ID of B lymphoblasts and B-ALL Flashcards by Jessica Tomsula

How can B-ALL be identified by

flow cytometry ?

positive:
- CD19, CD22, CD79a
- HLA-DR, CD38, and CD10
- TdT, CD34
negative:
- surface light chain expression
- negative or dim for CD45
SSC is low with minimally increased FSC (moderate)
Subset of B-ALLs are positive for:
- CD13, CD33, CD71 and/or CD123

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What is the expression pattern of

B cell markers in B-ALL ?

moderate CD19
- occasional cases have been reported with a reduced level
positive for CD79a and Pax-5
CD20
- most often is negative but ~20% of cases can be partial, dim or variable

IMP:

NK cells can sometimes show non-specific B cell marker staining so must exclude if evaluating for MRD

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What is the pattern of expression of

CD34 and TdT in B-ALL ?

CD34 is usually positive but
- ~30% of cases can be negative
- lack of CD34 or partial expression is often seen in BCR-ABL1 B-ALL
TdT
- is most often positive
~4% of B-ALL
- lack both TdT and CD34

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What is the pattern of expression of

CD71 and CD123 in B-ALL ?

CD71
- positive on a subset (10-32%) of B-ALL
- it is more often positive in T-ALL
CD123
- is often positive
- stronger expression is usually noted in B-ALL with mature phenotype

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What is the pattern of expression

of CD45 in B-ALL ?

may be completely negative but most cases show heterogeneous or variable expression
IMP
- moderate expression can be occasionally seen
- bright CD45 is never seen in B-ALL

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What is the pattern of expression

for CD10 in B-ALL ?

majority of B-ALL show bright CD10 expression
- BUT some cases may be CD10 negative
- BCR-ABL1 associated B-ALL is bright for CD10
- KMT2A associated B-ALL is negative for CD10

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What is the pattern of expression of

CD13, CD15, CD33, and CD65 in B-ALL ?

subsets of B-ALL may show myeloid marker expression
CD33 is more often positive than CD13
- IMP: co-expression of both is RARE
Aberrant expression of CD13 or CD33 is more common in BCR-ABL1 positive B-ALL
KMT2A B-ALL
- shows aberrant expression of CD15, CD33 and or CD65

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What are the immunophenotypic categories

often seen with B-ALL ?

Pro-B-ALL
Common ALL
Pre-B-ALL
Mature B-ALL

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What is the immunophenotype of

pro-B-ALL and Common ALL ?

Pro-B-ALL
- aka early pre-B-ALL
- TdT+, CD19+, CD10-
Common ALL
- CD10+

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What is the immunophenotype of

Pre-B-ALL and Mature B-ALL ?

Pre-B-ALL
- CD10+/-
- cytoplasmic IgM+
Mature B-ALL
- surface IgM+
- Note:
  - majority of mature B-ALL with expression of surface Ig are classified as Burkitt lymphoma in leukemic phase

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What are some differentiating flow findings

to help separate B-ALL from Hematogones ?

FSC vs. SSC properties of B-ALL
presence of aberrant CD13 or CD33
lack or very dim expression of CD45

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Which B cell lymphomas can show a lack

of surface Ig expression ?

Follicular lymphoma
CLL
DLBCL
rarely HCL

Note:

they may also show dim or moderate expression of CD45
positive CD20, lack of blastic markers (TdT and CD34) and lack of bright CD10
- favors a mature neoplasm

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What is the normal % of

hematogones in the marrow ?

~1%
number decreases with age but may be increased in BMs from patients on chemotherapy
patients with MDS and MPNs
- show a lack of hematogones
- or at least decreased numbers of them

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The phenotype of hematogones depends on their

developmental stage, how many stages are there and

what are their immunophenotypes ?

Usually all have very low SSC and variable FSC, smeary other markers

Stage I
- positive: TdT, CD10, CD19, CD34, CD38 and CD58
Stage II
- positive: CD10, CD19, CD38 and cytoplasmic IgM
- negative: CD34, CD58 and TdT
Stage III
- acquire dim (often variable) CD20
- progressive loss of CD10

Note: CD45 expression and intensity increases from Stage I–> III

IMP: Stage II are predominant form (~65%)

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How can CD34 and CD123 be used

to differentiate B-ALL from Hematogones ?

