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An Approach to Lymphoma Diagnosis
Anita M BorgesMumbai
How to make a lymph node diagnosis
o Carefully evaluate the H & E stained section
o Arrive at possible diagnoseso Correlate with clinical datao Order IHC only if necessaryo Interpret the immunostainso Put all the pieces together to make a
diagnosis
Evaluate the H&E
o All compartments of the nodeo Overall architectureo Distortions in architecture & content
q Focalq Partialq Diffuse
2X
Arrive at Possible Diagnoses
o Pattern diagnosis: Low Powerq Follicular, Diffuse, Sinus, Mixed,
o Cytological diagnosis: High Powerq Small cell, large cell, mitotically activeq Lymphoid, Histiocytic, Myeloid, Epithelial,
Melanocytic, etco Entity diagnosis: Observation &
Interpretation
Correlate with clinical data
o Ageo Site
q Posterior triangle neck
q Axillaq Groinq Mesenteric vs
paraaortico Localised vs
generalisedo Spleen, liver
o Hematological parameters
o Durationo Accompanying
symptoms/ signso Previous treatmento Drug historyo Co- morbidities esp
immune disorders
Order IHC only if necessary
o To confirm a diagnosis made on H&Eo To immunophenotypeo Uncertain diagnosis
Interpretation of Immunostains
o Knowledge of normal pattern of expression in different compartments and stages of differentiation.
o Expression in cell compartments,viz. nuclear, cytoplasmic, membrane.
o Variations under neoplastic & reactive conditons- aberrant expression and non-expression
o Clonality assessment only possible in some B cell lesions. T cell clonality requires gene rearrangement studies
CD20
CD3
MUM-1
CD20 CD5
BCL-2CD79A
Lambda Light Chain
CD23 CD30
Kappa Light Chain
Interpret Immunostains
o All assessments have to be made on the population of interest.
o Lymphomas very seldom show a pure population. Exceptions are some childhood lymphomas viz. Burkitt’s Lymphoma.
o Lymphoma cells may be a minority population.
o No place for percentages or +++ !!!
What IHC? Always use a panel.
n CD20, CD3, CD30, CD45, CD23, bcl2- General all purposen CD20 & CD3, CD5, +/- 79a, CD 30, Alk-I- Large cell diffuse NHLn CD30, CD15, CD45, CD20, CD3, +/-EMA/Oct2- Hodgkins Disn CD20, CD3, CD5, CD10, CD23, +/- cyclinD1- Small Cell NHLn CD20,CD3, CD10, bcl2, bcl6, - Follicular lesionsq CD3, CD5, CD7, CD2, CD4, CD8, CD10, CD23- PTCLo CD20, CD3, CD10, Tdt, CD99, CD34, bcl2, bcl6,Mib1 ALL,
Burkittso CD43, MPO, CD34, C-kit, Glyc C, CD61- Myeloid o CD138, Mum1, Kappa & Lamda light chains,- Plasma cell o CD1a, CD68, CD21, CD35, CD123, CK, HMB45,- Histiocytic,
metastatic
How to evaluate an Atypical Lymphoid process
o Rule out a malignant process!o Be familiar with reactive patterns of
proliferation
3 Questions
o Is this a lymphoma?o If it is- Is it HL or NHL?o What type?
Is this a lymphoma?
o Is this a benign/ reactive lymphoid process/
o Is this a malignant tumour other than lymphoma?
Common patterns in LN Histology that resemble lymphoma
o Polymorphous proliferations with or without large cells viz. Viral lymphadenopathyn Hodgkin’s lymphoma & PTCL
o Follicular/ nodular proliferations viz. Foll. Hyp.n Follicular lymphoma & NLPHD
o Focal areas of atypical cells with or without necrosis. viz. Kikuchi’s disease.n Partial involvement by NHL or HL
o Mitotically active proliferations viz. ALPSn High grade NHL eg LL or BL
o Diffuse or sinusoidal infiltration of undefined cells viz. Histiocytic disease, metastatic ca.n ALCL,
The Masqueraders
o Benign lesions that simulate lymphomas and vice versa.
o Tumors that simulate lymphomas
o Lymphomas that simulate other tumours
The Masqueraders
Case History
o 29 year old maleo Single lymph node swelling in the
parotid region of 4 weeks duration .o No other complaints or significant
clinical findings. o Normal hemat. parameters including
ESR o Block sent for review with a diagnosis
of mixed cellularity HL.
