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Clinical Diagnostic Cytometry

CD5+ LPD

Immunophenotyping LaboratoryRoyal Marsden Hospital HMDS

Ricardo MorillaSenior Clinical Scientist

Head of Immunophenotyping

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Clinical Diagnostic Cytometry

CD5+ LPDTwo groups:CD5+ and CD5- mature B-cell LPD

This distinction has only didactic value and there areMany important exemptions.

Such as not yet recognized CD5+ B-cell LPD.

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Traditional CD5+ LPD

Typical or atypical B-CLL / B-SLL

B-CLL in plasmacytoid transformation

MCLMBL

? B-PLL discrepancy in publications form neg to pos 50-70%. Some cases from pre-existing B-CLL others de novo others from leukemized MCL.

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Traditional CD5- LPDs

MZL HCL LPL FL and PC neoplasms

CD5+ 25% of SMZL in rare cases of FL and HCL

Unfavorable prognosis.

In HCL resistance to a-interferon and sensitive to 2-CdA

Others: Richter syndrome and 10% of DLBCL

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B-CLL / B-SLL

Lymphocytosis > 5000/ulAt least 5000 clonal B-lymphsBM infiltration >30%Lymphadenophaties

Clinical features vary:none weakness fevers night sweats

feeling tired weight loss

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Typical CLL

Morphology: Small lymphocytes less than 10% prolymphocytes and less than 15% lymphoplasmacityc cells

Typical smudge cells

Phenotype: Positive : CD5 CD19 CD23 CD43 CD200++ Dim: sIg CD20+/- CD22 +/-Negative : CD79b and CD10

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Prognostic markers in CLL:

Genetics Abnormalities: 17p 11q t12 del: 13q

Molecular markers: unmutated IGHV and NOTCH & SF3B1 mutations

Phenotypic markers:

CD38: Independent. Can vary course. Cut off 5% to 30%?Therapeutic target

CD49d: >30% shorter survival found in 34% CLL(ITGA4) α intregrin (α4β1)

Zap 70: ?surrogate for IGHV. Not easy to detect by FC? Quantitative mRNA more reliable

E. Monserrat BJH 2016 172 (48-55)N. Chiorazzi Haematology 2012 (76-87)

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Good prognostic markers in CLL

1 Mutated IGH V 2 Deletion 17p 3 CD38 >30%4 Trisomy 125 ZAP 70 >20%

Learning point

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Prognostics marker Incidence

%

Med Survival

years

IGH V mutated 24 24yrs

IGH V unmutated 45 8

CD38 >30% 30 <10

CD38<30% 70 >15

ZAP70 >20% 60 6-10

ZAP70<20% 30-40 >15

Deletion 13q 55 17

Deletion 11q 18 6-8

Trisomy 12 16 9-11

Normal cytogenetics 18 9-11

Deletion 17p 7 2-3

T L Parker, Discov Med 11(57):115-123, February 2011

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Cell markers in CLL

Matutes’ revised CLL score: 1997CD5 CD23 CD79b SmIG FMC7 (CD20)

Euroflow strategy BCLPD subgroupsLST & B-CELL1CLL from other B-LPD Leukaemia 2012 5 markers most contributed to 1st PC (principal component)

CD79b CD200 SmIG CD23 CD20

ERIC/ESCCA members harmonization (2016)Required markers: CD19 CD5 CD20 CD23 SmIGRecommended: CD43 CD79b CD81 CD200 CD10 ROR1

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Evaluation markers CD45 CD19 CD20

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

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CLL recommended markers CD200 CD43 CD79b

CD200

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

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CD43

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

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CD79b

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

CLL 5 FL B-NHL 0 MCL HCL CLL4 BNHL1 BNHL3 CD5+

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Immunophenotype: CLL Score: 5Prognostic markers:CD38 positive - Zap70 negativeFISH: loss of P53Molecular Biology: Unmutated

Learning point

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Diagnosis:

1. Atypical CLL2. Mantle cell lymphoma3. CLL with good prognosis4. CLL with bad prognosis5. Marginal Zone Lymphoma

Learning point

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69 Year old male Referred from external hospitalCLL diagnosis BM sample pre-treatmentHb 88 WBC 34 Plts 108 lymphs 1732% clonal B-cells Phenotype:Positive: CD5 CD19 CD20++ CD22 FMC7

weak Kappa expressionNegative: CD10 CD23 CD43 CD79b and CD200CLL Score: 2CD5+ CD19+ CD38+: 2%

What to do next?1 Morphology 2 Cytogenetics 3 Molecular

Learning point

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Phenotype:

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Morphology:

Small lymphs evenly condensed chromatin no nucleoli. Some nuclei appear to have

clefts or are folded

DD: CLL / CD5+ LPD.

Cytogenetics:

No clonal loss of: TP53 ATM or 13q14 No evidence of t(11;14)Probe CEN12 34% gain of Chromosome 12 Comment Trisomy 12 is a common finding in CLLBut also a frequent finding in other B-LPDs

Molecular:No TP53 mutations detected.

CEP12 13q14 13q34

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Diagnosis:

1 CLL2 MCL3 Atypical CLL4 CD5+ LPD5 MBL

Learning point

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Atypical CLL

Defined on morphology basesNuclear clefts or LP morphologyOr prolymphocytes 15 to 55Atypical phenotypeMost frequent in +12Unmutated IgPoor prognosis

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Monoclonal B-cell Lymphocytosis (MBL)Clonal B-cell population in PB

< 4999 lymphocytes/ulno evidence of B-LPD

Healthy individuals 3.5% >40 yoaDiveded in:

CLL like and CLL not like

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CLL like MBL:Reported in 0.6 to 3% of adult population

More common in the eldery andRelatives of CLL patients and

Patients with autoimmune cytopeniasIn most cases is stable 1% of

Subjects develop CLLPhenotype that of CLL

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Non CLL like MBLReported in 2% of adult population

More frequent in the elderyAnd patients infected with HCV

Heterogeneous phenotypeCD5 neg or MCL like atypical CLL like

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B-PLL

>55% prolymphocytesPhenotype: Positive : CD19 CD20 CD22 CD79b FMC7 CD5 & CD23 in 20-30% cases (WHO)CD38 in 45% cases

t(11;14) excluded

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MCL3 to 10% of NHLLN BM Spleen with/out PB involvementLymphadenophaty hepato & splenomegaly

Derived from the margin of the germinal center

Blastoid & Pleomorphic variantsare of clinical significance

Other variants: small cell and marginal zone like

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MCLPhenotype: Positive : CD5 CD19 CD20 CD22 CD79b FMC7Negative: CD10 BCL6 CD43 CD200

CD23 is negative or weakly posAberrant phenotypes CD5 neg CD10 posImmunohisto: BCL2 Cyclin D1

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66 year old male patient.

Hb:10.5 WBC: 9.5 (Lymphs:6.0) Plts:92Splenomegaly 21cm. Abdominal LN.

Abnormal lymphoid cells in PB film.

PB specimen for Immunophenotyping.

Learning point

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FISH study:using Vysis dual-fusion probes for CCND1(11) and IgH (14)

Normal nucleus

CCND1

IgH

Fusions

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Diagnosis:

1 CLL2 MCL3 Atypical CLL4 CD5+ LPD5 MBL

Learning point

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PB sample Lymphocytosis ? Reactive.

Hb 15.7 WBC 11.3 Plts 224 lymphs 5.8

Clonal B-cells with small size and CLL/SLL phenotypeRepresent 25%

Positive for: CD5 CD10 CD20+/- CD22 CD23 CD200 Lambda+/-Negative: FMC7 CD79b CD10CLL score 5

Morphology: mild lymphocytosis small lymphocytes with Condensed chromatin and no nucleoli478617

Learning point

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Diagnosis:

1 CLL2 MCL3 Atypical CLL4 CD5+ LPD5 MBL

Learning point

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Clinical Diagnostic Cytometry

CD5+ LPD

Many thanks