Discordant expression is seen in hematogones
- Less mature hematogones (dim CD45+)
  - express CD34
  - lack CD123
- more mature hematogones (moderate CD45+)
  - lack CD34
  - express CD123

IMP: blasts in B-ALL will have concordant expression of these markers

also hematogones do not express markers from different cell lines, eg CD13 and CD33

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What is the expression of TdT

in hematogones ?

hematogones have down-regulated TdT expression (from negative to dim)
B-ALL blasts usually have strong TdT but there are subsets that are negative

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What is the pattern of expression of CD10

in hematogones vs. B-ALL ?

much brighter inB-ALL
- but subsets of B-ALL can be negative for CD10

IMP: in contrast, CD38 expression is bright in hematogones and more dim in B-ALL

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What is the pattern of expression

for CD58 in B-ALL and hematogones ?

B-ALLs are positive for CD58
Hematogones
- this antigen is only positive in the very early forms
- majority of hematogones are negative

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How is T-ALL differentiated from

B-ALL by flow cytometry ?

positive for cCD3
- sCD3 is usually negative
positive for:
- CD2, CD5, and or CD7
- at least 1 blastic marker, TdT, CD34 and or CD1a
Subset are positive for
- CD71
- CD123
CD45 is usually brighter than B-ALL
B cell markers are negative
CD10 is positive on at least a subset
- but usually partial or dim
- not bright like in B-ALL

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What are some markers that B-ALL

and AML can have in common ?

AML cases can be positive for some B cell markers
- esp CD19 but less often CD79a
B-ALL blasts can be positive for some myeloid markers
- CD13, CD33
Both types of leukemia are positive for:
- CD38
- HLA-DR
- Can also be positive for:
  - TdT
  - CD71
  - CD123

How can AML be differentiated

from B-ALL?

AML is positive for CD117
- strong expression of CD13 and CD33
- strong MPO
- lack of CD10 expression
AML with monocytic differentiation is positive for:
- CD11b, CD11c, CD14, CD56, CD64 and CD123
Pure erythroid leukemia
- CD71
- Glycophorin A, Hemoglobin A
- lacks B cell markers and CD10

What is the definition of a mixed

phenotype acute leukemia (MPAL)?

the majority are myeloid/B and myeloid/T leukemias
they coexpress myeloid and lymphoid markers
MPALs are often positive for:
- CD34, TdT, and HLA-DR
Myeloid lineage
- confirmed by MPO (flow or IHC)
Monocytic lineage
- need positive expression of at least 2 markers
  - CD11c
  - CD14
  - CD64, Lysozyme
  - Non-specific esterase

How are the B and T cell lineages of an

MPAL confirmed ?

B cell lineage
- bright CD19 expression, if dim you need either cCD22 or cCD79a
- expression of CD10 can also be considered B cell lineage
T cell lineage
- surface or cCD3

In a B-ALL, what is there is MPO expression, does

that qualify for an MPAL ?

IF the B-ALL has a perfect immunophenotype with lack of CD13 and CD33
homogeneous expression of lymphoid markers
low level MPO
- This does NOT qualify for an MPAL

B-ALL vs. TdT+ B cell neoplasms with BCL2 and or MYC rearrangements ?

* occasionally mature B cell neoplasms can have blastic morphology and may represent a lymphoblastic transformation of a lower grade lymphoma like follicular * Suggested * TdT+ B cell lymphomas with BCL2 and or MYC rearrangment be classified as * High-grade TdT+ blastic B cell leukemia/lymphoma * they are very aggressive with poor prognosis

In B-ALL, what is the single most powerful clinical predictor of remission induction and duration as well as long term survival ?

* Total WBC count at the time of diagnosis * High WBC count is associated with extramedullary disease at diagnosis with high risk for relapse in the CNS and testes * Other important prognostic parameters include: * age at diagnosis * chromosomal abnormalities of the tumor * persistence of leukemia after induction

Which B-ALL types are often positive for t(9;22)?

* Common B-ALL * pre-B-ALL

Which B-ALL types are frequently associated with t(4;11) ?

* pro-B-ALL

What patients typically have a poor prognosis?

* adult patients with any of the following: * pre-B-ALL * t(4;11) * t(9;22) Note: the absence of these shows a better outcome after intense chemotherapy

What is true about CD10- pre-B-ALL ?

* high risk subgroup in adult patients * associated with high frequency of KMT2A aberrations and worse prognosis

What is true about pro-B and/or t(4;11) ALL ?

* associated with worse prognosis * but responds well to high dose cytarabine therapy and stem cell transplantation

Which B-ALL subgroup tends to have variable morphology ?