CD20 CD3
CD5
CD30
CD20
LCA
CD15 Negative
ALK-1 Negative
Diagnosis
o Atypical paracortical lymphoid proliferation with a T cell predominance associated with clustered B immunoblasts and epithelioid cells.
o Reactive process. o Serology for EBV, Toxoplasmosiso Drug history esp. antiepileptics viz.
Dilantin
Case History
o 28 year old maleo Anemia, splenomegaly, cervical,
axillary & inguinal adenopathy of 6 months duration.
o Anti-tuberculous treatment administered with no effect.
o Cervical node biopsied.
CD 20CD 20
CD 3 CD 3
CD 23
CD 30 p24
HIV associated Lymphadenopathy
Case History
o 31 year old female with multiple small firm lymph nodes on both sides of the neck associated with malaise and evening rise of temperature for the past 1 month.
o Blood investigations were normal except for an ESR of 40mm in the first hour.
o The chest X-ray showed a possible para-cardiac shadow.
CD 20 CD 20
CD 3 CD 3
CD4 CD8
CD68 CD 123
Diagnosis
o Histiocytic Necrotizing Lymphadenitis of Kikuchi-Fujimoto type.
o ZN stain for AFB is negativeo Comment: Every case of KFD must be
investigated investigated by microbiology and serology for mycobacteriosis & SLE.
o Paracortical location, apoptosis, CD3, CD8 predominant lymphoid proliferation, histiocytes (CD68) and plasmacytoid dendritic cells (CD123).
Case History (Courtsey Dr S Gujral)
o 9 month old male child admitted with fever and multiple boils over the scalp.
o First Admission: at 2 monthso Two siblings died of prolonged illnesses in
infancyo Poor weight gain, lump in abdomeno Generalised lymphadenopathyo Hepato-splenomegalyo Cevical lymphnode biopsiedo Total WBC count 89,000/ cmm
CD20 CD3
Mib1
TDT-- negative
Lymphocyte subset subpopulation
Lymphocytes subpopulation
% lymphocytes ALC/mm3 Normal range
1 Lymphocytes 92 89,884 2600-10,400
2 B lymphocyte (CD 19+) 8 7191 600-2700
3 CD3+/CD4+ T lymphocytes 4 3595 1000-4600
4 T lymphocytes (CD3+) 87 78,111 1600-6700
5 CD3+/CD8 T lymphocytes 7 6252 400-2100
6 CD3-16+56+ NK cells 4 3595 200-1200
7 CD3+ HLA-DR+ 73 65615 100-600
8 CD4+/CD25+ 1.5% of CD3
9 TCR alpha beta positive DNT cells 87% <1%
10 CD3 positive DNT cells 86%
11 CD2, CD5, CD7 86, 89, 88
12 CD34, Tdt Negative
Diagnosis
o Autoimmune Lymphoproliferative Syndrome (ALPS)
o Mitotically active T cell proliferation which is Tdt negative.
o Typical history. o Flow cytometry confirms the
Alpha/beta DN T population.
Case History
o 21 year old male o Axillary lymph node enlargement
Fite- Faraco
Hansen’s Disease Of the lymph node
FL Grade 1 / 2 Follicular hyperplasia
bcl2
What IHC is this?
o Tumors that simulate lymphomasn Non hematopoetic round cell tumoursn Non-Lymphomatous hematopoeitic
tumours.o Lymphomas that simulate other
tumoursn Those that resemble Carcinomas, adult-
type sarcomas, melanomas
The Malignant Masqueraders
Features commonly associated withLymphomas
o Patternless monotony and/or nodularityo Small blue cells/ round cellso Inconspicuous cytoplasmo Lack of pleomorphismo Many mitoses in high grade lesionso Crush artifactso Lack of cohesion o “Water through sieve” pattern of infiltrationo Large areas of infarctiono Starry sky pattern
Features often not associated with lymphomas
o Spindle or ovoid cellso Rosetteso Nuclear palloro Marked pleomorphism o Nesting or conspicuous clusteringo Sinusoidal spread within a lymph nodeo Sinusoidal stromao Intense desmoplasia
Masqueraders
o 3 year old male with a large mass in the upper retroperitoneum.
