Heme #2: Exam #3

Chronic Lymphocytic Leukemia

Chronic Lymphoproliferative Disorders

-clonal proliferation disorders of MATURE B or T cells

-insidious onset and an indolent course

-subclassification depends on morphology, immunophenotyping, cytogenetics, clinical features, and molecular testing

lymphoma

disorders that affect lymph nodes and other extramedullary sites

Chronic Lymphocytic Leukemia (CLL)

-most common chronic lymphoproliferative disorder

-generally a neoplasm of mature B lymphs but can involve T cells

-B-Cell CLL is what we focus on

->50 years old

-lymphocytosis >5

-small, mature lymphocytes with soccerball chromatin

-SMUDGE CELLS (basket cells) on PBS

-lymphoid accumulation crowds out normal bone marrow elements

-infiltration of malignant cells in the lymph nodes and spleen (so you get lymphadenopathy or splenomegaly)

-reduced humoral immunity

Small Lymphocytic Leukemia (SLL)

-synonymous wiht CLL but tends to involve lymph nodes and other lymphoid organs

-it will have the same tissue morph and immunophenotype but without the PBS and BM leukemia

Etiology of CLL

-no specific agent

-accumulation of dysfunctional mature lymphs in PBS and BM

-failure in apoptosis causing survival and expansion of the malignant clone

-normal BM elements are replaced causing anemia, thrombocytopenia, and neutropenia

Clinical Features of CLL

->50 in males more

-gradual onset

-routine physical CBC is where it is found

-lymphocytosis, lymphadenopathy, splenomegaly

Lab findings in CLL

-lymphocytosis >5

*mature lymphs with cracked chromatin/soccer ball look

*round to slighly indented nucleus

*scant cytoplasm

*indistinct or absent nucleoli

**prolymphs are sometimes seen

-SMUDGE CELLS are diagnostic for CLL

-N/N anemia sometimes

-normal to low reticulocytes

-thrombocytopenia sometimes

-WBC INCREASED

BM in CLL

-mature lymphs

BM INFILTRATION:

*Nodular

-distinct, randomly distrubuted lymphoid aggregates

*Insterstitial

-lymphs infiltrate interstitium without displacing fat cells

*Diffuse

-entire BM is replaced with lymphs

*Combination of some of these

Other lab results

-hypogammaglobulinemia in most patients

-IgA, G, M (GAM) decreased bc B cell disease and abnormal function

-leads to bacterial and viral infections

-can lead to autoimmunity like AIHA (autoimune hemol), ITP, pure red cell aplasia, etc.

-10-30% will develop AIHA so you may see spherocytes and polychromasia, nRBCs, positive DAT, increased bilirubin

**look for SPHEROCYTES and REPORT PRESENT OR NOT LIKE WE DO WITH SCHICHTOCYTES!!

Smudge Cells

-CLL

-fragile lymphs that break during slide prep

-can be removed by mixing 1 drop of albumin with one drop blood before PBS made

-this needs to be done for an accurate lymph count

-but albumin distorts red cells so need original smear too

-plt estimate on original slide too (could falsely lower plt count bc diluted)

Immunologic features of CLL

-can't distinguish B vs T cells on PBS

-monocloncal antibodies and flow helps

-these cells don't mature to the final stages of B cell maturity

-weak IgM/D with kappa or lambda light chains

-CD19+, 20, 22, 79a, 5 (USUALLY SEEN ON T CELLS), CD23, 43

-you may see reactive-like lymphs to flow is important on anyone over 50 with lymphocytosis >5

CLL genetic abnormalities

-50-60% have

-FISH!

-Trisomy 12 (poor prognosis)

-del or t(13q14) (longer survival)

-11q22-23 del (poor)

-17p13 del (biggest predictor of POOR!)

