REPORTING TEMPLATES

DIAGNOSIS 1-3. Bone marrow, right posterior iliac crest, biopsy, aspirate smears, clot: - Normocellular marrow with trilineage maturing hematopoiesis. PERIPHERAL BLOOD a. No circulating blasts identified. b. No overt dysplasia identified. c. No increase in lymphocytes or presence of atypical lymphoid cells. IMMUNOHISTOCHEMISTRY CD34 and CD117 show no increase in blasts (<5% total cellularity). Controls worked appropriately. FLOW CYTOMETRY No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T- cell lymphoma or leukemia is identified. CYTOGENETIC STUDIES: See separate report. MOLECULAR STUDIES: Not requested. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 9.81 K/uL, RBC 4.39 M/uL, Hgb 12.9 g/dL, Hct 39.6%, MCV 90.2 fl and platelets 251 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 67% neutrophils, 1% bands, 17% lymphocytes, 9% monocytes, 1% basophils and 5% eosinophils. Granulocytes, lymphocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. No large/atypical lymphoid cells are noted. BONE MARROW SMEAR Differential: Blasts 2% Promyelocytes 2% Myelocytes 4% Metamyelocyte 1% Neutrophils/Precursors 52% Lymphocytes 15% Monocytes 2% Eosinophils 4% Basophils 0% Plasma cells 0% Erythroid Precursors 20% Number of cells counted 500 ME ratio Normal Bone marrow aspirate smears are spicular and adequately cellular for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is normal. The blast count is not increased (2% on a 500-cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are seen and overall, they are normal in morphology. No atypical lymphoid cells are noted. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Normocellular for age (30%) M:E ratio: Normal Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, unremarkable morphology Lymphocytes: Interstitially scattered Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Siderocyte: No ring sideroblasts are identified. Clot: Adequate, shows similar features as biopsy

Subcortical biopsy shows higher than expected cellularity (50-60% cellularity) in the subcortical areas. There is trilineage maturing hematopoiesis with no increase in lymphocytes or plasma cells. Myeloid elements are mildly reduced and show full maturation with no overt dysplasia. Erythroid elements are relatively increased and show full maturation with no overt dysplasia. Megakaryocytes are overall adequate in number with occasional hyperchromatic forms. Blasts are not increased. Aspirate smears are aspicular, hemodiluted and markedly hypocellular, suboptimal for morphologic evaluation. The limited differential count may not accurately reflect the marrow composition.

DIAGNOSIS 1-3. Bone Marrow, biopsy, aspirate, clot and peripheral blood smears: - Cellular marrow with trilineage maturing hematopoiesis. - No morphologic or immunophenotypic involvement by B cell lymphoma. PERIPHERAL BLOOD a. No circulating blasts identified. b. No overt dysplasia identified. c. No increase in lymphocytes or presence of atypical lymphoid cells. IMMUNOHISTOCHEMISTRY CD3 immunostaining show small T cells scattered interstitially. CD20 and PAX5 highlight no increase in B cells. Controls worked appropriately. FLOW CYTOMETRY No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T- cell lymphoma or leukemia is identified. CYTOGENETIC STUDIES: See separate report. MOLECULAR STUDIES: Not requested. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 9.81 K/uL, RBC 4.39 M/uL, Hgb 12.9 g/dL, Hct 39.6%, MCV 90.2 fl and platelets 251 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 67% neutrophils, 1% bands, 17% lymphocytes, 9% monocytes, 1% basophils and 5% eosinophils. Granulocytes, lymphocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. No large/atypical lymphoid cells are noted. BONE MARROW SMEAR Differential: Blasts % Promyelocytes % Myelocytes % Metamyelocyte % Neutrophils/Precursors % Lymphocytes % Monocytes % Eosinophils % Basophils % Plasma cells % Erythroid Precursors % Number of cells counted 500 ME ratio Normal Bone marrow aspirate smears are spicular and adequately cellular for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is normal. The blast count is not increased (2% on a 500-cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are seen and overall, they are normal in morphology. No atypical lymphoid cells are noted. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Normocellular for age (~30%) M:E ratio: Normal Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, unremarkable morphology Lymphocytes: Interstitially scattered Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Siderocyte: No ring sideroblasts are identified. Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Siderocyte: No ring sideroblasts are identified.

DIAGNOSIS 1-3. Bone marrow, biopsy, aspirate, clot and peripheral blood smears: - Involved by patient's previously diagnosed follicular lymphoma (30% cellularity by CD20/PAX5 immunostains). - Hypercellular marrow with trilineage maturing hematopoiesis. PERIPHERAL BLOOD a. Occasional atypical cleaved lymphoid cells are identified. b. Normocytic normochromic anemia. c. No circulating blasts identified. d. No overt dysplasia identified. IMMUNOHISTOCHEMISTRY Immunostains for CD20 and PAX5 labels increased B cells (30% of total cellularity). CD3 stains background T cells. All controls worked appropriately. FLOW CYTOMETRY Abnormal B-cell population identified. No abnormal mature T-cell population detected. Flow cytometry reveals an abnormal mature B-cell population that shows aberrant expression of CD10 (dim), CD19 (bright), CD20 (bright), CD38 (dim) and lambda light chain restriction; with normal expression of CD45 and without significant CD5 expression. The abnormal B-cell population accounts for 48.24% of the total white cells. No immunophenotypic evidence of involvement by T-cell lymphoma or leukemia is identified. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 9.53 K/uL, RBC 2.72 M/uL, Hgb 7.0 g/dL, Hct 22.9%, MCV 84.2 fl and platelets 227 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 40% neutrophils, 2% immature granulocytes, 51% lymphocytes, 5% monocytes, 1% basophils and 1% eosinophils. Granulocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. RBC shows normocytic normochromic anemia. Few small atypical cleaved lymphoid cells are seen. BONE MARROW SMEAR Differential: Blasts % Promyelocytes % Myelocytes % Metamyelocyte % Neutrophils/Precursors % Lymphocytes % Monocytes % Eosinophils % Basophils % Plasma cells % Erythroid Precursors % Number of cells counted 500 ME ratio Normal Bone marrow aspirate smears are particulate and adequate for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is normal. The blast count is not increased (1% on a 500-cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are seen and overall, they are normal in morphology. An increase in atypical cleaved lymphoid cells is noted. BONE MARROW BIOPSY/CLOT Quality: Fragmented Cellularity: Cannot be ascertained M:E ratio: Normal Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, unremarkable morphology Lymphocytes: Aggregates of lymphoid cells noted, some paratrabecular in location Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Ring sideroblasts: Not increased.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears and peripheral blood smears: - Chronic lymphocytic leukemia (CLL/SLL) involving marrow (48% of cellularity on aspirate smears). - Hypercellular marrow (40%) with trilineage maturing hematopoiesis and mild polytypic plasmacytosis. COMMENT: No morphologic or immunophenotypic evidence of plasma cell neoplasm is seen. Plasma cells are polytypic by immunostains. IMMUNOHISTOCHEMISTRY Immunostains for CD20 and PAX5 label increased B cells. The neoplastic B cell are positive for LEF1, CD5 and CD23 (10-20% cellularity) and lambda light chain restricted. Cyclin D1 is negative. CD3 stains background T cells. Plasma cells are positive for CD138 and polyclonal by kappa and lambda immunostains. FLOWCYTOMETRY Abnormal B-cell population identified. No abnormal mature T-cell population detected. Flow cytometry reveals an abnormal mature B-cell population that shows aberrant expression of CD5 (dim), CD19 (bright), CD20 (moderate), CD23, CD38 (dim) and lambda light chain restriction; with normal expression of CD45 and without significant CD10 expression. The abnormal B-cell population accounts for 48.24% of the total white cells. No immunophenotypic evidence of involvement by T-cell lymphoma or leukemia is identified. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 13.53 K/uL, RBC 2.72 M/uL, Hgb 7.0 g/dL, Hct 22.9%, MCV 84.2 fl and platelets 227 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 40% neutrophils, 2% immature granulocytes, 51% lymphocytes, 5% monocytes, 1% basophils and 1% eosinophils. Granulocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. RBC shows normocytic normochromic anemia. Lymphocytes are small with clumped chromatin. No prolymphocytes or large transformed cells seen. Smudge cells are present. No circulating plasma cells seen. BONE MARROW BIOPSY (FORMAT 1) Quality: Adequate Cellularity: Hypercellular (50-60%) M:E ratio: Normal Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Normal in number and morphology Lymphocytes: Increased small lymphocytes Plasma cells: Scattered BONE MARROW BIOPSY (FORMAT 2) Quality: Adequate Cellularity: Hypercellular (70-80%) M:E ratio: Normal Blasts: Not increased Myeloid lineage: Reduced, shows full maturation Erythroid lineage: Reduced, shows full maturation Megakaryocytes: Reduced, normal in morphology Lymphocytes: Increased small lymphoid cells interspersed interstitially and in aggregates Plasma cells: Scattered BONE MARROW ASPIRATE SMEAR Differential: Myelocytes 4% Metamyelocytes 2% Neutrophils/Bands 10% Monocytes 1% Eosinophils1% Erythroid Precursors 10% Lymphocytes 72% Number of Cells Counted 500 M: E Ratio 1.8 Morphology example 1: Spicular and hypercellular smears show increased small lymphocytes with round to irregular nuclei and scant to moderate cytoplasm. Maturing trilineage hematopoiesis is present. Plasma cells are mildly increased. There is no increase in blasts. Erythroid and myeloid elements are adequate in proportion with progressive maturation and unremarkable morphology. Megakaryocytes are present with predominantly unremarkable morphology. Morphology example 2: Aspicular / hemodiluted aspirate smears. Touch imprints contain too few cells for evaluation. There is prominent increase in lymphoid cells which are comprised of small mature lymphocytes, present in the interstitial areas. Myeloid and erythroid elements are markedly reduced. No dysplastic features are identified. Megakaryocytes are not represented.