o Imaging was unable to pinpoint the organ of origin or the epicentre of the mass.
o An image guided needle core biopsy was obtained
CD 45 Mic-2
Desmin WT-1
Neuroblastoma
CK
synaptophysin
Antigens often preserved in crushed tissue
Masqueraders
o 12 year old male with multiple masses in the abdomen and pelvis.
o Open biopsy obtained.o Histology: Crushed round cell tumour,
possibly NHL.o Treated on a presumptive diagnosis of NHLo Tumour grew on chemotherapyo CT guided core biopsy of a non necrotic
lesion in the liver was obtained.
CD45
CK EMA
Desmin
DSRCT
Round cell Tumors in Childhood
o Desmin ERMSo Synaptophysin Neuroo CD45,CD3,CD20,CD10,Tdt, MPO CD34 Hemato CK/EMA, Desmin, Vim, Neural marker DSRCTo CD99 PNET
ERMS NHL
All RCTs in children must be immunophenotyped
CD45, if negative, then DSRCT panel + CD99
Masqueraders
o 53 year old male with multiple enlarged nodes on both sides of the neck and a definite nasopharyngeal bulge.
o FNAC of the node was done and reported as a large cell lymphoma. However a lymph node biopsy was advised for phenotyping.
DESMIN MYOGENIN
Masqueraders
o 37 year old, asymptomatic male with an anterior mediastinal mass detected on a chest film at a routine pre-employment health check.
o Hematological parameters normal.o Recalled recent discomfort on lying
flat at night.
CD3 TDT
CK
Thymoma
o The neoplastic epithelial cells may be obscured by numerous background T lymphoblasts,
o Germinomas and embryonal carcinomas may also masquerade as large cell lymphomas or Hodgkin’s Lymphomas.
Masqueraders
o 54 year old male with enlarged lymph nodes in the axilla.
o Chest X-Ray : Normalo Blood counts normal. Hemoglobin: 10g/ dlo ESR raised.o Empirical anti-tuberculous treatment for 3
months.o Surgical biopsy advised and performed by a
surgeon
CD20 CD3
CD43 MPO
Extra-medullary Myeloid cell tumoro Eosinophils, recognisable granulocytic
precursors, in a background of ‘immature cells’ help in suspecting the diagnosis.
Small Round Cell Tumours in Adultso Small cell carcinoma CK TTF1o Neuroendocrine tumour CK
Synapto Chromogr
o NHL CD45 CDso EMMT C-kit, CD43
MPO o Plasmacytoma CD138 k l
Pan CK & CD45, If both are negative, think plasma cell lesion.If CD45+, but B&T cell markers neg., think EMMT
Masqueraders
o 48 year old femaleo Bilateral cervical lymphadenopathy
Melanoma?Carcinoma?
S100P, HMB45, Melan A – NegPan CK –Neg
EMA - Expressed
CD30
ALK-1
CD45
CD3
CD20
Masqueraders
o 56 year old male with multiple firm lymph nodes on one side of the neck.
o No other significant history or clinical findings
o Radiograph of the chest was normal.
CD 30 CK
ALCL
o Epitome of a lymphoma masquerading as carcinoma, melanoma or even a sarcoma
o The hallmark embryo-like nucleus is not always present.
o Cells often in sinuseso CD45 may be +/-o EMA may be expressed but not CKo CD30+/- ALK-1 clinches the
diagnosiso Caution: CD 30 is often expressed in GCTs
Masqueraders
o 43 year old female with a solitary enlarged axillary lymph node.
CD45
CD20
Approach to lymphoma Diagnosis
o Is it HL or NHL?o What type?
Hodgkin’s Lymphoma
o Nodular lymphocyte predominant HL (NLPHL)
o Classical HLn NS- Types 1&2n Mixed cellularityn Lymphocyte richn Lymphocyte depleted
Hodgkin’s Disease Immuno-phenotype- N LP
o R-S cells: CD20+ CD30- CD15- CD45+ EMA+o Background Lymphocytes: B cell predominant
CD 20 CD 20
Hodgkin’s Disease Immuno-phenotype- CHL
o R-S Cell: CD30+ CD15+/- CD20- CD45- EMA-o Background lymphocytes: T predominance
CD30 CD15
Non Hodgkin’s lymphoma
o Familiarity with WHO 2008 classification
o Based on Morphology, immuno-phenotype, genotype, clinical features.
o Simple way to make sense of complexity
o Good, bad, ugly.
Adult Multiple relapses.
Treatment does not alter eventual outcome.
Cure is never achieved
CNS involvement rare
Three groups:• Indolent disseminated
CLL/SLL, MCL• Indolent Extranodal
MALT• Indolent Nodal- MZL
Fatal in months if left untreated.
Curable with current therapeutic modalities
Large cells correspond to cells transformed by Ag.
Types:•Diffuse large B cell lymphoma •Anaplastic large cell lymphoma•Peripheral T cell lymphoma
Acute lymphomas’ often seen in children.
Fatal in weeks if untreated. Respond well.
Widely disseminated. CNS involvement.Primitive ‘blastic’ cells.Very high proliferation.Viral aetiology ( EBV & HTLV-1)
•Burkitt’s, Adult lymphoma/ leukemia•T-lymphoblastic,
NHL- The Good, The bad, & The UglyIndolent
Aggressive Highly Aggressive
What IHC? Always a panel.
n CD20, CD3, CD30, CD45, CD23, bcl2- General all purpose
n CD20 & CD3, CD5, +/- 79a, CD 30, Alk-I- Large cell diffuse NHL
n CD30, CD15, CD45, CD20, CD3, +/-EMA/Oct2- Hodgkins Dis
n CD20, CD3, CD5, CD10, CD23, +/- cyclinD1- Small Cell NHL
n CD20,CD3, CD10, bcl2, bcl6, - Follicular lesionsq CD3, CD5, CD7, CD2, CD4, CD8, CD10, CD23- PTCLo CD20, CD3, CD10, Tdt, CD99, CD34, bcl2, bcl6,Mib1
ALL, Burkittso CD43, MPO, CD34, C-kit, Glyc C, CD61- Myeloid o CD138, Mum1, Kappa & Lamda light chains,-
Plasma cell o CD1a, CD68, CD21, CD35, CD123, CK, HMB45,-
Histiocytic, metastatic
Summary
o RCTs often masquerade as lymphomas esp in core biopsies of extra-nodal lesions, or when appearances are distorted by bad preps.
o Good histology, careful morphological examination, clinical findings, hematological data and imaging will help to determine the panel of immuno-markers.
o All RCTs must be immunophenotyped.
Summary
o CD 45 is a robust antigen which is very informative in the differential diagnosis of RCTs in childhood.
o A primary panel of immunostains for a childhood RCT should include the following: CD45, CD99, desmin, synatophysin, pan CK/ EMA, with or without vimentin.
Summary
o If a lesion resembles a large cell lymphoma but does not mark like one, EMMT, plasmablastic lesions, undifferentiated carcinoma and melanoma must be considered.
o Pleomorphism, abundant cytoplasm, sinusoidal spread, cell cohesion, and non-expression of CD45 do not negate the diagnosis of lymphoma.
o CD30 must be correlated with panCK expression.
o EMA is expressed in ALCL, L&H cells, plasma cell neoplasms etc.
Summaryo Many problematic cases are paracortical
proliferations because T cell clonality cannot be assesed on IHC.
o CD20 & CD3 very useful to evaluate pattern & content.
o CD30 cannot be interpreted outside the context of CD20, CD45, CD15 and the milieu, as immunoblasts are CD30 +.
o Interpretation of lymphoid content as percentages of B & T cells is meaningless. The population of interest is important.
o IHC does not solve all problems. Simple stains sometime do.
Summary
o Hodgkin’s Lymphoma has 2 main variants
o NHLn History very importantn WHO 2008 can be put into a clinical
perspective by conceptualizing NHLs as Good, bad & ugly.
n Clonality assessment by IHC only possible for some B cell neoplasms.