CLL Survival

-4-5 years

-many pts asymptomatic (indolent)

-treatment not started until pt has a lot of lymphocytosis, splenomegaly/lymphadenopathy, anemia, neutropenia, thrombocytopenia, autoimmune events, infections

-sometimes CAN be aggressive 1-2 year survival

-prognosis based on lymphcyte doubling time, bone marrow infiltration patterns (diffuse is worse), and chromosome abnormalities

-not curable with current tx but long survival ish

CLL transformation

-can progress when a new pop of malignant lymphs develops

-increases resistance to therapy

-prolymphocytic leukemial, diffuse large B-cell lymphoma (Richter syndrome), or acute leukemia (not often)

Richter's Syndrome

-CLL converts

-progressive lymphoma

-fever, weight loss, organomegaly, worsening cytopenias

-very poor response to chemo

(CLL): ZAP-70

-this and CD38 are usually positive when there is unmuated Ig variable regions

-more high risk

-NEGATIVE PROGNOSIS!!! for CLL

PLL (prolymphocytic leukemia)

-proliferation of malignant PROlymphs (b or t)

-CLL can transform or it can occur separately

-extremely rare but more in 60+ year olds with males

B-PLL

-WBC >100

-Absolute lymphocytosis

-marked splenomegaly NO lymphadenopathy

-anemia and low plts

->55% cells are prolymphs

Prolymphs

-9-18 microns (large cells)

-round or indented nuclei

-mature, condensed between blast and lymphcyte chromatin

-0-1 prominant nucleoli

-large amounts of basophilic cytoplasm

-NO GRANULES!

-punched out looking nuceloli surrounded by a halo of condensed chromatin

B-Cell PLL

-sIg+, 19,20,22,79a,79b,5

-NO CD23

T-Cell PLL

-2,3,5,7,tdt-, 4+,8- mostly (could be both pos or both neg)

PLL genetics

-chromosome t or inv 14 q11 q32-

-t 11;14 q13q32 (worse prog bc response poor to tx, short survival)

CLL vs OTHERS

-acute lymphoblastic leukemia (ALL)

-reactive lymphocytosis

-PLL

-non-hodgkin lymphoma (mantle cell lymphoma)

-t-cell large granular lymphocytic leukemia

-sezary cell lymphoma

-hairy cell leukemia

Hairy Cell Leukemia!!!

Hair Cell Leukemia summary

-uncommon chronic B-cell lymphoproliferative disorder

-irregular HAIRLY cytoplasmic projections on the malignant cells

-2% of leukemias

-middle aged males 4-7x more than females

-52 is median age very rare in less than 30 year olds

causes of HCL

-ionizing radiation

-organic chemicals

-farming

-wood working

-EBV

origin of HCL

-clonal expansion of mature B cells with light chain resitricted surface Ig expression

-antigen-experienced memory B cell from the post-germinal center

-usually have some somatic mutation in their Ig variable genes

-single disease entity that is very distinct from other B-cell malignancies

Clinical Findings of HCL

-1/4 asymptomatic

-abdominal pain/fullness

-massive splenomegaly

-hepatomegaly

-lymphadenopathy

-fatigue, weakness, weight loss, night sweats, fever

-bleeding/bruising bc low platelets

-recurrent infection bc neutropenia

Lab Findings in HCL

-pancytopenia bc of splenomegaly and BM failure

-SPLEEN CAUSES PANCYTOPENIA BC:

*sequesters 90% of platelets

*30% of RBCs, and 65% of granulocytes

-LEUKOPENIA! with neutropenia and monocytopenia

-WBC are <4

-infections are the leading COD with HCL

-N/N anemia and plts 20-100

-azotemia (uremia) is a poisonous condition caused by failure of the kidneys to remove urea from the blood

-hypergammaglobinemia (TOO MANY ANITBODIES)

-abnormal liver function tests!!

PB and BM of HCL

PB:

-nucleus is large, oval, reniform (kidney shaped round or slightly clefted), eccentric but may be central

-chromatin homogenous and relatively fine

-cytoplasm is abundant pale with FINE PROJECTIONS (hairy)

-can be vacuolated

-HC are not definitive diagnosis!! bc it can look like splenic marginal zone lymphoma!!