DIAGNOSIS 1. Bone marrow, left posterior iliac crest, biopsy: - Chronic lymphocytic leukemia (CLL/SLL) extensively involving marrow (90% of cellularity). - Hypercellular marrow (90-95%) with patchy foci of relatively uninvolved marrow with trilineage maturing hematopoiesis. COMMENT: Suggest a marrow aspirate submitted for morphologic and genetic/cytogenetic evaluation. Scattered CyclinD1 positive nuclei are seen within the infiltrate, the significance of which is unclear. BONE MARROW BIOPSY Histologic sections show hypercellular marrow (90-95%) with an extensive lymphoid infiltrate with a nodular and diffuse architecture, composed of small lymphocytes with clumped chromatin and patchy proliferation centers with medium size lymphoid cells. Focal relatively uninvolved marrow shows trilineage hematopoiesis and megakaryocytes with few atypical forms. Reference: Giné E, Martinez A, Villamor N, López-Guillermo A, Camos M, Martinez D, Esteve J, Calvo X, Muntañola A, Abrisqueta P, Rozman M, Rozman C, Bosch F, Campo E, Montserrat E. Expanded and highly active proliferation centers identify a histological subtype of chronic lymphocytic leukemia ("accelerated" chronic lymphocytic leukemia) with aggressive clinical behavior. Haematologica. 2010 Sep;95(9):1526-33. doi: 10.3324/haematol.2010.022277. Epub 2010 Apr 26. PubMed PMID: 20421272; PubMed Central PMCID: PMC2930954. IMMUNOHISTOCHEMISTRY Immunostains show the lymphoid infiltrate is positive for CD20, PAX5, CD5, CD23 and LEF1. Scattered nuclei show CyclinD1 positivity.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Chronic lymphocytic leukemia (CLL/SLL) extensively involving marrow (60-70% of cellularity). - Hypercellular marrow (70%) with patchy foci of uninvolved marrow with trilineage maturing hematopoiesis. COMMENT: Numerous ring sideroblasts are seen on submitted iron stain. The aspirate smears are paucispicular and hemodilute and show erythroid and myeloid elements with subtle mild dyspoietic changes. Therefore, a myelodysplastic syndrome or other myeloid neoplasm with ring sideroblasts cannot be ruled out. No CBC is available and the morphologic findings are not definitively diagnostic. Even though by itself it would not rule out a myeloid neoplasm, reported cytogenetic studies showed a normal karyotype. Correlation with CBC, review of a peripheral blood smear and comprehensive genomic testing would be helpful in further characterization. If clinically indicated, a new marrow and/or peripheral blood sample may be submitted for further evaluation. In addition, non-neoplastic causes of ring sideroblasts should be considered in differential diagnosis which includes alcohol, Isoniazid and Chloramphenicol treatment and lead poisoning. Correlation with clinical findings is required.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: -Involvement by T lymphoproliferative disorder consistent with hepatosplenic T-cell lymphoma. - Hyperellular marrow with trilineage maturing hematopoiesis. PERIPHERAL BLOOD a. Circulating atypical lymphoid cells. b. No overt dysplasia. c. No circulating blasts. COMMENT: Morphologic and immunophenotypic findings are suggestive of T cell lymphoproliferative disorder, most likely Hepatosplenic T cell lymphoma. Clinical correlation and gene rearrangement studies is recommended. IMMUNOHISTOCHEMISTRY Immunohistochemical studies performed outside show there is a marked increase in small CD3+ T cells (30-40% of marrow cellularity). The T cells have a mixed intrasinusoidal and interstitial distribution, and do not form discrete aggregates. Most of the T cells express neither CD4 or CD8. CD5 is also negative in the majority of T cells. The T cells express normal-intensity CD2 and CD7. A small subset of T cells express granzyme B, but most are negative. Similarly, rare lymphocytes express faint CD56, but most are negative. The T cells are negative for CD30, ALK1, CD57, PD-1 and TdT. There are <1% Cd34+ blasts, 2-3% CD117+ immature myeloid cells, and a few scattered CD117+ mast cells which do not form aggregates. Factor VIII highlights widely scattered megakaryocytes, which appear morphologically within normal limits and include a few small hypolobated megakaryocytes. CD138 reveals 5% plasma cells, which are predominantly widely scattered and form a few perivascular clusters. The plasma cells express a polytypic mixture of kappa and lambda light chain, with a slight excess of lambda-positive plasma cells and an estimated kappa: lambda ratio of 1:1.5. EBER in situ hybridization shows faint nonspecific background staining similar to the negative control slide and is negative in the T cells. In nutshell the neoplastic cells express: CD3, CD2, CD7, CD4, TCR delta, TIA1 and do not express: CD5, CD8, CD56, TCRbeta, Granzyme B. FLOWCYTOMETRY Abnormal T-cell population identified. No abnormal mature B-cell population detected. Flow cytometry reveals an abnormal CD3 positive T-cell population with loss of expression of CD5 and PD1. T cells show normal expression of CD2, CD3, CD4(partial), CD7, CD45; and without CD8 or CD56. The abnormal population represents 60% of the total white cells. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 3.53 K/uL, RBC 2.72 M/uL, Hgb 7.0 g/dL, Hct 22.9%, MCV 84.2 fl and platelets 107 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 51% neutrophils, 2% immature granulocytes, 40% lymphocytes, 5% monocytes, 1% basophils and 1% eosinophils. Granulocytes and monocytes are normal in morphology. Platelets are normal in size and degree of granularity. RBC shows normocytic normochromic anemia. Lymphocytes are small and mature. BONE MARROW ASPIRATE SMEAR Differential: Myelocytes 4% Metamyelocytes 2% Neutrophils/Bands 10% Monocytes 1% Eosinophils1% Erythroid Precursors 10% Lymphocytes 72% Number of Cells Counted 500 M: E Ratio 1.8 BONE MARROW BIOPSY Histologic sections show hypercellular marrow (90-95%) with an extensive intrasinusoidal and interstitial distribution of atypical lymphoid cells, and they do not form discrete aggregates. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is normal. The blast count is not increased (2% on a 500-cell count). Plasma cells are not increased in number. Megakaryocytes are seen and overall, they are normal in morphology.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Involvement by hairy cell leukemia (10-15% of marrow space). - Cellular marrow with trilineage maturing hematopoiesis. PERIPHERAL BLOOD a. Circulating atypical lymphoid cells b. No overt dysplasia. c. No circulating blasts. IMMUNOHISTOCHEMISTRY The atypical lymphoid cell aggregate is positive for CD20, CD11c, CD25 and Annexin A1. COMMENT Morphology and immunophenotype is consistent with bone marrow involvement with classic hairy cell leukemia. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 2.81 K/uL, RBC 4.39 M/uL, Hgb 12.9 g/dL, Hct 39.6%, MCV 90.2 fl and platelets 51 K/uL. The manual leukocyte differential is 67% neutrophils, 1% bands, 2% atypical lymphocytes, 15% lymphocytes, 9% monocytes, 1% basophils and 5% eosinophils. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. Granulocytes and monocytes are normal in morphology. Platelets are normal in size and degree of granularity. Few large/atypical lymphoid cells are noted. The atypical lymphocytes are medium sized with oval nuclei, ground glass chromatin, inconspicuous nucleoli and abundant pale cytoplasm with circumferential hairy projections. BONE MARROW BIOPSY Histologic sections show variably cellular marrow (less than 10% to 50%) with erythroid predominance, myeloid and erythroid elements with maturation, megakaryocytes are adequate with mostly unremarkable morphology. There is patchy interstitial involvement of bone marrow biopsy with atypical lymphoid cells with abundant cytoplasm. BONE MARROW ASPIRATE SMEAR Hypospicular and hypocellular smears show maturing trilineage hematopoiesis with normal M:E ratio, myeloid and erythroid elements with progressive maturation, no increase in blasts and adequate megakaryocytes with unremarkable morphology.

- Extensive involvement by hairy cell leukemia (80%) - Residual erythroid and megakaryocytic hematopoiesis and mild dyserythropoiesis. - Absent iron stores. COMMENT: Iron stores are absent on iron stain of the aspirate smear. However, the patient is noted to have macrocytic anemia (MCV 99) which likely represents a combination of etiologies for his anemia (including extensive involvement by hairy cell leukemia). Mild dyserythropoiesis is also seen which can be seen in iron deficiency as well as other conditions such as secondary to therapy or in myeloid neoplasms. Correlation with evaluation of serum iron status, careful clinical work up (for the possibility of blood loss) and cytogenetic studies will be helpful in comprehensive evaluation of this patient’s anemia.

DIAGNOSIS 1.Bone marrow, left posterior iliac crest; biopsy and aspirate smear: - B acute Lymphoblastic leukemia, See comment. - Adequate iron stores. PERIPHERAL BLOOD a. Circulating blasts seen. b. No overt dysplasia. FLOWCYTOMETRY Immunophenotyping of peripheral blood by flow cytometry shows predominant a B cell population (about 96% of all the cells analyzed). These B cells have small-intermediate nuclear size (based on forward-scatter signal) and show expression of CD10, CD19, CD38, HLA-DR, CD34, CD13, and TdT. They are negative for CD20, CD2, CD3, CD4, CD8, CD5, CD7, CD56, CD117, MPO, CD64, and CD33. COMMENT The immunophenotype results and morphology are consistent with precursor B lymphoblastic leukemia. Bone marrow aspirate was sent for chromosome analysis, FISH panel for ALL, and PCR for bcr/abl1. PERIPHERAL SMEAR CBC Results: WBC 26.0 K/CMM Hi RBC 3.72 M/CMM Low Hgb 10.9 g/dL Low Hct 32.7 % Low MCV 87.9 fL Normal MCH 29.4 pg Normal MCHC 33.4 g/dL Normal RDW 15.7 % Hi Platelet 122 K/CMM Low MPV 8.3 fL Normal Segs 30.0 % Low Bands 2.0 % Normal Lymphocytes 20.0 % Normal Atypical Lymphs 0.0 % Normal Monocytes 4.0 % Normal Metamyelocytes 1.0 % Normal Promyelocytes 1.0 % Hi Blasts 42.0 % Hi NRBC: 0% PERIPHERAL BLOOD Numerous blasts are seen. Blasts are small with increased N/C ratio, fine chromatin, indistinct nucleoli and scant cytoplasm. No Auer rods are seen. Normocytic hypochromic anemia with a few NRBCs, mild polychromasia. White blood cells are increased in number. Granulocytes are decreased with normal morphology. Lymphocytes, monocytes are normal in morphology. Platelets are decreased with normal morphology. BONE MARROW ASPIRATE SMEAR Differential (%) Lymphoblasts: 79% Promyelocytes: 1% Myelocytes: 1% Metas: 1% Bands & PMN's: 4% Eos: 0% Baso: 0% Monos: 0% Lymphs: 0% Plasma cells: 1% Erythroids: 5% Cellularity: 95% Megakaryopoiesis: Markedly decreased Erythropoiesis: Markedly decreased Granulopoiesis: Markedly decreased Lymphocytes: Decreased mature lymphocytes; marked increase in lymphoblasts. Blasts are small with increased N/C ratio, fine chromatin, indistinct nucleoli and scant cytoplasm. No Auer rods are seen. BONE MARROW BIOPSY Histologic sections show diffuse distribution of lymphoblasts occupying 90% of marrow space. Rare megakaryocytes seen. Occasional myeloid lineage cells seen. Special stain Iron content (biopsy and clot section): Adequate iron stores. Iron Content (aspirate): Adequate iron stores.

