignant) Educational Immature or abnormal cell Educational The identified cell is a lymphocyte, correctly identified by 96.5% of participants. Lymphocytes are small cells, 7 to 15 μm in diameter with a high N:C ratio, round or oval nuclei, dense or clumped chromatin, no clearly visible nucleoli, and a small amount of pale to medium basophilic agranular cytoplasm. Nuclei in some may be slightly indented. A few small lymphocytes may have a small, pale chromocenter that can be mistaken for a nucleolus. The lymphocytes of CLL are often very slightly larger than normal lymphocytes, and may be unusually monomorphic, lacking the subtle morphologic heterogeneity of normal benign lymphocytes. Nevertheless, on a cell by cell basis, it is not possible to reliably distinguish CLL cells from normal lymphocytes by morphology alone. For this reason, it is recommended that for proficiency testing purposes the lymphoid cells of CLL be classified as lymphocytes, rather than lymphoma cells. The small lymphocytes in this case are also quite monotonous in appearance, although a few minor variations are seen, such as occasional notched nuclei. In scanning the blood film, one can see that some lymphoid cells have slightly irregular or frayed cytoplasmic borders. This is a common artifact of blood film preparation and should not be mistaken for the unusual and very characteristic extended fine cytoplasmic projections seen in either hairy cell leukemia or splenic lymphoma with villous lymphocytes. 23

19 EHE1-26 Participants Identification No. % Evaluation Monocyte Educational Lymphocyte, reactive Educational Monocyte, immature (promonocyte, monoblast) Educational Neutrophil, toxic Educational Plasma cell with inclusion (Dutcher body, Russell body) Educational Blast cell Educational Lymphocyte Educational Lymphoma cell (malignant) Educational Plasma cell, abnormal Educational Immature or abnormal cell Educational The indicated cell is a monocyte, correctly identified by 87.8% of participants. Monocytes are slightly larger than neutrophils, with indented or convoluted nuclei, and moderate grey-blue cytoplasm that may contain vacuoles and scant fine azurophilic granules. The chromatin is condensed and mature but less dense than that of neutrophils. Nucleoli are absent or indistinct. The current case shows that monocytes and prolymphocytes have similar size and amount of cytoplasm, but the monocytes nuclear shape, chromatin pattern, lack of nucleoli, and vacuolated cytoplasm make these readily distinguishable from the prolymphocytes. 24

20 EHE1-27 Participants Identification No. % Evaluation Lymphocyte Educational Lymphocyte, reactive Educational Lymphoma cell (malignant) Educational Blast cell Educational Lymphocyte, large granular Educational Immature or abnormal cell Educational The cell identified by the arrow is lymphoid, but is difficult to classify. This challenging cell was intentionally chosen for this extended hematology exercise as an example of a cell with features intermediate between a small mature lymphocyte and a prolymphocyte. The difficulty in classifying the cell is reflected in the survey results, in which 59.9% of participants identified the cell as a lymphocyte, and 15.1% identified it as a lymphoma cell. The arrowed cell is slightly larger than most small lymphocytes, but is not quite as large as the prolymphocytes to the right. Compared to small lymphocytes, the arrowed cell has slightly greater cytoplasm and slightly more open chromatin. Toward the 4 o clock position in the nucleus is an area suggesting a small chromocenter or nucleolus. These features are similar to those of the adjacent prolymphocytes, but the chromatin is not sufficiently open nor the nucleolus sufficiently prominent to definitively classify this cell as a prolymphocyte or lymphoma cell. This cell was included to illustrate that unambiguous classification of every individual cell on a blood film is not always possible. 25