BM:

-hypercellular mostly

-fibrotic so dry tap bc hairy cells infiltrate the marrow

-honeycomb apperance due to the cytoplasm making a halo around the nucleus

-hairy cells can look like a FRIED egg

-reticulin stain is always increased reticulin fibers

Cytochemical Features of HCL

-TRAP positive

-acid phosphatase isoenzyme #5 still works with tartrate added

-with and without tartrate serves as + and - control

-you need 40+ granules in at least 2 cells to be called + *maybe just 1 cell

-staining will be red/purple granules in the cytoplasm

-Neutrophils negative

Immunophenotyping in HCL

-bright CD19

-20,22

-also for uncommon ones like CD11c usually on monocytes and neutrophils

-CD25 usually on activated T-cells

-Extensive co-expresion of CD11c + CD22 and CD103 are highly sensitive for HCs

-

HCL Variant

-more aggressive form with median age of 71

-cell is smaller, round nucleus, prominent nucleoli, higher N:C ratio, basophilic cytoplasm with rare projections

-WBC may be >50

-cytopenias and splenomegaly

-CD25 and maybe CD103 negative

-poor prognosis

treament of HCL

-usually not tx until significantly symptomatic

-watch and wait usually

-tx started with significant neutropenia, anemia, low plts, symptomatic splenomegaly, recurrent infections, night sweats, fever

TX NOW:

-splenectomy for normal counts

-interferon increases prognosis by inducing apoptosis in nonadherent hairy cells

-nuceloside or purine analogs target lymphs and are cytotoxic to resting and dividing cells (pentostatin, cladribine)

-many relapse but can use the same tx again

-with each tx though the disease free time is shortened

Experimental TX:

-monoclonal antibodies against CD20 (rituxan)

-BL22 or anti-CD22 anitbody

-anti-CD25 antibody

prognosis of HCL

-median survival rate of 4-7 years before interferon and nucleoside analogs. now 10+ years

-infection is the greatest risk of death

Starting Plasma Cell Neoplasms!!!!!!!!

plasma cell neoplasms general info

-M-spike on protein or urine electrophoresis (bc of monoclonal antibodies being made

-multiple myeloma, plasmacytoma, MGUS, waldenstrom's macroglobulinemia, primary amyloidosis

plasma cells are

-fully mature B cells that have undergone activation by antigens

-4% of BM and NOT usually in PBS

-in response to infections, inflammatory or malignant conditions

-coarse chromatin 8-20 microns in size; ECCENTRIC nucleus with 80s prom dress cytoplasm. no granules. deeply basophilic cytoplasm

-have haf a HOF or clearing of golgi apparatus

types of antibodies made by plasma cells

-GAMED

-each has 2 light chains and 3 heavy chains

HEAVY CHAINS:

-alpha, gamma, Mu (micro), epsilon, delta

LIGHT CHAINS:

-kappa, lambda

plasmacytoid lymphocyte

-between lymph and plasma cell

-15-20 micorns, chromatin less clumpled, visible single nucleoli, eccentric or central nucleus but deeply basophilic cytoplasm

Multiple Myeloma

-most common plasma cell neoplasm

-numerous lytic bone lesions

-median age 66 with male predominance

-~20% asymptomatic

-bone pain or fracture due to lesions

-fatigue, thirst, nausea, infection, weakness, numbness, renal insufficiency

-bone radiographs diagnose osteolytic lesions, osteoporosis, etc.

cause of MM

not well known but:

-atomic bomb survivors, organic solvents, toxins in textile industry, chemicals, pesticides, herbicides

pathophysiology of MM

-acceleration of plasma cell growth due to genetic alterations that prevent normal differentiation and apoptosis

(IL-6 is secreted with accelerates plasma cell growth and inhibits apoptosis)

-activation of bone resorption factors or osteoclasts

(OAF (osteoclast activating factor) is secreted and stimulates osteoclasts to absorb bone); serum calcium increases due to bone loss)

-production of abnormal monoclonal protein

(as production of plasma cells increases, the production of immunoglobulin increase)

osteoclasts

-large cells up to 100microns

-low N:C ratio

-several, separate round nuclei

-blue, slighly granular cytoplasm

plasma cell tumors may go to

other parts of the body besides BM

PBS in MM

-N/N anemia

-ROULEAUX (STACKING) OF RBCs

-abnormal proteins in the blood cause them to stick

-plasma cells may be seen

-cytopenias may form bc plasma cells are replacing normal BM cells

-increased protein caused by excess Igs causes blood smears to have a blue ish background!!!