* t(1;19) * blasts are larger with more irregular nuclei * basophilic cytoplasm and pronounced vacuoles

What is the immunophenotype profile of pro-B-ALL ?

* aka early pre-B-ALL * CD10- * CD19+, TdT+

What is the immunophenotype of Common B-ALL ?

* CD10+ * CD19+ * CD34+ * TdT+

What is the immunophenotype of Pre-B-ALL ?

* CD10+/- * cytoplasmic IgM+ * CD34+/- * TdT+/-

What is the immunophenotype of mature B-ALL ?

* surface IgM+ * CD34- * TdT- Note: if there is a MYC gene rearrangement these are considered to be Burkitt lymphoma in leukemic phase

What is the immunophenotype of BCR-ABL1 B-ALL ?

* bright CD10 * CD11b+/-, **CD13 (dim)+/-** * **CD15+/-** * CD19+, CD20- (rarely+) * CD22+, **CD25+**, **CD33+(dim, but may be negative)** * CD34+/- (may be partial) * CD38+/- * CD45+(dim)/- * CD66c+, TdT+/-

What is the immunophenotype of MLL alterred B-ALL ?

* **CD10- (rare cases may be partially +)** * **CD15+(rare cases are negative)** * CD19+, CD22+, CD33+/- * CD34+, CD65s+/- * TdT+/- Note: * Occasional cases may show a mature B cell immunophenotype with clonal surface light chains, blastic morphology and lack of CD10, CD20, CD34 and TdT

What marker can be used to differentiate B-ALL from lymphomas of follicle center origin ?

* CD10 * usually quite bright in B-ALL * only moderate intensity in non-B-ALL * ex: follicular lymphoma

What is the association of myeloid antigen expression on B-ALL ?

* associated with a worse prognosis * seen in: * BCR-ABL1 * Balanced t(12;17) * extra copy of ETV6-RUNX1 fusion * usual markers expressed * CD33 \>CD13

How does the expression of CD20 and CD45 in B-ALL correlate with outcome?

* CD45 * intensity greater than 75th percentile * CD20 * intensity greater than 25th percentile * adverse prognostic indicator, particularly with younger patients * Rituximab does improve the outcome * both significantly correlated with poor outcome independently

What are poor prognostic factors in adult B-ALL ?

* elevated total WBC count (\>50 x10^9/L) * t(9;22) * t(4;11) * t(1;19) * hypodiploidy * -7, +8 * expression of myeloid antigens (CD13 and CD33) * persistent MRD * older age

What are factors associaated with better prognosis in adult B-ALL ?

* age \<30 years old * WBC count \<30,000 * t(10;14) * complete remission in 4 weeks

In adult patients with BCR-ABL1 + B-ALL, what additional cytogenetic abnormalities are frequently seen?

* 67% of patients with B-ALL with BCR-ABL1 show additional cytogenetic aberrations including: * additional philadelphia chromosome * -7 * +8 * +X * del(9p)

What is a very important risk predictor for pediatric ALL patients?

* MRD status is an independent prognostic factor that used for risk stratification * monitored by flow cytometry * IMP: hematogones are absent during the first few weeks of induction therapy, therefore, anything immature that is present likely represents residual disease * quantitative real time PCR looking for fusion transcripts

What finding by flow cytometry is predictive of an impending CNS relapse in B-ALL?

* sample free of RBCs * TdT+ cells or cells with aberrant immunophenotype with negative cytology

What is the differentiatl diagnosis for B-ALL ?

* Burkitt lymphoma, DLBCL * Follicular lymphoma * Mantle cell lymphoma, blastoid variant * PTCL * Plasma cell myeloma * Plasmablastic lymphoma * High-grade TdT+ blastic B cell lymphoma/leukemia with BCL2/MYC gene rearrangements * AML, MPAL * BPLL, TPLL * Metastatic carcinoma/Small round blue cell tumor * Hematogones

What are some IHC markers for Small cell carcinoma and Ewing's sarcoma ?

* Small cell carcinoma * panck, CD56, TTF1, high Ki67 * Ewing's sarcoma * CD99, FLI-1, Caveolin1, Leu7 * Chromogranin, NSE and synaptophysin

What are some IHC findings for Rhabdomyosarcoma, Neuroblastoma and Desmoplastic small round cell tumor ?

* Rhabdomyosarcoma * MyoD1, myogenin, desmin, muscle specific actin * Neuroblastoma * NSE, chromogranin A * Desmoplastic small round blue cell tumor * keratins, EMA, desmin, NSE