21 Interpretive Questions - EHE Identify the on ecorrect statement regarding CLL. Response No. % (A) CLL occurs exclusively in the elderly (B) CLL is an indolent and asymptomatic disorder with consistently long survival (C) The typical lymphocytes of CLL are large with abundant cytoplasm, partially immature chromatin, and a single prominent nucleus. (D) Large cell transformation of CLL is termed Richter syndrome Intended Response: Large cell transformation of CLL is termed Richter syndrome. The correct answer is (D) Large cell transformation of CLL is termed Richter syndrome. Pathologist Dr. Maurice Richter described the first recognized case of large cell lymphoma arising in transformation in a patient with known CLL. (A) is incorrect. Although the majority of patients with CLL are older than 65 years at diagnosis, there are nevertheless a significant number of younger patients with CLL as well. (B) is incorrect. The clinical course of CLL is quite variable, and while some patients have a prolonged and indolent course, in others the disease is more aggressive with more complications, more rapid progression, and shorter survival. (C) is incorrect. The typical lymphocytes in CLL are small with condensed chromatin, absent nucleoli, and scant cytoplasm. The description provided in (C) is that of prolymphocytes, which should be less than 2% of lymphoid cells in typical CLL. 2. Complications of CLL include all of the following except: Response No. % (A) Pure red aplasia (B) Weight gain (C) Recurrent infections (D) Immune thrombocytopenia (E) Night sweats Intended Response: Weight gain The correct answer is (B) weight gain. The complications of CLL arise from the progressive proliferation of lymphoid cells, and from a dysfunctional immune system. The progressive burden of disease will lead to a high peripheral blood white count, which may lead to the risk of leukostasis. Patients may also develop progressive lymphadenopathy and splenomegaly, and progressive marrow infiltration will impair normal hematopoiesis. The burden of disease is also related to the likelihood of experiencing constitutional symptoms, which include unexplained weight loss (not weight gain), fevers, fatigue, and drenching night sweats. Immune related complications include hypogammaglobulinemia, recurrent infections, autoimmune hemolytic anemia, autoimmune thrombocytopenia, and pure red cell aplasia. 26

22 Interpretive Questions - EHE1-28 (con t.) 3. Which of the following mechanisms is postulated to be involved in at least some cases of transformation in CLL? Response No. % (A) Mutations arising within a myeloid stem cell (B) New mutations driven by CMV infection (C) Autoimmune-driven transformation (D) Newly acquired mutations within the CLL clone Intended Response: Newly acquired mutations within the CLL clone The correct answer is (D) newly acquired mutations within the CLL clone. The mechanisms involved in transformation are not completely known, and it is likely that different cases of transformation involve more than one mechanism. There is evidence to support the theory that some cases of transformation arise due to the acquisition of new mutations in the existing CLL clone. Other studies suggest that some cases of apparent transformation reflect a new second lymphoma unrelated to the CLL. (A) is incorrect. CLL is a lymphoid neoplasm that does not arise from myeloid stem cells. (B) is incorrect. EBV has been implicated in some cases of CLL transformation, but not CMV. (C) is incorrect. Autoimmune phenomena are common complications in CLL, but these processes and the associated autoantibodies are unrelated to histologic transformation. 4. B-cell prolymphocytic leukemia (PLL) always occurs as a trasnformation from CLL. Response No. % (A) True (B) False Intended Response: False The correct answer is (B) false. Although prolymphocytic transformation of CLL may appear morphologically indistinguishable from B-PLL, de novo B-PLL is a distinct entity that does not arise from a preexisting CLL. 27

23 Discussion on Prolymphocytic Transformation of CLL Introduction Chronic lymphocytic leukemia (CLL) is a neoplastic proliferation of small lymphocytes that usually involves the peripheral blood and bone marrow, as well as the spleen and lymph nodes. In a small minority, the disease primarily involves lymph nodes without significant blood or marrow involvement. This non-leukemic variant is termed small lymphocytic lymphoma (SLL), but CLL and SLL are considered the same disease entity. Epidemiology and Clinical Presentation CLL is the most common leukemia in western countries. The median age at diagnosis is 72 years, with close to 70% of patients being older than 65 years at the time of diagnosis. Because of this, CLL is considered a disease of the elderly. Nevertheless, there are a significant number of younger patients with CLL as well. It is twice as common in men as in women. Initially, patients may present with painless lymph node swelling or constitutional symptoms such as significant fatigue, unintentional weight loss, persisting fevers, or drenching night sweats. Twenty-five percent of patients are asymptomatic, and CLL is diagnosed following the discovery of a lymphocytosis on blood work done for other reasons. Diagnosis of CLL CLL/SLL is a clonal neoplasm of mature B lymphocytes. In the absence of extramedullary (ie, outside the blood or bone marrow) tissue involvement, cytopenias, or disease-related symptoms, the diagnosis requires 5 x 10 9 /L monoclonal lymphocytes with a CLL phenotype in the peripheral blood. The phenotype of CLL cells is usually readily determined by flow cytometry, which will show a population of kappa- or lambda-light chain restricted B- cells (positive for CD19, dim expression of CD20, and CD22) with co-expression of CD5 and usually CD23. Surface light chain expression is often dim. Some cases express CD38 or Zap-70, which are thought to be adverse prognostic markers. Patients with <5 x 10 9 /L clonal lymphocytes with this phenotype and without cytopenias or disease-related symptoms are referred to as having monoclonal B lymphocytosis (MBL). It is currently unclear whether or not MBL represents an oligoclonal expansion of benign lymphocytes or represents an early form of overt CLL. The typical morphology of CLL cells on blood smears is that of small lymphocytes with clumped chromatin that may have a cracked appearance, absent nucleoli, and scant cytoplasm (Figure 1). An individual CLL cell may be difficult to distinguish from a normal lymphocyte. CLL cells are fragile, resulting in many smudge or basket cells during blood smear preparation. 28