-elevated ESR meaning quicker settling because of rouleax

-as plasma cells take over, low plts and anemia, and neutropenia occur

BM in MM

->30% plasma cells usually in sheets/large aggregates

-could be abnormal looking with fine chromatin, nuceloli, or intracellular inclusions with Igs

-mott cells, flame cells could be seen

-normal lymph and plasma cells decreases so less antibodies around meaning more risk of infection

Flame Cells

-reddish purple cytoplasm

-more Ig than usual for sure

Mott Cells

-plasma cell filled with globules containing Ig called RUSSELL BODIES

-grape cells

Dutcher Bodies

-inclusion in plasma cells seen in MM sometimes that have GLYCOGEN!!!

-usually in neoplastic plasma cells

serum protein electrophoresis for MM

-monoclonal proteins can be confirmed

-Ig, alumbin, and minor proteins

-ALBUMIN, alpha 1, 2, beta, ,gamma (where MM is)

-M SPIKE = multiple myeloma

-these monoclonal proteins have only ONE light chain (kappa or lambda) and one heavy chain (GAMED)

-can see the m spike in serum or URINE

Bence Jones proteins

-excess free light chains in the urine

-serum electrophoresis is normal

-Bence-Jones Proteinuria

-small proteins that can be filtered by the kidney but cause kidney damage

immunophenotyping in MM

-lack sIg

-no 19,20

-have 138, 38, 56+ (usually)

cytogenetics in MM

-hard because plasma cells don't readily divide to produce metaphase cells

-FISH!

-many chromsome abnormalities are found (not gonna learn)

prognosis and tx of MM

-poor prognosis

-determine prognosis with labeling index test

-plasma cell labeling index (PCLI) is a fluorescent slide technique to identify plasma cells in the PBS and BM and report proliferation index as a prognostic indicator of myeloma

FAVORABLE:

-t(11;14)

-hyperdiploidy

UNFAV:

-del(17q)

-monosomy 13

other factors:

-renal failure, Hg levels, calcium levels, B2 microglobulin levels, lytic bone lesions, % of plasma cells, amount of M-component

tx of MM

-radiation for relief of bone pain

-chemo

-autologous BM transplant

-stem cell transplant

-thalidomide

-interferon alpha

-relapse occurs in almost all patients

-infection is one of the major COD

plasmacytoma

-localized plasma cell tumors causing monoclonal spikes of SPEP

-lesions could develop of bones and develop into MM

-respiratory tract plasmacytomas have a good prognosis

plasma cell leukemia

-a complication seen in MM

-as plasma cells take over BM they spill into PBS causing increased calcium and plasmacytomas with bone pain

DIAGNOSED:

->2 or 20% plasma cells

-primary is diagnosed in the leukemic phase

-secondary represents transformation of MM

-aggressive leukemia with poor prognosis

Monocloncal Gammopathy of Undetermined Significance (MGUS)

-finding an M spike doesn't always mean MM

-benign monoclonal gammopathy is this

-small M spike with small amounts of urine light chains

-<10% plasma cells in BM with normal morph

-usually asymptomatic and these are NOT THERE:

*no lytic bone lesions

*no anemia

*no hypercalcemia

*renal insufficiency NOT there

-25% can go to MM

-tx usually unecessary unless symtomatic, not usually their COD is this

Waldenstrom's macroglobulinemia

-plasma cell disorder with an overproduction of monoclonal IgM

-lymphadenopathy, hepatosplenomegaly, hyperviscosity syndrome (decreased blood flow bc of excess protein so confusion, chest tightness, blurred vision, headache, numbness of fingers)

-no lytic bone lesions or bone pain though

-anemia and pallor

-sharp narrow M-spike on SPEC

PB/BM

-mixed population of lymphs, plasma cells, and plasmcytoid lymphs

M-spike

-IgM heavy chain with kappa or lambda light chain

-large molecule IgM so increases viscosity

-IgM can also coat platelets causing inteference with clotting facotrs so thrombotic complications

Tx:

-plasmapheresis where blood is removed, plasma is drawn off and discarded, and blood is returned

-NOT CURABLE, chemo, alkylators,

-4 years survival

-cryoglobulins are abnormal proteins that precipitate or gel when cooled to 0 degreesC, and can form in these patients

amyloidosis

-when light chains (kappa or lambda) accumulate

-amyloid: fibrous protein that deposits in the organ

-that organ will have loss of function

-commonly see weight loss and fatigue

-heart: CHF

-kidneys: nephrotic syndrome or renal failure

-Gut: malabsorption

-Tongue: macroglossia

-Nerves: peripheral neuropathy

-biopsy of affected organ can diagnose

-congo RED stain

-amyloids will have apple green birefringence with polarized light

-poor survival and treat underlying cause but COD is cardiac disease, renal failure, infection, hemorrhage

PCLI

-plasma cell labeling index

-MM patients

-measures the proliferative rate of BM plasma cells using either BM or PBS

-flow cytometry can separate plasma cells and allow for specific identification and confirmation

-measured as a % of plasma cells in the SYNTHETIC phase (synthesis phase S phase of the cell cycle where they are actively making DNA)

-tests for antibodies against 5-bromo-2'-deoxyuridine which are found in actively dividing cells

-High PCLI (>=3%) means more dividing cells and a poor prognosis

-Low PCLI (<3 is a better prognosis)

plasma cell proliferation assay (PCPRO)

-uses flow cytometry to isolate plasma cells for evaluation for clonality and analyze the DNA content of the cells

-BM in ACD, Heparin or EDTA anticoagulant is used

-isolate using CD38,138, 19 and 45

-DAPI stains the DNA and allows the DNA content to be individually quantitated based on fluoresence

Cell Phases:

-G0G1: normal resting cells and have normal DNA (2n)

-Sphase: proliferating and preparing to divide, making DNA and goes to 4n

-G2M: growth right before division; cells have 4N and are ready to go into mitosis

STARTING LYMPHOMA LESSON!!

tissue equivalent to CLL and the immunophenotype for it

*this is dif from mantle cell lymphoma why

SLL (small lymphocytic lymphoma)

-same immunophenotype as CLL (5,19,20,22,23)

-dif from mantle because it has CD23 and not FMC-7

cells found in follicular lymphoma

-BUTT cells (large cells with very irregular nuclear outlines and a deep indentation/cleft of the nuclear membrane)

-most common to circulate in the peripheral blood (leukemic phase)

preceding H.pylori infection can lead to

MALT (extranodal marginal zone lymphoma of mucosa associated lymphoid tissue)

which lymphoma represents 1/3 of all pediatric lymphoma

*what does the biopsy look like

-BURKITTs

-starry sky (sky is blue nuclei of the lymphs and the stars are pale staining macrophages)

LGL Leukemia (large granular lymphocytic)

*PBS

*2 categories

PBS:

-can look like CLL but is technically T lymphs

-the lymphs with have abundant cytoplasm with AZUROPHILIC GRANULES in the cytoplasm

2 categories:

*????????????? email her!

LGL vs reactive lymphocytosis

-LGL is a leukemia so patients also have anemia, low plts, and neutropenia

-you need molecular 2,3,5,7,8,16,57 positive too in flow negative for 4 and 56

Sezary syndrome

*cell

*markers

*diagnostic criteria

-T-cell with very irregular, convuluted nuclear outlines

-CD3,4 positive but lacks CD7

-CD4:CD8 ratio >10

-patients have erythroderma (red skin) and lymphadenopathy

-sezary cells have nuclei with marked convultions (not perfectly round); kinda cerebriform (brain like)

-Mycosis fungoides (MF) is similar

-most common cutaneous T-cell lymphoma

-diffuse skin involvement and must have a + skin biopsy

-Hgb normal

-Plt Normal

-increased WBC maybe with eosinophilia

-minimal BM involvement

tissue equivalent of ALL

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Classic Hodgkin Lymphoma vs nonHodgkin

Reed-Sternberg cell (R-S) cell which has 2+ nuclear lobes containing inclusion-like nucleoli and an area of perinucleolar clearing imparting an owl's eye appearance

lymphoma cells

-deep indentations, clefts, folds

-vacuoles maybe

NewBorn smears

-increased lymphs (funny looking or 'kid' lymphs)

-normal absolute count is 2-11 on newborn

-normal absolute count up to a month is 2-17

-immature neutrophls

-polychromasia

-macrocytic reds

-MCV 98-120 is normal

-hyposplenic picture because just not fully working yet (target cells, HJ bodies, acanthocytes)

-thrombocytopenia is NOT NORMAL, schistocytes, spherocytes, and blasts are all NOT NORMAL!