DIAGNOSIS 1-3. Bone marrow left posterior iliac crest, biopsy; aspirate and peripheral blood smears: - No diagnostic morphologic or immunophenotypic evidence of previously diagnosed B- lymphoblastic leukemia/lymphoma. See comment. - Mildly hypocellular marrow with erythroid predominant maturing trilineage hematopoiesis. COMMENT Correlation with the forthcoming cytogenetic and molecular studies remains important for minimal residual disease assessment. PERIPHERAL BLOOD a. No circulating blasts seen. b. No overt dysplasia. IMMUNOHISTOCHEMISTRY CD34 shows no increase in blasts (<5% overall). TdT highlights few scattered cells overall <3%, however a focal area with approximately 5-7% TdT-positive cells are present, which are scattered without cluster formation or diagnostic morphologic abnormalities. FLOW CYTOMETRY No abnormal immature B cell population is detected. Immature B cells with normal immunophenotype by this method account for 1.9% of WBC following erythroid lysis. Sensitivity of residual disease analysis is reduced due to absence of the original immunophenotype performed by the same methodology. The testing assumes expression of CD19 by abnormal B cells. If the patient has received therapy known to affect surface CD19 expression, the sensitivity of the test may be markedly reduced. No abnormal mature B or T cell population is identified. CYTOGENETIC STUDIES Cytogenetic analysis will be reported separately. See separate report. MOLECULAR STUDIES Molecular analysis will be reported separately. See separate reports. PERIPHERAL BLOOD CBC: WBC 3.0 L [4.0-11.0 K/mcL] RBC 2.60 L [3.80-5.00 M/mcL] HGB 7.8 L [11.2-15.4 g/dL] HCT 24.2 L [34.3-46.0 %] MCV 93 [80-98 fL] MCH 30.0 [27.0-33.0 pg] MCHC 32.2 [31.0-36.5 g/dL] RDW 15.7 H [12.2-15.1 %] Platelets 79 L [160-400 K/mcL] Neutrophil 77.0 H [32.5-74.8 %] Mono 8.0 [0.0-12.3 %] Eos 0.0 [0.0-4.9 %] Baso 0.0 [0.0-1.5 %] Lymph 15.0 [12.2-47.4 %] Morphology: WBC: Leukopenia with absolute lymphopenia. No circulating blasts seen. RBC: Normochromic normocytic anemia. Platelets: Thrombocytopenia. BONE MARROW BIOPSY Quality: Marginally adequate, subcortical, partially aspirated, fragmented, and with intramedullary hemorrhage Cellularity: Difficult to assess accurately due to the quality of the biopsy, appears approximately hypocellular for age (20-30%) M:E ratio: Decreased Blasts: Not increased Myeloid lineage: Decreased and exhibits maturation Erythroid lineage: Relatively increased and exhibits maturation Megakaryocytes: Adequate in number with occasional loose clusters, "bare nuclei morphology", and hypolobation Lymphocytes: Few, scattered Plasma cells: Rare, scattered BONE MARROW ASPIRATE SMEAR Differential: Blasts 1% Promyelocytes 2% Myelocytes 9% Metamyelocytes 7% Neutrophils/Bands 26% Monocytes 2% Eosinophils 1% Erythroid Precursors 43% Lymphocytes 9% Number of Cells Counted 500 M:E Ratio 1.1 Morphology: Adequate, spicular and cellular aspirate smears with maturing trilineage hematopoiesis and no expanded blast population. The M:E ratio is decreased. Myeloid elements are relatively decreased, present at various stages of maturation, and show no significant dysplasia. Erythroid cells are relatively increased, present at various stages of maturation and show occasional late-stage mitoses. Megakaryocytes are seen without overt atypia. Few small mature- appearing lymphocytes are seen.

DIAGNOSIS 1. Bone marrow, left posterior iliac crest; biopsy and aspirate smear: - Acute myeloid leukemia. - Adequate iron stores. COMMENT The Immunophenotype results and morphology are consistent with acute myeloid leukemia. Bone marrow aspirate was sent for cytogenetics and molecular studies for further categorization of acute myeloid leukemia. PERIPHERAL BLOOD a. Circulating blasts seen. b. No overt dysplasia. FLOWCYTOMETRY The blasts have abnormal expression of CD56 with normal expression of CD117, HLADR, CD13, CD33, CD34, CD38, CD45 (dim), CD71, CD117, CD123 and HLA-DR without CD2, surface CD3, cytoplasmic CD3, CD5, CD7, CD8, CD10, CD11b, CD14, CD15, CD16, CD19, CD20, CD25, CD56, CD64, cytoplasmic CD79a or surface light chains. MPO expression is present on a subset of blasts. CD34 positive myeloid blasts represent 90% of WBC. PERIPHERAL SMEAR CBC Results: WBC 26.0 K/CMM Hi RBC 3.72 M/CMM Low Hgb 10.9 g/dL Low Hct 32.7 % Low MCV 87.9 fL Normal MCH 29.4 pg Normal MCHC 33.4 g/dL Normal RDW 15.7 % Hi Platelet 122 K/CMM Low MPV 8.3 fL Normal Segs 30.0 % Low Bands 2.0 % Normal Lymphocytes 20.0 % Normal Atypical Lymphs 0.0 % Normal Monocytes 4.0 % Normal Metamyelocytes 1.0 % Normal Promyelocytes 1.0 % Hi Blasts 42.0 % Hi NRBC: 0% PERIPHERAL BLOOD Numerous blasts are seen. Blasts are intermediate size with increased N/C ratio, fine chromatin, indistinct nucleoli and scant cytoplasm. Auer rods are seen. Normocytic hypochromic anemia with a few NRBCs, mild polychromasia. White blood cells are increased in number. Granulocytes are decreased with normal morphology. Lymphocytes, monocytes are normal in morphology. Platelets are decreased with normal morphology. BONE MARROW ASPIRATE SMEAR Differential (%) Blasts: 79% Promyelocytes: 1% Myelocytes: 1% Metas: 1% Bands & PMN's: 4% Eos: 0% Baso: 0% Monos: 0% Lymphs: 0% Plasma cells: 1% Erythroids: 5% Cellularity: 95% Megakaryopoiesis: Markedly decreased Erythropoiesis: Markedly decreased Granulopoiesis: Markedly decreased Lymphocytes: Decreased mature lymphocytes; Myeloid blasts: Marked increase in myeloblasts. Blasts are intermediate size with increased N/C ratio, fine chromatin, indistinct nucleoli and scant cytoplasm. Auer rods are seen.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Persistent/relapsed acute myeloid leukemia with monocytic differentiation (blasts 79%). - Hypercellular marrow with extensive involvement by acute leukemia and absent hematopoiesis. BONE MARROW BIOPSY&CLOT Histologic sections show inadequate small biopsy with limited hypercellular and crushed marrow spaces and clot sections with hypercellular marrow (100%) with extensive involvement by sheets of large blasts with folded nuclei and no hematopoietic elements. BONE MARROW ASPIRATE SMEAR Spicular and hypercellular smears show sheets of blasts with monocytic morphology and rare background myeloid elements. Differential: Blasts/Promonocytes 79% Promyelocytes 5% Myelocytes 3% Metamyelocytes 1% Neutrophils/Bands 2% Monocytes 7% Eosinophils 1% Lymphocytes 2% Erythroid precursors 0% Number of Cells Counted 500

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Hypocellular marrow with maturing trilineage hematopoiesis. - No morphologic or immunophenotypic evidence of involvement by leukemia. IMMUNOHISTOCHEMISTRY No CD34 positive blasts seen in core biopsy. Controls were appropriate. BONE MARROW BIOPSY Histologic sections show hypocellular marrow (20-30%) with erythroid predominance; myeloid and erythroid elements with maturation, megakaryocytes are few and show hyperchromatic nuclei. BONE MARROW ASPIRATE SMEAR Morphology: Hypospicular and hypocellular smears show crush artifact and maturing trilineage hematopoiesis with normal M:E ratio, myeloid and erythroid elements with progressive maturation, no increase in blasts and adequate megakaryocytes with unremarkable morphology.

DIAGNOSIS 1-2. Bone marrow, left posterior iliac crest; biopsy and aspirate smear: - Hypercellular marrow with myeloid hyperplasia, mildly left shifted maturation, dysmyelopoiesis, reduced erythroid and megakaryocytic elements with mild dyspoietic changes and absolute peripheral blood monocytosis. See comment. COMMENT The findings are highly suggestive of a myeloid neoplasm such as chronic myelomonocytic leukemia (CMML). Suggest correlation with cytogenetic and genomic studies and clinical findings. BONE MARROW BIOPSY Histologic sections show hypercellular marrow (80%) with increased M:E ratio, myeloid elements are increased with left shifted maturation, reduced erythroid elements with maturation, megakaryocytes are mildly reduced with increased pleomorphism. No increase in blasts is seen. BONE MARROW ASPIRATE SMEAR Spicular and hypercellular smears show trilineage hematopoiesis with increased M:E ratio, mildly left shifted myeloid maturation, dysmyelopoiesis, reduced erythroid elements with megaloblastic changes and increased pronormoblasts, no increase in blasts, reduced megakaryocytes with increased pleomorphism and occasional dysplastic forms with widely separated nuclei.

DIAGNOSIS 1. Bone marrow, left posterior iliac crest; biopsy and aspirate smear: - Mildly hypercellular marrow with normal M:E ratio, mild atypical megakaryocytic hyperplasia and reported JAK2 mutation consistent with myeloproliferative neoplasm. See comment. COMMENT The morphological findings including an increase in atypical megakaryocytes. Increased platelet count together with the absence of marrow fibrosis and erythrocytosis, and reported JAK2 mutation, is concerning for a possible diagnosis of essential thrombocythemia (ET). However, the pre polycythemic phase of polycythemia vera (PV) cannot be ruled out. Suggest correlation with laboratory studies including an erythropoietin level, follow up of hemoglobin levels and additional genetic testing (such as BCR-ABL1) to further rule out other myeloproliferative neoplasms. IMMUNOHISTOCHEMISTRY CD71 highlights the erythroid precursors in adequate numbers. CD61 highlights increased number of singly scattered megakaryocytes and includes enlarged forms. CD34 and CD117 show no increase in blasts. FLOW CYTOMETRY There is no flow cytometric evidence of B- or T-cell lymphoma or acute leukemia. The study shows a relative normal T:B ratio for bone marrow. The T-cell population expresses pan-T-cell antigens (CD2, CD3, CD5, and CD7) in a non-aberrant fashion. The CD4:CD8 ratio is normal. The B cells are a minor population. They express pan-B-cell antigens (CD19 and CD20) in a non-aberrant fashion. CD10 and CD5 are not significantly expressed by the B-cells. Staining for light chains does not reveal monotypia. The NK-cell population is normal in number. No significant increase in blasts is noted. Maturing granulocytic / monocytic elements exhibit no diagnostic antigenic aberrancies. No abnormal plasma cell population is detected. CYTOGENETIC STUDIES Normal female karyotype. No consistent numerical or structural chromosome abnormalities were observed within the limits of the technology used in this analysis. MOLECULAR STUDIES + JAK2 mutation. PERIPHERAL BLOOD CBC: 01/11/20xx WBC 13.3 HGB 15.2g/dL HCT 47.8% MCV 92.7fL RDW 16.3% Platelets 454K/uL Differential: Lymphocytes 14.8% Monocytes 5.7% Granulocytes 79.5% Morphology: No granulocytic dysplasia. Lymphocytes are small and mature. Red blood cells are morphologically unremarkable. Platelets are increased. Few large platelets seen. BONE MARROW BIOPSY Quality: Adequate Cellularity: Mildly hypercellular (50-60%) M:E ratio: Increased Blasts: Not increased Myeloid lineage: Adequate with complete maturation Erythroid lineage: Adequate with progressive maturation Megakaryocytes: Mildly increased and show enlarged atypical forms Lymphocytes: Present Plasma cells: Present BONE MARROW ASPIRATE SMEAR Differential: PMNs and precursors 72% Myelocytes 4% Promyelocytes 1% Nucleated red blood cells 12% Eosinophils 1% Lymphocytes 10% Number of Cells Counted 200 Special stain Iron stain performed on the aspirate smear and biopsy sections shows trace to absent reticuloendothelial iron stores and decreased sideroblastic iron. No ring sideroblasts are noted.

The morphologic, immunohistochemical and special stain findings are most compatible with a myeloproliferative neoplasm, especially in light of the history of splenomegaly. Although the main significant peripheral blood finding now is anemia, the patient did have mild thrombocytosis approximately seven to eight years ago and more recently had a mild degree of peripheral blood basophilia. Given the presence of megakaryocytic atypia of this degree, the primary consideration would be primary myelofibrosis (PMF). There is some overlap between PMF and other myeloproliferative neoplasms, especially essential thrombocythemia (ET). Testing for BCR/ABL and JAK2 mutations would be helpful, as would testing for MPL and CALR if indicated. Myelofibrosis can occur in myelodysplastic syndromes but is uncommon and aside from atypical megakaryocytes there is little morphologic evidence of dyspoiesis.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest; biopsy and aspirate smear: - Mildly hypercellular bone marrow with maturing trilineage hematopoiesis and mild atypical megakaryocytic hyperplasia; see comment. COMMENT: In a patient with a reported history of a JAK2 mutation and erythrocytosis, the findings are consistent with a myeloproliferative neoplasm. However, complete evaluation is not possible without a concurrent CBC and peripheral blood smear. Based on the most recent CBC (dated --), the patient would not meet the diagnostic criteria for any MPN based on WHO criteria, though based on the CBC reported in clinical notes (dated --), the patient’s hematocrit of 48.7% is suggestive of polycythemia vera. However, per clinical notes, the patient was also recently phlebotomized, which also confounds diagnosis. Correlation with molecular genetics (JAK2, MPL and CALR mutations in particular), cytogenetics and laboratory findings (serum EPO levels) is recommended.

DIAGNOSIS Bone marrow aspirate, biopsy, clot and peripheral blood smears: 1. Hypercellular marrow with maturing trilineage hematopoiesis with granulocytic and megakaryocytic hyperplasia (with clustering), no increase in blasts, no reticulin fibrosis. See comment. PERIPHERAL BLOOD a. Leukocytosis. b. Thrombocytosis. c. Normocytic normochromic anemia. d. No circulating blasts. e. No overt dysplasia. COMMENT The bone marrow is hypercellular with megakaryocytic hyperplasia and clustering, granulocytic proliferation, along with peripheral blood leukocytosis, thrombocytosis and anemia. The features warrant a workup to rule out a myeloproliferative neoplasm, especially a pre fibrotic stage of Primary Myelofibrosis. A thorough MPN molecular panel comprising of JAK2, CALR, MPL, BCR-ABL and other clonal molecular markers as ASXL, EZH2, TET2, IDH1/2, SRSF2, SF3B1 etc should be performed to establish clonality. A diagnosis of myeloproliferative neoplasm cannot be rendered unless clonality is established. IMMUNOHISTOCHEMISTRY CD34 and CD117 show no increase in blasts (<5% cellularity). Reticulin stain show no fibrosis. Controls worked appropriately. FLOW CYTOMETRY No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T-cell lymphoma or leukemia is identified. CD34 blasts amount for <1% of total white blood cells. CYTOGENETICS MDS-FISH shows no MDS related cytogenetic abnormalities per report by XXXX. See comment. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 21.6 K/uL, RBC 2.83 M/uL, Hgb 7.5 g/dL, Hct 25.1%, MCV 88.7 fl and platelets 715 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 84% neutrophils, 1% bands, 9% lymphocytes, 6% monocytes. Granulocytes, lymphocytes, and monocytes are normal in morphology. Platelets are normal in size and degree of granularity. BONE MARROW ASPIRATE SMEAR Differential: Blasts % Promyelocytes % Myelocytes % Metamyelocyte % Neutrophils/Precursors % Lymphocytes % Monocytes % Eosinophils % Basophils % Plasma cells % Erythroid Precursors % Number of cells counted 500 ME ratio Normal Bone marrow aspirate smears are spicular and adequately cellular for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is increased. The blast count is not increased (2% on a 500-cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are increased with clustering. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Hypercellular for age (~80%) M:E ratio: Increased Blasts: Not increased Myeloid lineage: Predominant, shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Increased in number with clustering. Megakaryocytes are large with hyperchromatic bulbous cloud–like nuclei Lymphocytes: Interstitially scattered Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Siderocyte: No ring sideroblasts are identified. Reticulin: No reticulin fibrosis seen (MF:0/3)

DIAGNOSIS 1. Bone marrow, left iliac crest, core biopsy and aspirate smears: - JAK2 V617F positive myeloproliferative neoplasm with fibrosis, MF-2 to focal MF-3, see comment. - Hypercellular marrow with myeloid and megakaryocyte hyperplasia with megakaryocyte clustering and atypia. COMMENT Per outside report molecular studies detected a JAK2 p.V617F mutation. The overall findings are consistent with a JAK2 positive myeloproliferative neoplasm with moderate to focally marked fibrosis. The differential diagnosis includes primary myelofibrosis, and post-essential thrombocythemia myelofibrosis or post- polycythemia vera myelofibrosis, in the proper clinical context. In addition, there are scattered cells identified on the aspirate smear consistent with mildly increased mast cells with slightly atypical morphology; however, the aspirate is limited and a complete work-up is unavailable on the submitted material. IMMUNOHISTOCHEMISTRY CD34 stain shows no increase in blasts (<5%). CD34 stain highlights increased vessels and dilated sinusoids. FLOW CYTOMETRY Per report, In the sample analyzed, there is no evidence for abnormal myeloid maturation or an increased blast population. There is no evidence for a lymphoproliferative disorder. Immunophenotypic Analysis: Viability 7AAD: 95% There is a mixed population of maturing myeloid cells, B cells and T cells. No abnormal myeloid maturation is seen. There is no increase in CD34 positive blasts, and they comprise 1.4% of the total cells. The B-cells (0.3% of total) are polytypic and the T-cells (3.0% of total) show no pan T-cell antigen deletion. CYTOGENETIC STUDIES Per report, No BCR/ABL1 fusion was detected by FISH study in a recent blood sample. MOLECULAR STUDIES Per report, Result Summary: Abnormal Markers identified in sample: ASXL1 p.Tyr591* JAK2 p.Val617Phe One unclear variant in DNMT3A (p.Arg635Trp). A nonsense mutation in ASXL1 (p.Tyr591*) was detected in this patient's sample. Of note, this low level ASXL1 mutation was verified by confirmation testing. JAK2 p.Val617Phe (V617F) mutation detected in this patient's sample. An unclear variant in DNMT3A (p.Arg635Trp) was detected in this patient's sample. A comprehensive review of the patient's medical record (including oncology notes, laboratory results and imaging reports) was performed. PERIPHERAL BLOOD Per report, CBC (9/18/2018): WBC 17.65 K/uL HGB 13.7 g/dL HCT 42.7 % MCV 87.5 fL RDW 20.3 % ANC 14.76 K/uL Platelets 249 K/uL Morphology: Per report, the peripheral blood smear shows leukoerythroblastosis. BONE MARROW BIOPSY Quality: Adequate (small, fragmented). Cellularity: Hypercellular (approximately 90%). M:E ratio: Increased. Blasts: Not increased. Myeloid lineage: Increased, exhibits full maturation. Erythroid lineage: Decreased, exhibits full maturation. Megakaryocytes: Increased (up to 15 per high power field), with focal loose clustering and atypical forms (hyperchromatic and hyperlobated). Lymphocytes: Scattered. Plasma cells: Scattered. Special stains: Reticulin stain shows moderate to focally marked increase in reticulin fibrosis (MF-2 to focal MF-3). Iron: No iron particles are present in decalcified core biopsy. BONE MARROW ASPIRATE SMEAR Per report, Differential: Myeloblasts <1% Promyelocytes <1% Myelocytes 9% Metamyelocytes 10% Neutrophils/Bands 69% Monocytes 4% Eosinophils 1% Basophils <1% Lymphocytes 5% Plasma Cells <1% Pronormoblasts <1% Normoblasts 2% M:E ratio 46.5=1 Morphology: The aspirate smears are aspicular, hypocellular and hemodilute. There is no increase in blasts. Myeloid elements are increased in proportion with full maturation and unremarkable morphology. Erythroid elements are decreased in proportion with full maturation and no significant dysplasia. Megakaryocytes are not seen. Scattered cells with pyknotic round nuclei and abundant cytoplasm with variably prominent granules, consistent with mildly atypical mast cells. Per report Special stain: Iron stain is non-contributory.

FORMAT 1 DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Myeloproliferative neoplasm with marked osteosclerosis and fibrosis. - Peripheral blood with leukoerythroblastic picture (5% blasts) and thrombocytosis. BONE MARROW BIOPSY Histologic section shows markedly osteosclerotic and fibrotic marrow with hypercellularity (90%; cellularity may not be truly representative due to marked fibrosis), marked atypical megakaryocytic hyperplasia, erythroid and maturing myeloid elements present and dilated sinusoids. Reticulin stain shows marked myelofibrosis (MF3). Trichrome is pending. Differential: Markedly hypocellular touch imprints are available for evaluation and show few osteoblasts and rare myeloid elements; no aspirate smears provided. Iron: Non-contributory due to lack of spicules and erythroid elements. FORMAT 2 DIAGNOSIS 1. Bone marrow, right iliac crest; biopsy and aspirate (MS17-1139, 2/6/17, 18 Stained Slides): - Myeloproliferative neoplasm with myelofibrosis and osteosclerosis. COMMENT The overall morphologic findings in light of reported CALR type 1 mutation, are those of primary myelofibrosis. However, correlation with tandem CBC, clinical findings (organomegaly), laboratory studies and evaluation of peripheral blood smear is required for definitive characterization. It is noted that the biopsy is dated XX/XX/XXXX. We also noted the current CBC shows leukocytosis (14.4), thrombocytosis (628), anemia (11.5), absolute peripheral blood monocytosis (13%, 1.9) and circulating myeloid precursors. It is likely that the presence of monocytosis may represent progression of the underlying myeloproliferative neoplasm. Even though, the current CBC findings raise the possibility of a myelodysplastic/myeloproliferative neoplasm such as CMML, the overall findings support a myeloproliferative neoplasm (primary myelofibrosis) in this marrow biopsy. Correlation with historic/original CBC’s and peripheral blood smears and new genetic/genomic studies is recommended to further understand this myeloid neoplasm. BONE MARROW BIOPSY Hypercellular marrow (80-90%) with fibrosis, and hematopoiesis with normal M:E ratio, myeloid and erythroid maturation present, atypical megakaryocytic hyperplasia with clustering and enlarged megakaryocytes with bulbous and hyperchromatic nuclei. Special stains: Stainable iron is present. There is significant increase in reticulin fibers (at least 2+ and likely 3+). Trichrome stain will be helpful in further characterization. BONE MARROW ASPIRATE SMEAR Morphology: -Aspicular and hypocellular smears show maturing myeloid elements and erythroid precursors. No dysplastic changes are seen in limited cellularity available. Megakaryocytes are not seen. Platelet clumps are present. Special stain Iron: Stainable iron is non-contributory due to lack of spicules. Ring sideroblasts: Absent PERIPHERAL BLOOD Not available IMMUNOHISTOCHEMISTRY Immunohistochemical studies received at MSKCC show: CD3 and CD20: Scattered small T and B cells. CD138: Less than 5%, Interstitial and perivascular pattern. CD34: Less than 1% scattered cells. CD117: Scattered mast cells and myeloid and erythroid precursors. CD61: Increased megakaryocytes of variable size; in clusters or aggregates. Myeloperoxidase: 70-80% positive cells. CD71: 20-30% positive cells in clusters. FLOW CYTOMETRY Per report, No diagnostic immunophenotypic abnormalities detected. COMMENTS This is a possible hemodiluted specimen. No immunophenotypic evidence of a lymphoproliferative disorder, acute leukemia, increase in blasts, or plasma cell neoplasm is identified. Myeloproliferative neoplasms and myelodysplastic syndromes may not show antigenic abnormalities on myeloid cells and cannot be ruled out by flow cytometry.

DIAGNOSIS 1-3. Bone Marrow left iliac crest; core biopsy, aspirate, clot and peripheral blood smears: - JAK2 positive myeloproliferative neoplasm with moderate to severe reticulin fibrosis (MF 2-3+/3) and increase in blasts (13% by aspirate differential). See comment. PERIPHERAL BLOOD a. Leukocytosis with left shift. b. Circulating blasts seen (8%). c. No overt dysplasia. d. Normocytic normochromic anemia. COMMENT Per clinical charts, initially in 20XX the patient presented with increased platelets (1428 k/ul), increased WBC counts (13.05 k/ul) and mildly increased hemoglobin (16.1 g/dl). There was no bone marrow biopsy at that moment of time, however a JAK2V617 F mutation was positive. The patient has been treated with phlebotomies and hydroxyurea since then. Currently the patient has normal hemoglobin and platelet counts with very high WBC levels, left shift and increase in blasts, with increased myelofibrosis. Current biopsy represents a progression of his initial Myeloproliferative neoplasm and the differential could include Post ET myelofibrosis, post PV myelofibrosis, overt myelofibrotic phase of PMF. No BCR-ABL fusion transcripts were detected per the report from Date xx, ruling out CML. The increase in blasts (13%) currently denotes that the patient's myeloproliferative neoplasm is in an accelerated phase at this time. Correlation with molecular and cytogenetic studies is recommended. IMMUNOHISTOCHEMISTRY CD34 and CD117 show an increase in blasts (10-15% of total cellularity). The controls worked appropriately. FLOW CYTOMETRY Abnormal myeloid blast population detected. Flow cytometry identifies an abnormal myeloid blast population expressing CD13, CD33, CD34, CD38, CD45, CD71, CD117, and HLA-DR without CD3, cy CD3, CD11b, CD14, CD15, CD19, CD20, CD56, CD123, cy-MPO, TdT, CD64 and aberrantly expressing CD2, CD5, and CD7. CD34 positive myeloid blasts represent 6% of WBC. Flow cytometry is generally not a sensitive methodology in the context of myeloproliferative disorders, correlation with molecular studies for JAK2, CALR and MPL mutation status and/or BCR-ABL and morphology is advised if clinically indicated. No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T-cell lymphoma or leukemia is identified. CYTOGENETIC STUDIES: Pending. MOLECULAR STUDIES: Pending. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 69.01 K/uL, RBC 3.93M/uL, Hgb 9.9 g/dL, Hct 34.1%, MCV 86.8 fl and platelets 176 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 8% blasts,2% metamyelocytes, 1% myelocytes, 50% neutrophils, 25% bands, 5% lymphocytes, 3% monocytes, 1% basophils and 5% eosinophils. Granulocytes, lymphocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. BONE MARROW ASPIRATE SMEAR Bone marrow aspirate smears are spicular and adequately cellular for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is increased. The blast count is increased (13% on a 500-cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are seen and overall show no overt dysplasia. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Hypercellular for age (~90%) M:E ratio: Increased Blasts: Increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Increased in number, occasional hypolobated forms seen Lymphocytes: Interstitially scattered Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Absent. Siderocyte: No ring sideroblasts are identified. Reticulin: Moderate to severe reticulin fibrosis seen (MF 2-3+/3)

DIAGNOSIS 1-2. Bone marrow, biopsy, aspirate smears and peripheral blood: - Markedly hypercellular marrow (up to 95%) with left shifted myeloid hyperplasia and dwarf megakaryocytes consistent with myeloid neoplasm. - Peripheral blood with leukocytosis, circulating myeloid precursors and rare nucleated red blood cells. See comment. COMMENT The overall morphologic findings support a diagnosis of chronic myeloid leukemia (CML). However, genetic confirmation of the presence of t(9;22)/BCR-ABL1 fusion gene transcript is required for a definitive diagnosis of CML. Suggest correlation with pending genetic studies. IMMUNOHISTOCHEMISTRY CD34 and CD117 show no increase in blasts (<5% cellularity). FLOW CYTOMETRY Abnormal myeloid blast population detected. Flow cytometry identifies an abnormal myeloid blast population expressing CD13, CD33, CD34, CD38, CD45, CD71, CD117, and HLA-DR without CD3, cy-CD3, CD11b, CD14, CD15, CD19, CD20, CD56, CD123, cy-MPO, TdT, CD64 and aberrantly expressing CD2, CD5, and CD7. CD34 positive myeloid blasts represent 6% of WBC. Flow cytometry is generally not a sensitive methodology in the context of myeloproliferative disorders, correlation with molecular studies for JAK2, CALR and MPL mutation status and BCR-ABL and morphology is advised if clinically indicated. No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T-cell lymphoma or leukemia is identified. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 35.57 K/uL, RBC 5.25 M/uL, Hgb 16.0 g/dL, Hct 50.9%, MCV 97.1fl Platelets 291 K/uL. Peripheral Blood Manual Differential (100 cells): Neutrophils: 60% Bands: 4% Metamyelocytes: 5% Myelocytes: 10% Promyelocytes: 0% Blasts: 1% Eosinophils: 1% Basophils: 2% Lymphocytes: 12% Monocytes: 5% Morphology: WBC: Occasional circulating blasts. Predominantly mature neutrophils with left shift. RBC: Normocytic nomochromic RBC. Occasional nucleated RBC. Platelets: Adequate in number with unremarkable morphology. BONE MARROW ASPIRATE SMEAR Spicular and hypercellular smears show trilineage hematopoiesis with increased M:E ratio, mildly left shifted myeloid maturation, reduced erythroid elements, no increase in blasts, and mildly increased megakaryocytes with small and dwarf forms. BONE MARROW BIOPSY/CLOT Histologic sections show hypercellular marrow (95%) with increased M:E ratio, myeloid elements increased with left shifted maturation, reduced erythroid elements with maturation, megakaryocytes are mildly increased and show numerous small dwarf forms. No increase in blasts is seen. Clot has similar features as the biopsy. Iron stain does not show an increase in ring sideroblasts.

DIAGNOSIS 1. Bone marrow; core biopsy and aspirate: - Markedly hypercellular marrow (up to 100%) with atypical/dysplastic megakaryocytic hyperplasia, left shifted myeloid and erythroid maturation and reported moderate reticulin fibrosis. See comment. COMMENT Molecular genetic testing shows BCR-ABL1 p210 fusion gene transcript supporting the diagnosis of CML. The hypercellular marrow shows left shifted erythroid and myeloid elements and atypical/dysplastic megakaryocytic hyperplasia. Reported cytogenetic studies showed presence of chromosomal abnormalities in addition to t(9;22). Overall, the findings suggest the progression of patient’s known CML. However, for better characterization, an optimal evaluation including CBC findings, a peripheral blood smear and optimal marrow aspirate smear or touch imprints is required.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Acellular/markedly hypocellular marrow with rare focal erythroid and myeloid elements and rare megakaryocytes. COMMENT The findings are those of an aplastic/hypoplastic marrow. No definitive morphologic evidence of myelodysplasia is seen. Prolonged pancytopenia in this patient might be due to reported radiotherapy and/or an idiosyncratic/allergic reaction to systemic therapy. On the other hand, other possible etiologies such as nutritional deficiencies (serum vitamin B12, folate, and/or copper levels), ethanol toxicity, infections (in particular viral etiologies) or autoimmune illnesses need to be investigated. Suggest close patient follow up, correlation with cytogenetic studies and repeat marrow studies if there is a clinical indication after serial observation of CBC. BONE MARROW BIOPSY Histologic sections show acellular to markedly hypocellular marrow (less than 5%) with rare focal erythroid island, rare focal myeloid elements and megakaryocytes absent. Osteoclastic activity is seen.

DIAGNOSIS 1-3. Bone marrow, right posterior iliac crest, biopsy, aspirate and peripheral blood smears: - Normocellular marrow with trilineage maturing hematopoiesis. - No diagnostic morphologic and immunophenotypic evidence of involvement by lymphoma. COMMENT No diagnostic morphologic evidence of myelodysplasia is seen. Prolonged leukopenia and thrombocytopenia in this patient might be due to idiosyncratic / allergic reaction to systemic therapy or ongoing treatment. On the other hand, infections (in particular viral etiologies), autoimmune illnesses or immune mediated peripheral destruction/sequestration associated etiologies need to be investigated. Suggest close patient follow up, correlation with cytogenetic studies and repeat marrow studies if there is a clinical indication after serial observation of CBC. PERIPHERAL BLOOD a. No circulating blasts. b. No overt dysplasia. IMMUNOHISTOCHEMISTRY - CD34 and CD117 show no increase in blasts (<5% cellularity). - CD138 show no overt increase in plasma cells (<10% cellularity). Controls worked appropriately. FLOW CYTOMETRY No abnormal mature B- or T-cell populations detected. No immunophenotypic evidence of involvement by B- or T- cell lymphoma or leukemia is identified. CYTOGENETICS: MDS-FISH pending. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 2.81 K/uL, RBC 4.39 M/uL, Hgb 12.9 g/dL, Hct 39.6%, MCV 90.2 fl and platelets 51 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 67% neutrophils, 1% bands, 17% lymphocytes, 9% monocytes, 1% basophils and 5% eosinophils. Granulocytes, lymphocytes and monocytes are normal in morphology. Platelets are normal in size and degree of granularity. No large/atypical lymphoid cells are noted. BONE MARROW ASPIRATE SMEAR Bone marrow aspirate smears are spicular and adequately cellular for evaluation. The myeloid and erythroid series proceed to maturation. There is no significant dysmyelopoiesis or dyserythropoiesis. The myeloid to erythroid ratio is normal. The blast count is not increased (2% on a 300 cell count). Lymphocytes and plasma cells are not increased in number. Megakaryocytes are seen and overall they are normal in morphology. No atypical lymphoid cells are noted. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Normocellular for age (~30%) M:E ratio: Normal Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, unremarkable morphology Lymphocytes: Interstitially scattered Plasma cells: Interstitially scattered Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Siderocyte: No ring sideroblasts are identified.

DIAGNOSIS - Cellular marrow with trilineage maturing hematopoiesis. - No diagnostic morphologic or immunophenotypic evidence of myeloid neoplasm. COMMENT Reported pancytopenia in this patient might be due to multiple therapies. However, other possible etiologies such as nutritional deficiencies (iron, vitamin B12/folate, copper levels), ethanol toxicity, infections (in particular viral etiologies) or autoimmune illnesses need to be investigated. Suggest close patient follow up, correlation with cytogenetic and molecular genetic studies and repeat marrow studies if there is a clinical indication after serial observation of CBC. The submitted report references a concurrent flow cytometry study (original report not submitted) which identified a minute abnormal population of plasma cells with CD56 and CD117 expression, but no definitive light chain expression. While not definitively diagnostic, this finding is suspicious for low level involvement by a plasma cell neoplasm. Confirmation with repeat biopsy and flow cytometry is recommended.

Few lymphoid aggregates are identified likely representing reactive etiology. No diagnostic evidence of myelodysplastic syndrome is seen. Prolonged pancytopenia in this patient might be due to an idiosyncratic/allergic reaction to systemic therapy. Other possible etiologies such as nutritional deficiencies (serum vitamin B12, folate, and/or copper levels), ethanol toxicity, infections (in particular viral etiologies) and autoimmune illnesses need to be investigated. Suggest close patient follow up, correlation with cytogenetic studies and repeat marrow studies if there is clinical indication after serial observation of CBC.

No direct hematopoietic cause of the patients macrocytic anemia is identified. Non-hematopoietic causes to consider include medications, nutritional deficiencies, liver disease, thyroid abnormalities and much less likely B12/folate deficiency given the patients normal values. No leukemia or lymphoma is identified. Clinical and cytogenetic correlation is recommended.

No direct hematopoietic cause for the patient's pancytopenia is appreciated. Hypolobated megakaryocytes are present with an exuberant background megaloblastic erythroid population and no increase in blasts by CD34. Overall, the findings are nonspecific with a differential including both an early evolving low grade myelodysplastic syndrome as well as a wide variety of reactive causes. Non-hematopoietic causes to consider include medications, nutritional deficiencies, liver disease, thyroid abnormalities and B12/folate deficiency. Clinical correlation and correlation with cytogenetic studies including FISH is recommended. If pancytopenias persist, a repeat bone marrow biopsy may be warranted if clinically indicated.

The morphologic findings in the context of pancytopenia are suspicious for myelodysplastic syndrome, which would be confirmed if characteristic cytogenetic abnormalities are present. If absent, the differential diagnosis would expand to include drug and toxin exposure, immunologic disorders, nutrient deficiencies and other causes. If cytogenetic and FISH analysis are negative, a repeat bone marrow biopsy in several months is recommended for further assessment.

Morphologic features of myelodysplasia are not present at this time. This does not exclude an evolving myelodysplastic syndrome, for which correlation with cytogenetics, clinical history and continued follow-up is recommended. Additional causes of myelosuppression like toxin, infection and underlying illness should be excluded.

The bone marrow demonstrates significant dyserythropoiesis. No significant dysplasia is identified in the myeloid or megakarocytic lineages and there is no increase in blasts. In conjunction with normal B12, folate and methylmalonic acid levels, and provided other secondary causes are ruled out (other nutritional deficiencies, medication related changes, etc), the findings can be consistent with a low-grade myelodysplastic syndrome (myelodysplastic syndrome with single lineage dysplasia). No high-grade features are identified. Correlation with pending MDS FISH studies is recommended.

Considerations include drug/medication effect, toxin exposure, post infection change, autoimmune disease and paroxysmal nocturnal hemoglobinuria.

DIAGNOSIS 1-3. Bone marrow, right posterior iliac crest, biopsy, aspirate and peripheral blood smears: - Small abnormal myeloblasts detected by flow cytometry (0.37% of WBC), consistent with persistent low-level involvement of myeloid neoplasm. - Acellular marrow with virtually absent trilineage hematopoiesis. COMMENT Bone marrow biopsy is acellular and shows virtually absent trilineage hematopoiesis, precluding a morphologic evaluation of dyspoiesis. There is no dysgranulopoiesis seen on peripheral blood smear. Small abnormal myeloblasts detected by flow cytometric studies is consistent with low level persistent involvement by previously diagnosed myeloid neoplasm (hypocellular MDS). IMMUNOHISTOCHEMISTRY CD34 and CD117 show no increase in blasts (<5% cellularity). FLOW CYTOMETRIC ANALYSIS Abnormal myeloid blast population detected. Flow cytometry identifies an abnormal myeloid blast population having abnormal expression of CD7 (partial), CD11b (partial), CD13 (dim), CD33 (uniform bright), CD38 (dim to intermediate), CD117 (subset absent), CD123 (uniform intermediate), with normal expression of CD4, CD34, CD45, CD71 and HLA-DR without CD2, CD5, CD14, CD15, CD16, CD19, CD25, CD56 or CD64. CD34 positive myeloid blasts represent 0.37% of WBC. Mostly mature granulocytes and monocytes seen in the sample, suggestive of hemodilution. Sensitivity of analysis for myeloid neoplasm is reduced. Results may not be indicative of bone marrow composition. CYTOGENETIC STUDIES Cytogenetic analysis will be reported separately. See separate report. MOLECULAR STUDIES Molecular analysis will be reported separately. See separate report. The interpretation of bone marrow biopsy results is based in part on the decalcification procedure performed. PERIPHERAL BLOOD CBC: WBC 3.6 L [4.0-11.0 K/mcL] RBC 2.75 L [3.95-5.54 M/mcL] HGB 8.0 L [12.5-16.2 g/dL] HCT 24.4 L [37.5-49.3 %] MCV 89 [80-98 fL] MCH 29.1 [27.0-33.0 pg] MCHC 32.8 [31.0-36.5 g/dL] RDW 15.6 H [12.2-15.1 %] Platelets 27 L [160-400 K/mcL] Neutrophil 71.0 [32.5-74.8 %] Mono 16.0 H [0.0-12.3 %] Eos 3.0 [0.0-4.9 %] Baso 0.0 [0.0-1.5 %] Myelocyte 1.0 H [0.0-0.0 %] Metamyelocyte 3.0 H [0.0-0.0 %] Lymph 5.0 L [12.2-47.4 %] Variant Lymph 1.0 [%] Nucleated RBC 4 [/100(WBCs)] Morphology: WBC: No circulating blasts. RBC: Anisocytosis and polychromasia; few nucleated RBCs. PLT: Unremarkable morphology. BONE MARROW BIOPSY Quality: Fragmented and hemorrhagic biopsy Cellularity: Essentially acellular M:E ratio: NA Blasts: Absent Myeloid lineage: Rare to absent Erythroid lineage: Rare to absent Megakaryocytes: Absent Lymphocytes: Few scattered Plasma cells: Rare Special stains: Reticulin stain shows no increase in reticulin fibrosis. BONE MARROW ASPIRATE SMEAR Differential Not Performed. Aspirate smears and touch imprint are aspicular, hemodilute and hypocellular, precluding an optimal morphologic evaluation and an accurate differential count. Few scattered maturing myeloid and erythroid elements are present. Megakaryocytes and blasts are not seen.

DIAGNOSIS - Normocellular marrow with erythroid predominant maturing hematopoiesis, myeloid dysplasia and increased blasts (15-20%), consistent with at least high-grade myelodysplasia (MDS-EB2) and worrisome for evolving acute myeloid leukemia. COMMENT Suggest correlation with clinical findings, current CBC and if indicated a new bone marrow biopsy/aspiration for evaluation. The findings in this patient with history of prostate cancer treated with radiation therapy might represent therapy related myeloid neoplasm. IMMUNOHISTOCHEMISTRY CD34 and CD117 highlight the blasts are increased (~15%, with focal areas approaching 20%). Glycophorin A and MPO highlight the erythroid predominance. MPO also highlights the clusters of blasts. CD42b highlights the adequate megakaryocytes. CD3 and CD20 highlight scattered T and B cells. AE1/AE3: Negative. PERIPHERAL BLOOD Per report, CBC (6/27/2018): WBC 1.70K/uL RBC 4.31M/uL HGB 13.4g/dL HCT 40.1% MCV 93.0fL MCH 31.1pg MCHC 33.4g/dL RDW 14.4% Platelets 214K/uL Differential: Neutrophils 53.0% Lymphocytes 26.6% Eosinophils 12.000% Monocytes 7.26% Basophils 1.17% Peripheral smear not available for evaluation BONE MARROW BIOPSY Histologic sections show normocellular marrow (40%) with erythroid predominance, myeloid maturation left shifted, increased interstitial clusters of immature cells, and megakaryocytes adequate with mostly unremarkable morphology. BONE MARROW ASPIRATE SMEAR Differential: Neutrophils/Precursors 32% Erythroid Precursors 40% Promyelocytes 2% Blasts 15% Lymphocytes 5% Monocytes 2% Eosinophils 4% Basophils 0% Plasma cells 0% Number of cells counted 500 ME ratio Decreased: 1:1 (excluding blasts) Special stain Iron stores are increased. No ring sideroblasts identified. Reticulin stain shows no increase in fibers. Morphology: Spicular and cellular smears show maturing trilineage hematopoiesis with reduced M:E ratio, erythroid predominance, myeloid elements with left shifted maturation, dysplastic granulocytes, increased blasts (15%) and adequate megakaryocytes with unremarkable morphology. Granulocytes are hypolobated and hypo granular. The blasts are medium size with fine chromatin, prominent nucleoli and moderate cytoplasm. Rare Auer rods are seen.

DIAGNOSIS 1-2. Bone marrow, right posterior iliac crest, biopsy and aspirate smears: - Normocellular marrow with trilineage maturing hematopoiesis and dysplastic megakaryocytes. COMMENT Dysplastic megakaryocytes and rare ring sideroblasts are seen that may represent persistent myeloid neoplasm. Suggest correlation with genetic studies. BONE MARROW BIOPSY Histologic sections of subcortical and fragmented biopsies with aspiration artifact show normocellular marrow (40%) with normal M:E ratio, myeloid and erythroid elements with maturation, few megakaryocytes are present and increased iron stores. BONE MARROW ASPIRATE SMEAR Spicular and cellular smears show maturing trilineage hematopoiesis with normal M:E ratio, myeloid and erythroid elements with progressive maturation, mild increase in immature cells and adequate megakaryocytes with dysplastic hypolobated forms and forms with separated nuclei.

DIAGNOSIS 1. Bone marrow, left iliac crest; biopsy: - Hypocellular marrow with fibrosis, osteosclerosis, markedly reduced myeloid precursors, erythroid predominance, increased dysplastic/atypical megakaryocytes, and reported increased iron stores consistent with myeloid neoplasm. See comment. COMMENT The overall findings in the current marrow sample are consistent with myeloid neoplasm with overlapping morphologic features of myelodysplastic syndrome and myeloproliferative neoplasm. Peripheral blood findings, morphologic evaluation of the peripheral blood smear and the reported dilute aspirate smear and if available touch imprints along with clinical findings (splenomegaly) and laboratory studies would be needed for complete evaluation and further characterization. The patient is known to be status post-treatment metastatic renal cell carcinoma as per previous clinical notes. The findings may represent a therapy related myeloid neoplasm. Suggest a repeat marrow sample submitted for further morphologic evaluation (including touch imprints made from bone marrow core biopsies as fibrosis is expected), flow cytometric analysis and comprehensive genomic profiling for further characterization.

Format 1 DIAGNOSIS 1,2,3- Bone marrow, biopsy; aspirate and peripheral blood smears: 1. Plasma cell neoplasm, - 7-10% involvement by CD138 immunohistochemistry. - 10% involvement by aspirate differential. - Minute abnormal plasma cell population by flow cytometry. 2. Cellular marrow with trilineage maturing hematopoiesis. 3. No evidence of involvement by lymphoma. 4. Peripheral blood within normal limits. IMMUNOHISTOCHEMISTRY CD138 labels an increase in plasma cells (7-10% of total cellularity), which show lambda predominance by kappa and lambda immunostains. FLOW CYTOMETRY Minute abnormal plasma cell population detected. No abnormal mature B- or T-cell populations detected. The abnormal population represents very few events which is below limit of accurate quantitation. Flow cytometry studies generally underestimate the number of plasma cells, as compared to morphologic assessment. CYTOGENETIC STUDIES: See separate report. MOLECULAR STUDIES: Not requested. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 8.01 K/uL, RBC 4.43 M/uL, Hgb 14.0 g/dL, Hct 41.5 %, MCV 93.7 fl and platelets 228 K/uL. Examination of the erythrocytes reveals no significant anisocytosis or poikilocytosis. The manual leukocyte differential is 60% neutrophils, 1% bands, 30% lymphocytes, 9% monocytes and 0% eosinophils. Granulocytes, lymphocytes and monocytes are normal in morphology. Platelets are normal in number, size and degree of granularity. No circulating plasma cells seen. BONE MARROW ASPIRATE SMEAR Spicular and cellular smears showing maturing trilineage hematopoiesis, with no increase in blasts on 200 cell differential count. Plasma cells on the aspirate differential count is 10%.Plasma cells show occasional nuclei showing nucleoli and few binucleated forms. Myeloid elements exhibit progressive maturation with no significant dysplasia. Erythroid elements show progressive maturation with no significant dysplasia. Megakaryocytes are adequate in number with predominantly unremarkable morphology. The lymphoid cells present are mostly small and maturing-appearing. BONE MARROW BIOPSY/CLOT Quality: Biopsy inadequate with fragmentation and aspiration artifact. The following results are from the clot specimen. M:E ratio: Within normal limits Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, occasional hypolobated forms Lymphocytes: Interstitially scattered Plasma cells: Increased Other: No extraneous cells identified Special stains: All controls are adequate. Iron: Present, adequate. Ring sideroblasts: No ring sideroblasts are identified. FORMAT 2 DIAGNOSIS 1-3. Bone marrow, right posterior iliac crest, biopsy; aspirate and peripheral blood smears: - Plasma cell neoplasm, -5-10% involvement by CD138 immunohistochemistry. - 8% involvement by aspirate differential. -5.8% involvement by 10-color flow cytometry. - Cellular marrow with erythroid predominant trilineage maturing hematopoiesis and erythroid dyspoiesis. - No evidence of involvement by lymphoma. COMMENT Erythroid dyspoiesis might be secondary to treatment. However, correlation with cytogenetic studies is recommended for further evaluation. BONE MARROW BIOPSY Quality: Adequate, hemorrhage, bone part Cellularity: 20-30% at the preserved areas Myeloid lineage: Orderly maturation, decreased Erythroid lineage: Relatively increased and show orderly maturation Megakaryocytes: Adequate Plasma cells: Increased BONE MARROW ASPIRATE MORPHOLOGY Aspirate differential: Blasts 3% Promyelocytes 3% Myelocytes 8% Metamyelocytes 1% Neutrophils/Bands14% Monocytes 1% Eosinophils 1% Erythroid Precursors 48% Plasma Cells 8% Lymphocytes 13% Number of Cells Counted 500 M:E Ratio 0.6 Morphology: Marrow spicules: Spicular Cellularity: Slightly hypocellular Plasma cell morphology: Small to large intermediate in size with condensed chromatin. Inconspicuous nucleoli and perinuclear hoff Myeloid lineage: Orderly maturation, decreased Erythroid Lineage: Orderly maturation with dysplastic changes, increased Megakaryocytes: Present Other findings: Smudge/naked nuclei IMMUNOHISTOCHEMISTRY CD138 immunostain highlights plasma cells, 5-10% of cellularity. Kappa and lambda immunostains show the plasma cells are non-contributory due to high back ground. BCMA highlights 100% of plasma cells, (2-3+) intensity, membranous/ Golgi pattern. CD20, PAX5 and CD3 highlight singly scattered B and T cells. FLOW CYTOMETRY Abnormal plasma cell population detected. No abnormal mature B-cell population detected. Flow cytometry reveals an abnormal plasma cell population having abnormal expression of CD19 (absent), CD27 (variable, absent to bright), CD45 (major subset absent), CD56, CD81 (absent), CD117, and monoclonal lambda cytoplasmic light chain restriction; with normal expression of CD38 and CD138; and without CD20 expression. The abnormal population represents 5.8% of the total white cells by flow cytometry, and almost certainly will be an underestimate in comparison with morphology. Abnormal plasma cells represent 89.8% of total plasma cells in the sample. No immunophenotypic evidence of involvement by B-cell lymphoma or leukemia is identified. Hodgkin lymphoma would not be detected by these studies. PERIPHERAL BLOOD CBC: WBC 3.8 L [4.0-11.0 K/mcL] RBC 3.35 L [3.95-5.54 M/mcL] HGB 11.3 L [12.5-16.2 g/dL] HCT 33.4 L [37.5-49.3 %] MCV 100 H [80-98 fL] MCH 33.7 H [27.0-33.0 pg] MCHC 33.8 [31.0-36.5 g/dL] RDW 18.8 H [12.2-15.1 %] Platelets 86 L [160-400 K/mcL] Neutrophil 43.0 [32.5-74.8 %] Mono 4.0 [0.0-12.3 %] Eos 0.0 [0.0-4.9 %] Baso 0.0 [0.0-1.5 %] Myelocyte 4.0 H [0.0-0.0 %] Metamyelocyte 2.0 H [0.0-0.0 %] Lymph 47.0 [12.2-47.4 %] Nucleated RBC 1 [/100(WBCs)] Morphology: Rouleaux formation CYTOGENETIC STUDIES Cytogenetic analysis will be reported separately. See separate report. MOLECULAR STUDIES Molecular analysis will be reported separately. See separate report.

DIAGNOSIS 1-3. Bone marrow, biopsy, aspirate and peripheral blood smears: 1. Cellular marrow with trilineage maturing hematopoiesis. 2. Plasmacytosis, ~5% by CD138 immunohistochemistry and aspirate counts, with lambda predominance. See comment. 3. Negative for carcinoma. COMMENT The findings are suggestive of monoclonal gammopathy of unknown significance. Close clinical follow up is advocated. IMMUNOHISTOCHEMISTRY CD138 labels plasma cells (~5% cellularity) which show lambda predominance by kappa and lambda immunostains. Pancytokeratin AE1/AE3 and PAX8 are negative. CD10 labels lymphoid cells. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 14.80 K/uL, RBC 5.38 M/uL, Hgb 12.9 g/dL, Hct 43.1%, MCV 80.1 fl Platelets 428 K/uL. Peripheral Blood Manual Differential (100 cells): Neutrophils: 65% Bands: 1% Metamyelocytes: 1% Myelocytes: 1% Promyelocytes: 0% Blasts: 0% Eosinophils: 2% Basophils: 1% Lymphocytes: 20% Monocytes: 9% Morphology: WBC: No circulating blasts. Predominantly mature neutrophils. RBC: Normocytic normochromic RBC with no significant abnormalities. Platelets: Adequate in number with unremarkable morphology. No circulating plasma cells seen. Rouleaux formation present. BONE MARROW ASPIRATE SMEAR Spicular and cellular smears showing maturing trilineage hematopoiesis, with no increase in blasts on 200 cell differential count. Plasma cells on the aspirate differential count is 5%. Myeloid elements exhibit progressive maturation with no significant dysplasia. Erythroid elements show progressive maturation with no significant dysplasia. Megakaryocytes are adequate in number with predominantly unremarkable morphology. The lymphoid cells present are mostly small and maturing appearing. Mild increase in plasma cells is seen with occasional nuclei showing nucleoli. BONE MARROW BIOPSY/CLOT Quality: Adequate Cellularity: Variably cellular, overall normocellular (~30-50%) M:E ratio: Within normal limits Blasts: Not increased Myeloid lineage: Shows full maturation Erythroid lineage: Shows full maturation Megakaryocytes: Adequate in number, occasional hypolobated forms Lymphocytes: Interstitially scattered Plasma cells: Mildly increased, interstitially scattered Other: No extraneous cells identified Clot: Adequate, shows similar features as biopsy Special stains: All controls are adequate. Iron: Present, adequate. Ring sideroblasts: No ring sideroblasts are identified.

DIAGNOSIS Bone marrow aspirate, clot section and biopsy: 1. Systemic Mastocytosis, C-KIT D816V mutation positive. See comment. 2. Normocellular bone marrow with no increase in blasts. (70% bone marrow cellularity: 10% mast cell involvement). COMMENT #1 Mast cells show atypical expression of CD2 and CD25. The atypical mast cells represent 0.5% of WBC. The finding meets one criterion for diagnosis of systemic mastocytosis, but is not diagnostic in isolation. Correlation with morphologic evaluation and potentially molecular genetic studies for c-KIT D816V mutation as well as tryptase level will be advised. COMMENT #2 Atypical, spindled, mast cell aggregates (>15 mast cells) are present with aberrant expression of CD25. PCR on bone marrow showed a c-KIT D816V mutation. The findings fulfil the diagnostic criteria of Systemic Mastocytosis. No background hematologic process (myelodysplasia, myeloproliferative disorder, leukemia) is identified. PERIPHERAL BLOOD a. No circulating blasts identified. b. No monocytosis or dysplasia is identified. FLOW CYTOMETRY Atypical mast cell population present. Mast cells show atypical expression of CD2 and CD25. The atypical mast cells represent 0.5% of WBC. This finding meets one criterion for diagnosis of systemic mastocytosis, but is not diagnostic in isolation. Correlation with morphologic evaluation and potentially molecular genetic studies for c-KIT D816V mutation as well as tryptase level is advised. No abnormal mature B or T cell populations detected. No abnormal myeloid blast, monocyte or maturing myeloid population identified. No immunophenotypic evidence of involvement by B or T-cell lymphoma or leukemia is identified. CD34 positive myeloid blast population with apparent normal immunophenotype is 0.03% of WBC. CYTOGENETIC STUDIES Cytogenetic analysis will be reported separately. See separate report. MOLECULAR STUDIES Bone marrow is positive for c-KIT D816 mutation by PCR. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 6.48 K/uL, RBC 4.23M/uL, Hgb 13.0g/dL, Hct 38.4%, MCV 90.8fl and platelets 186 K/uL. Peripheral Blood Manual Differential (100 cells): Neutrophils: 60% Bands: 1% Metamyelocytes: 0% Myelocytes: 0% Promyelocytes: 0% Blasts: 0% Eosinophils: 1% Basophils: 1% Lymphocytes: 33% Monocytes: 4% nRBC: 0% Examination of the peripheral blood reveals normocytic normochromic RBC . Leukocytes are predominately mature neutrophils without dysplasia. No circulating blasts are identified. Lymphocytes are small and mature appearing. Platelets are morphologically unremarkable. BONE MARROW ASPIRATE SMEAR The bone marrow aspirate smears are spicular, cellular and adequate for evaluation. There is multilineage hematopoiesis with adequate maturation. Blasts are not increased. An increase in mast cells with spindled/atypical morphology are identified. Myeloid and erythroid elements are present in normal proportions. There is mild dysplasia of the myeloid lineages. Megakaryocytes are quantitatively and qualitatively unremarkable. There is no lymphocytosis or increase in plasma cells. BONE MARROW BIOPSY/CLOT Examination of the clot section and decalcified bone marrow core biopsy reveals a normocellular marrow (70%), interstitial aggregates composed of predominately round mast cells with focal spinding (greater than 15 mast cells per aggregate). The aggregates are variably surrounded by small lymphocytes. Myeloid and erythroid elements are present in normal proportions. Megakaryocytes are morphologically unremarkable without clustering. Immunohistochemistry (clot): All controls are adequate. Mast cells are highlighted by CD117 and CD2 (focal and weak) with aberrant expression of CD25. The surrounding lymphocytes are admixed CD2 positive T cells. Special stains: All controls are adequate Iron: Present. Siderocyte: No ring sideroblasts are identified

DIAGNOSIS Bone marrow aspirate, clot section and biopsy: 1. Atypical mast cell population involving a normocellular bone marrow with mild eosinophilia and no increase in blasts (40% bone marrow cellularity: 10% mast cell involvement). See comment. COMMENT An atypical, focally spindled, mast cell population is present with aberrant expression of CD25. In the context of a patient with a prior diagnosis of systemic mastocytosis, the current findings are consistent with persistent involvement by the same process. No background hematologic process (myelodysplasia, myeloproliferative disorder, leukemia) is identified. PERIPHERAL BLOOD 1. Mild eosinophilia. 2. No circulating blasts identified. FLOW CYTOMETRY No monoclonal B-cell population, phenotypically aberrant T-cell population or increased blast population identified. No mast cell population seen. PERIPHERAL SMEAR Results from the automated CBC are as follows: WBC 6.14 K/uL, RBC 4.34M/uL, Hgb 13.1g/dL, Hct 41.1%, MCV 94.7fl and platelets 231 K/uL. Peripheral Blood Manual Differential (100 cells): Neutrophils: 56% Bands: 1% Metamyelocytes: 0% Myelocytes: 0% Promyelocytes: 0% Blasts: 0% Eosinophils: 7% Basophils: 1% Lymphocytes: 23% Monocytes: 12% nRBC: 1 Examination of the peripheral blood reveals RBCs with poikilocytosis including target cells and rare nucleated RBCs. Leukocytes are predominately mature neutrophils without dysplasia. Eosinophils are mildly increased. No circulating blasts are identified. Lymphocytes are small with scattered reactive forms. Platelets are morphologically unremarkable. BONE MARROW ASPIRATE SMEAR Bone marrow aspirate smear differential (300 cells): Blasts: 1% Promyelocytes: 1% Myelocytes: 8% Metamyelocytes: 13% Bands/neutrophils: 33% Lymphocytes: 10% Monocytes: 7% Eosinophils: 8% Basophils: 0% Plasma cells: 0% Erythroid: 19% M:E ratio: 3.5:1 The bone marrow aspirate smears are spicular, cellular and adequate for evaluation. There is multilineage hematopoiesis with adequate maturation. Blasts are not increased. Scattered mast cells with spindled morphology are identified. Myeloid and erythroid elements are present in normal proportions. Eosinophils are mildly increased without atypia. There is no dysplasia of the myeloid or erythroid lineages. Megakaryocytes are quantitatively and qualitatively unremarkable. There is no lymphocytosis or increase in plasma cells. BONE MARROW BIOPSY/CLOT Examination of the clot section and decalcified bone marrow core biopsy reveals a normocellular marrow (40%) with numerous interstitial aggregates composed of predominately round mast cells with focal spinding (greater than 15 mast cells per aggregate). The aggregates are variably surrounded by small lymphocytes. Myeloid and erythroid elements are present in normal proportions. Megakaryocytes are morphologically unremarkable without clustering. IMMUNOHISTOCHEMISTRY (CLOT) All controls are adequate. Mast cells are highlighted by CD117 and CD30 (focal and weak) with aberrant expression of CD25. There is no aberrant expression of CD2. The surrounding lymphocytes are admixed CD3 positive T-cells as well as CD20 positive B-cells. Special stains: All controls are adequate. Iron: Decreased storage iron. Siderocyte: No ring sideroblasts are identified. Reticulin: Mild focal grade 1 myelofibrosis.

The mast cells are positive for CD25 and partially positive for CD2. CD3 highlights the T cells. These immunophenotypic findings along with increased number of mast cells with cytologic atypia are highly suspicious for a mast cell neoplasm and the overall findings may represent a systemic mastocytosis with an associated hematological neoplasm. However, the mast cells do not form large aggregates, which is a major criterion required by a WHO diagnosis of systemic mastocytosis. In the absence of this major criterion, genetic studies for evaluation of KIT mutation are essential for a full evaluation and characterization of this atypical mast cell proliferation.

CLINICAL INFORMATION 43-year-old male with a PMH significant for pancytopenia and CNS vasculitis with concern for HLH. DIAGNOSIS Bone Marrow iliac crest; biopsy, aspirate smears and peripheral smears: Normocellular for age (60%) marrow with hemophagocytosis, See comment. No evidence of malignancy. Adequate iron stores. Peripheral blood with pancytopenia. COMMENT A moderate number of histiocytes in bone marrow are found with hemophagocytosis. The following findings in this patient support the diagnosis of Hemophagocytic Lymphohistiocytosis (HLH): pancytopenia, fever, markedly elevated ferritin (19,273), elevated triglyceride (256), hemophagocytosis in bone marrow. PERIPHERAL SMEAR CBC result (Dated XX) WBC 1.8 K/CMM Low RBC 3.33 M/CMM Low Hgb 9.8 g/dL Hct 29.6 % Low MCV 88.9 fL Normal MCH 29.5 pg Normal MCHC 33.2 g/dL Normal RDW 16.7 % HI Platelet 56 K/CMM Low MPV 10.0 fL Normal Segs 85.6 % Normal HI Lymphocytes 5.9 % Low Monocytes 4.9 % Normal Basophils 0.5 % Normal Segs-Bands # 1.5 K/CMM Lymphocytes # 0.1 K/CMM Low Monocytes # 0.1 K/CMM Normal PERIPHERAL BLOOD Erythrocytes: Normocytic hypochromic anemia with anisopoikilocytosis, minimal polychromasia White cells: Decreased in number Granulocytes: Left shift with bandemia Lymphocytes: Normal morphology Monocytes: Normal morphology Platelets: Decreased in number with normal morphology BONE MARROW ASPIRATE SMEAR Differential (%) Myeloblasts: 0% Promyelocytes: 1% Myelocytes: 6% Metas: 4% Bands & PMN's: 62% Eos: 3% Baso: 0% Monos: 4% Lymphs: 8% Plasma cells:0% Erythroids: 12% M:E ratio 7:1 BONE MARROW BIOPSY/CLOT Cellularity: 60%, normocellular for age Megakaryopoiesis: Adequate with normal maturation Erythropoiesis: Adequate with normal maturation. Iron Content (aspirate): Adequate iron stores Granulopoiesis: Adequate with normal maturation, no increase in blasts Lymphocytes: Normal number and morphology Other: No evidence of granuloma, fibrosis or abnormal cellular infiltrates Others: A moderate number of histiocytes with phagocytosis are seen in aspirate and biopsy. Iron content (biopsy and clot section): Adequate iron stores.

CLINICAL INFORMATION 3-year-old male with retroperitoneal mass suspicious of Neuroblastoma. DIAGNOSIS Bone Marrow, Right iliac crest biopsy, aspirate and peripheral smear: - Diffuse Neuroblastoma involvement in bone marrow, see comment. - Decreased iron stores. COMMENT : The morphology and immunostain findings are consistent with neuroblastoma. PERIPHERAL BLOOD - Moderate normocytic anemia IMMUNOHISTOCHEMISTRY Immunohistochemical stains, with adequate controls, are performed on biopsy for CD45, CD56, CD99, Desmin, Myogenin, and Neurofilement. The tumor cells are positive for CD56 and Neurofilement. They are negative for CD45, CD99, Desmin, Myogenin. COMMENT: The morphology and immunostain findings are consistent with neuroblastoma. PERIPHERAL BLOOD CBC result WBC 4.4 K/CMM Normal RBC 2.93 M/CMM Low Hgb 8.3 g/dL Low Hct 23.4 % Low MCV 79.8 fL Normal MCH 28.2 pg Normal MCHC 35.3 g/dL Normal RDW 15.6 % High Platelet 168 K/CMM Normal MPV 7.6 fL Normal Segs 33.0 % Normal Lymphocytes 52.0 % Normal Monocytes 9.0 % Normal Basophils 2.0 % High Eosinophils 3.0 % Normal Erythrocytes: Normocytic normochromic anemia with mild polychromasia. White cells: Normal in number and morphology Granulocytes: Normal in number and morphology Lymphocytes: Normal in number and morphology Monocytes: Normal in number and morphology Platelets: Normal number and morphology BONE MARROW ASPIRATE SMEAR There are many tumor cells in aspirate and very few hematopoietic cells. Differential counts are not performed. Cellularity: 90%, with diffuse tumor infiltration Megakaryopoiesis: Decreased with normal maturation Erythropoiesis: Decreased with normal maturation Iron Content (aspirate): Decreased iron stores Granulopoiesis: Decreased with normal maturation Lymphocytes: Decreased with normal morphology Others: Tumor cells in clumps BONE MARROW BIOPSY/ CLOT SECTION Diffuse infiltration of tumor cells in rosetting pattern. Iron content (biopsy and clot section): Decreased iron stores