24 Discussion on Prolymphocytic Transformation of CLL Figure 1: Peripheral blood smear with typical findings in CLL including small mature lymphocytes with condensed nuclear chromatin and frequent smudge cells (arrows). CLL lymphocytes may be difficult to distinguish from normal lymphocytes. Note that, as in normal lymphocytes, the nuclei of these lymphocytes are about the same size as a red blood cell. Compare this image to the prolymphocytes in Figure 2. Prolymphocytes in CLL and Prolymphocytic Leukemia (PLL) In contrast to the small lymphocytes of CLL with dense chromatin and absent nucleoli, prolymphocytes are distinctly larger cells with more abundant cytoplasm and more open and partially immature chromatin, and their nucleus characteristically contains a single prominent nucleolus (Figure 2). Prolymphocytes may be seen in the peripheral blood in CLL, but these usually comprise 2% or less of lymphocytes. If prolymphocytes comprise the majority of lymphoid cells (always more than 55% and usually more than 90%) in a new presentation (i.e., no previous diagnosis of CLL), the diagnosis is prolymphocytic leukemia (PLL) rather than CLL. The diagnostic category of de novo PLL may be a B-cell or T-cell neoplasm, excludes cases of CLL with prolymphocytic transformation (discussed below), and is considered to be distinct from CLL. The flow cytometry phenotype of B prolymphocytes may be the same as that seen in CLL, although CD5 and CD23 are less frequently expressed in de novo PLL. 29

25 Discussion on Prolymphocytic Transformation of CLL Figure 2. Typical prolymphocytes. Compared to normal lymphocytes and typical CLL cells, which have nuclei about the same size as a red blood cell, prolymphocytes (arrows) are large cells with more abundant cytoplasm and a distinct prominent nucleolus. The nuclear chromatin is not as condensed as in a normal mature lymphocyte. Compare these cells to the CLL cells in Figure 1. Transformation of CLL In a small percentage of patients with CLL, less than 10%, and typically when in advanced stage, the disease undergoes transformation into another form of lymphoma, usually a more aggressive disease. This is termed Richter syndrome or Richter transformation, named after Dr. Maurice Richter, who published the first case report describing this transformation in By far the most common form of transformation is into an aggressive large B-cell lymphoma. Uncommon forms of transformation include Hodgkin lymphoma, plasma cell myeloma, prolymphocytic transformation, and, very rarely, acute lymphoblastic leukemia/lymphoma. The pathogenesis of transformation is not entirely known, and there may be more than one mechanism. Some studies support the concept that transformation occurs though the development of new genetic abnormalities within the existing CLL clone. Other studies have shown that some transformed lymphomas are genetically distinct and seemingly unrelated to the underlying CLL. Similar to the development of a non-hematologic tumour, these may reflect the development of an independent lymphoma in an immunocompromised host. Epstein barr virus (EBV) infection has also been implicated in some cases of transformation. 30

26 Discussion on Prolymphocytic Transformation of CLL There is no established criterion for the percentage of prolymphocytes required for the diagnosis of prolymphocytic transformation in CLL. Initial case reports used their own criteria and did not distinguish between cases of established CLL with the new development of prolymphocytes, or de novo cases with prolymphocytes present. In a 1981 paper, Kjeldsberg defined transformation as 15% or more prolymphocytes in cases of CLL that previously contained only rare prolymphocytes. The WHO 2008 classification no longer recognizes the entity of mixed CLL/PLL, which was defined as CLL with between 10-55% prolymphocytes. However, some cases of disease progression in CLL show an increase in proliferation activity with increased numbers of prolymphocytes in the peripheral blood. In the current case, the percentage of prolymphocytes is so high as to appear indistinguishable from de novo prolymphocytic leukemia. The most relevant finding for identification of prolymphocytic transformation may be a significant and progressively increasing proportion of prolymphocytes in a patient with CLL in whom no significant proportion of prolymphocytes had previously been identified. This is usually accompanied by other clinical or laboratory indicators of a change in disease status to a more aggressive form. Histologic transformation has a poor clinical outcome, with a median patient survival of just a few months. Response rates to chemotherapy are poor, and those who respond are at high risk for disease recurrence shortly after treatment. Prognosis and Complications Although CLL is often thought of as a relatively benign and indolent leukemia, its natural history and prognosis are quite variable. Some patients are fortunate to be asymptomatic for decades, while others have a more aggressive course with significant morbidity and mortality. Molecular and cytogenetic/fish studies have been shown to be have prognostic utility in CLL. For example, patients with del(13q14) have a good prognosis while those with del(11q22-23) and del(17p) have a poor prognosis. In addition, molecular studies demonstrating somatic hypermutation of immunoglobulin (IGHV) genes is indicative of a better prognosis than cases with unmutated IGHV genes. The complications of CLL arise from the progressive burden of disease as well as from defects of the immune system. The progressive burden of disease leads to increasing lymph node swelling, enlargement of the spleen and sometimes liver, and progressive marrow infiltration, resulting in anemia and then neutropenia and/or thrombocytopenia. If lymphocytosis in the peripheral blood becomes extreme (more than 400 x 10 9 /L), there is a risk of leukostasis, in which the marked increase in white blood cells impairs blood flow in the microcirculation, leading to ischemic tissue injury, necrosis, and bleeding. Immune defects of CLL occur because CLL lymphocytes do not function properly as immune effector cells. The complications of defective immunity include not only impaired immunoglobulin production leading to recurrent infections, but also autoimmune phenomena such as autoimmune hemolytic anemia, immune thrombocytopenia, or red cell aplasia, in which autoantibodies attack erythroid progenitors in the bone marrow. Patients with CLL also have a slightly increased risk of second malignancies, which may be hematologic or solid tumours. There is speculation regarding the roles of defective immunity and the effects of chemotherapy in contributing to these secondary cancers. 31

27 Discussion on Prolymphocytic Transformation of CLL Treatment Conventional chemotherapy is not curative in CLL, but can for a time reduce the burden of disease, reduce symptoms and control complications. Therapy is usually not required in early stage disease, and is reserved for treatment of disease progression or complications. Therefore, the most common strategy for treatment of CLL is to avoid chemotherapy if the patient is asymptomatic, and to use periodic chemotherapy judiciously in order to maintain control of the disease. A select few patients are young enough to tolerate high dose chemotherapy and stem cell transplantation, a therapy which has significant risk and toxicity but which offers a chance for prolonged disease-free survival. Summary In the case presented, a patient with a long standing history of CLL presents with newly progressive cytopenias, which is worrisome for disease progression or transformation. Other presentations worrisome for transformation include new constitutional symptoms, or new and rapidly progressive lymphadenopathy or hepatosplenomegaly. Examination of the patient s peripheral blood smear demonstrates that the vast majority of lymphoid cells are prolymphocytes, which is a distinct change from previous, and diagnostic for prolymphocytic transformation of CLL. Luke R Shier, MD, FCAP Kyle T. Bradley, MD, FCAP Hematology and Clinical Microscopy Resource Committee Recommended Reading: 1. Dighiero G, Hamblin TJ. Chronic lymphocytic leukemia. Lancet. 2008;371: Robak T. Second malignancies and Richter s syndrome in patients with chronic lymphocytic leukemia. Hematol. 2004;9: Kjeldsberg CR, Marty J. Prolymphocytic transformation of chronic lymphocytic leukemia. Cancer. 1981;48: Gribben JG. How I treat CLL up front. Blood. 2010;115: