Precursor B Lymphoblastic Leukemia/Lymphoma (B-ALL / LBL) Precursor T Lymphoblastic Leukemia/Lymphoma (T-ALL / LBL) Early T cell Precursor (ETP) ALL

1. Precursor Lymphoid Neoplasms
By:
Ahmed Makboul Ahmed
M.B.B.Ch, M.Sc
Assistant Lecturer, Clinical Pathology Department, South Egypt Cancer Institute

2. B-Lymphoblastic Leukemia/Lymphoma

3. TERMINOLOGY:
Synonyms:
• B-cell acute lymphoblastic leukemia/lymphoma (B-ALL/B-LBL).
Definition:
• It is a neoplasm of precursors (lymphoblasts) committed to B-cell lineage.
• When blood and BM are extensively involved, acute lymphoblastic leukemia (B-ALL) is
appropriate.
• When disease is confined to mass with absent or minimal blood and BM involvement, term
lymphoblastic lymphoma (B-LBL) is used.
• If patient presents with mass lesion and blood and BM involvement, 25% blasts in BM
defines leukemia (B-ALL).
• Blasts express immature markers [CD34 and TdT (terminal deoxynucleotidyl transferase)]
and B-cell markers (CD19, CD10, CD79a, subset CD20).

4. CLASSIFICATION:
The 2016 WHO Classification of B-Lymphoblastic Leukemia/Lymphoma
I. B-Lymphoblastic Leuekemia/Lymphoma, not otherwise specified (NOS)
II. B-Lymphoblastic Leukemia/Lymphoma with recurrent genetic abnormalities:
1. B-ALL with t(9;22)(q34.1;q11.2); BCR-ABL1 2. B-ALL with t(v;11q23.3); KMT2A (MLL) rearranged
3. B-ALL t(12;21)(p13.2;q22.1); ETV6-RUNX1 4. B-ALL with hyperdiploidy
5. B-ALL with hypodiploidy:
o Haploid (23-29 chromosomes).
o Low hypodiploid (33-39 chromosomes).
o Hypodiploid (40-43 chromosomes).
o Near diploid (44-45).
6. B-ALL with t(5;14)(q31.1;q32.1); IL3-IGH
7. B-ALL with t(1;19)(q23;p13.3); TCF3-PBX1 (E2A-PBX1)
8. B-ALL with intrachromosomal amplification of chromosome 21 (iAMP21) (Provisional entity).
9. B-ALL with translocations involving tyrosine kinase or cytokine receptors (BCR-ABL1-like ALL) (Provisional
entity).

5. ETIOLOGY/PATHOGENESIS:
Etiology:
• Causes of ALL remain largely unknown.
• B-ALL arises in either hematopoietic stem cell or B-cell progenitor.
Pathogenesis:
1. Genetic factors:
• There is some evidence which supports that ALLs are congenital.
• Genetic abnormalities may arise in constitutively activated oncogenes, tyrosine kinase activity or
altered transcriptional regulation.
• Syndromes with increased incidence of ALL e.g. Down syndrome.
• Syndromes with increased incidence of malignancies including B-ALL:
oLi-Fraumeni syndrome, Neurofibromatosis type 1 (NF1), Ataxia telangiectasia.

6. 2. Environmental factors linked to increased incidence:
• Exposure to intrauterine ionizing radiation.
• Exposure to ionizing radiation.
• Exposure to pesticides.
• Post chemotherapy.

7. CLINICAL ISSUES:
Epidemiology:
• B-ALL is the most common childhood neoplasm.
• 80-85% of ALL is of B-cell origin.
• 75% of cases present before age 6.
• Slight male predominance.
Site:
• Bone marrow is the primary site.
• Blood is typically involved.
• CNS, lymph nodes, liver, spleen, and testis in males are often involved.
• Skin, soft tissue, bone, and lymph nodes are primary sites of involvement in B-LBL.

8. Clinical Presentation:
Constitutional symptoms:
• Fever.
• Night sweats.
• Weight loss.
Symptoms related to anemia, thrombocytopenia, or neutropenia:
• Fatigue (due to anemia).
• Bleeding tendency (due to thrombocytopenia).
• Neutropenic fever, recurrent infections (due to neutropenia/leucopenia).
Signs and symptoms related to leukemic infiltrate:
• Bone pain.
• Arthralgias.
• CNS symptoms.
• Frequent hepatosplenomegaly.
• Lymphadenopathy may be present.

9. Laboratory investigations:
1. Complete blood picture (CBC):
• Variable white blood cell (WBC) count:
o50% of patients have WBC < 10 x 10⁹/L (normal or low).
o30% of patients have WBC between 10-50 x 10⁹/L.
o20% of patients have WBC > 50 x 10⁹/L.
o20% of patients may have pre-leukemic state with cytopenia 2-9 months before
development of overt leukemia.
• Anemia and thrombocytopenia almost always present.
• Rarely patients present with asymptomatic eosinophilia.

10. Prognosis:
Overall prognosis:
• Excellent; complete remission rate > 95% in children and 60-85% in adults.
• Cure rate is 80% in children and < 50% in adults.
• Infants with KMT2A (MLL) gene rearrangement generally have poor prognosis.
• Adolescents and young adults (AYA) have better outcome when treated with
pediatric protocols than with adult protocols.
• CNS and testis are sanctuary for leukemic cells and are common relapse sites.

11. Prognostic factors:
I. Clinical & biological:
Favorable Unfavorable
Age 1-10 years < 1 or > 10 years
Gender Female Male
WBC count < 50 x 10⁹/L > 50 x 10⁹/L
Immunophenotype Common B-ALL Lack of CD10 expression
CNS disease No CNS disease Presence of CNS disease

12. Prognostic factors:
II. Cytogenetic abnormalities:
Favorable Intermediate Unfavorable
Hyperdiploidy (> 50
chromosomes, especially with
trisomy 4, 10, 17).
t(5;14); (IL3-IGH) t(9;22); (BCR-ABL)
t(12;21) (ETV6-RUN1). t(1;19); (TCF3-PBX1) KMT2A rearrangement
Normal karyotype B-ALL with iAMP21
Any other abnormality that is
not included in favorable or
unfavorable catagories.
Hypodiploidy (< 45)
BCR-ABL1 like B-ALL
Complex abnormalities

13. 1. Peripheral blood:
oBlasts are usually present in PB.
oBlasts vary in size from small to large.
oBlasts may have coarse azurophilic cytoplasmic granules.
oEosinophilia often seen in B-ALL with t(5;14).
L1 blasts L2 blasts
Size: Small sized blasts. Large sized blasts.
Cytoplasm: Scant cytoplasm. Moderate cytoplasm and may
have vacuoles.
Nucleus: o Condensed chromatin.
o Indistinctive nucleoli.
o Dispersed chromatin.
o Variable nucleoli.
MICROSCOPIC PATHOLOGY:

14. L1 Blasts L2 Blasts

15. 2. Bone marrow:
• Hypercellular BM, usually extensively replaced by lymphoblasts.
• Blast morphology is similar to morphology in PB.
• BM core biopsy shows extensive replacement by lymphoblasts.
• BM core biopsy may show starry-sky pattern due to high number of mitotic figures.
• Patchy or diffuse preserved hematopoietic cells.
• Rarely BM necrosis; ALL is most common neoplasm to present as diffuse necrosis in
BM.

17. 1. Flow cytometric immunophenotyping:
• Degree of differentiation determines immunophenotype:
• CD34 and CD20 expression variable.
• CD45 can be negative but is typically weak.
• Approximately 20-30% of cases also express myeloid associated antigen markers CD15, CD13, and CD33.
o CD13 expression is often seen in B-ALL with t(12;21).
o CD13 and CD33 often expressed in B-ALL with t(9;22).
Stage Immunophenotype
Earliest stage blasts (Pro-B) o Express CD19, cytoplasmic CD79a, cytoplasmic CD22, and TdT/CD34.
o Such ALLs occur in infancy.
Intermediate stage blasts (Common-B) o Express CD10, CD19, surface CD79a, surface CD22, and TdT/CD34.
Most mature stage blasts (Pre-B) o Express CD19, CD10, surface CD79a, surface CD22, cytoplasmic µ
chains (c-µ), CD34 often negative.
ANCILLARY TESTS:

19. 2. Genetic testing:
a). Conventional cytogenetic analysis (Karyotyping):
• Routine karyotype is required on all new B-ALL cases.
• Abnormalities are found in majority of B-ALL/B-LBL cases:
ot(12;21)(p13;q22) ETV6-RUNX1 (a.k.a. ETV6-RUNX1); most common fusion in
pediatric ALL (this abnormality is not detected by karyotyping “cryptic” and should
be detected by FISH or RT-PCR).
ot(1;19)(q23;p13.3) TCF3-PBX1 (E2A-PBX1).
ot(9;22)(q34.1;q11.2) BCR-ABL1.
ot(v;11q23.3) KMT2A rearranged; especially ALL in neonates/infants.
ot(5;14)(q31;q32) IL3-IgH, associated with eosinophilia.
oHyperdiploidy.
oHypodiploidy.

20. b). Fluorescence in situ hybridization (FISH) studies:
• For Adult B-ALL (Required by American Society of Hematology & CAP):
ot(9;22) (BCR-ABL1).
oKMT2A (MLL) gene rearrangement.
• For Childhood B-ALL (Required by Children's Oncology Group (COG) protocol):
ot(9;22) (BCR-ABL1).
oAneuploidy for chromosomes 4, 10, 17.
oKMT2A gene rearrangement.
ot(12;21) to detect fusion gene as well as detect copy number of RUNX1 gene to
exclude iAMP21.

21. BCR-ABL ETV6-RUNX1 KMT2A (MLL)

22. c). PCR:
• Almost all cases have clonal rearrangement of IGH gene.
• RT-PCR is performed if a gene fusion is detected, such as BCR-ABL1, to establish
baseline and to follow-up minimal residual disease.
• Cancer gene panels are performed in selected institutions &/or selected patients.

23. DIFFERENTIAL DIAGNOSIS:
1. Hematogones:
• Definition:
oHematogones are normal B-lymphocyte precursors.
oNormally, they are found in small numbers in most BM specimens.
oThey occur in larger numbers in some healthy infants and young children and in a variety of
diseases in both children and adults.
• Causes of hematogones hyperplasia:
oRegeneration following chemotherapy or BM transplantation.
oAutoimmune disorders.
oCongenital cytopenias.
oNeoplasms.
oViral infections.
oImmunodeficiency states.
• In some cases, they constitute 5% to more than 50% of bone marrow cells.

24. • Increased hematogones cause diagnostic challenges because they share morphologic, and some
immunophenotypic, features with B-lymphoblasts.
• Distinguishing hematogones from residual/recurrent B-lymphoblasts after treatment for B-
lymphoblastic leukemia/lymphoma can be challenging.
• Recognition of the typical maturational profile for hematogones and lack of aberrant antigen
expression are two useful tools to help with this distinction.

25. Hematogones have 3 stages of maturation by immunophenotyping:
• Stage 1 hematogones:
oExpress CD34, TdT.
oExpress high levels of CD10 and CD38
oModerate level of CD22
oWeak CD45
oAbsence of CD20 and surface immunoglobulin.
• Intermediate stage 2 hematogones:
oDownregulate CD34 and TdT completely
oDownregulate CD10 partially
oIncreasing expression of CD22 and CD20.
oModerate CD45.
oSome weak surface immunoglobulin may become evident.
• Stage 3 hematogones:
oUpregulate CD20 expression reaching the intensity of mature B cells
oCD10 and CD38 are slightly downregulated.
oIncreasing expression of polytypic surface immunoglobulin light chains.

26. Stage 1
Stage 1Stage 2
Stage 2
Stage 3
Stage 3

27. Stage 1
Stage 2
Stage 3
ALLHematogones

28. 2. Burkitt Lymphoma:
• Burkitt lymphoma (BL) is mature, high-grade B-cell lymphoma.
• Lymphoma cells are large with unique punctate cytoplasmic vacuoles.
• BL expresses surface immunoglobulin and lacks immature markers CD34 or TdT.
• Presence of MYC rearrangement (most common is t(8;14); MYC-IGH).
3. Other leukemias:
• Immunophenotyping is helpful to distinguish B-ALL from other leukemias.

29. MYC
IGH
MYC-IGH
MYC-IGH

30. Genetic Subtypes of B-Lymphoblastic Leukemia

31. t(12;21)(p13.2;q22.1) (ETV6-RUNX1)
Frequency:
• 25% in children.
• Rare in adults.
Unique findings:
• Common in children, not seen in infants, rare in adults.
• Translocation arises in utero, leukemia may develop later.
• Typically, CD20 negative, frequently expresses myeloid associated antigen CD13.
• Cryptic translocation. FISH and molecular tests are always required for detection.
Prognosis:
• Very favorable with high cure rate.
• Relapse usually occurs later than other types.

32. B-ALL with
t(12;21)

33. Hyperdiploidy (> 50 but < 60 chromosomes)
Frequency:
• 25% in children.
• Rare in adults.
Unique findings:
• Common in children, not seen in infants, rare in adults.
• CD45 is often negative
Prognosis:
• Favorable, particularly with trisomy 4, 10 and 17.

34. Hypodiploidy
Frequency:
• All hypodiploidy B-ALL together account for 5%.
• Seen in both children and adults.
Unique findings:
• No unique morphologic, immunophenotypic, or cytochemical features.
Prognosis:
• Poor prognosis.

35. B-ALL with
hypodiplody

36. t(9;22)(q34.1;q11.2) (BCR-ABL1)
Frequency:
• 2 – 4% in children.
• 25% in adults.
Unique findings:
• More common in adult ALL cases.
• Rarely associated with T-ALL.
• Frequent expression of myeloid associated antigens CD13 & CD33.
• CD25 is highly associated with t(9;22) B-ALL.
• Blasts may show coarse azurophilic cytoplasmic granules.
Prognosis:
• Worst prognosis among patients with ALL.

37. B-ALL with BCR-ABL1

38. t(v;11q23.3) (KMT2A rearrangement)
Frequency:
• Most common in infants < 1 year.
• Less common in older children, increased incidence with age into adulthood.
Unique findings:
• May occur in utero.
• Typically presents with very high WBC count.
• High frequency of CNS involvement at diagnosis.
• Often CD10(-) and CD15(+).
• MLL/KMT2A gene has many fusion partners with AF4 on chromosome 4q21 in majority of cases (t (4;11)).
• ENL gene on chromosome 19p13 and AF9 on chromosome 9p22 are common in remaining cases.
Prognosis:
• Poor prognosis; particularly in infants < 6 months of age.

39. B-ALL with KMT2A
rearrangement

40. t(5;14)(q31.1;q32.1) (IL3-IGH)
Frequency:
• Rare; < 1% of cases.
• Seen in both adults and children.
Unique findings:
• Typically associated with increased circulating non-neoplastic eosinophils. It should be
suspected in the presence of unexplained eosinophilia.
• Blast count may be < 20%.
• Patient may present with asymptomatic eosinophilia.
Prognosis:
• Intermediate prognosis.

41. B-ALL with t(5;14)

42. t(1;19)(q23;p13.3) (E2A-PBX1)
Frequency:
• 6% of cases.
• Less common in adults.
Unique findings:
• Often has pre-B-cell phenotype [CD19(+), CD10(+)], cytoplasmic µ chain [cµ] (+).
• Absent or subset CD34 expression.
Prognosis:
• Intermediate prognosis with intensive therapy.

43. B-ALL with t(1;19)

44. iAMP21
Frequency:
• 2% of cases.
Unique findings:
• No unique morphologic, immunophenotypic, or
cytochemical features.
• Identified using FISH probes for RUNX1 gene:
o5 or more copies of RUNX1 per cell.
o3 or more copies of RUNX1 per chromosome.
• Pathogenesis not thought to depend on RUNX1.
Prognosis:
• Poor prognosis.

45. BCR-ABL1-like ALL
Frequency:
• 10% among children with standard risk ALL.
• 27% among young adults.
Unique findings:
• No unique morphologic, immunophenotypic, or cytochemical features.
• Negative for BCR-ABL1 translocation.
• Have translocations or genetic alterations affecting other tyrosine kinases (e.g. PDGFRB)
or CRLF2 or EPOR.
• Often difficult to diagnose without specialized testing including gene expression profiling.
Prognosis:
• Poor prognosis.

46. Break-apart probe for PDGFRB gene confirms involvement of
PDGFRB.
BCR-ABL1-like ALL

47. T-Lymphoblastic Leukemia/Lymphoma

48. TERMINOLOGY:
Synonyms:
• T-cell acute lymphoblastic leukemia/lymphoma (T-ALL/T-LBL).
Definition:
• Revised 2016 WHO definition of T-lymphoblastic leukemia/lymphoma:
oIt is a neoplasm of lymphoblasts committed to T-cell lineage.
oT-ALL and T-LBL are separated based on numbers of lymphoblasts in BM:
ØT-ALL when > 25% blasts in BM.
oBlast percentage requirement to establish diagnosis of T-ALL:
ØIn contrast to AML, there is no agreement on lower limit of blast percentage
required to diagnose T-ALL.

49. CLINICAL ISSUES:
Epidemiology:
T- ALL T-LBL
Incidence o 15% of all childhood ALL.
o 25% of adult ALL.
85% of all lymphoblastic
lymphomas.
Age at diagnosis < 10 years in 1/2 of childhood T-ALL/t-LBL.
Gender More common in adolescent males.

50. Clinical Presentation:
T- ALL T-LBL
PB & BM
involvement
o Commonly manifests with high leukocyte
count with circulating blasts.
o Aleukemic presentation is uncommon.
o Bone marrow is always involved.
o Relative sparing of trilineage hematopoiesis in
bone marrow compared to B-ALL.
o Bone marrow involvement at diagnosis in about
20% of cases.
Mediastinum o Concurrent mediastinal/thymic or other mass
lesion often present.
o Frequently manifests with rapidly growing
anterior mediastinal/thymic mass.
o Pleural &/or pericardial effusions often present.
o Respiratory symptoms and superior vena cava
syndrome may be seen.
Lymphadenopathy o Lymphadenopathy is common. o LN involvement may be present.
Extranodal sites o Hepatosplenomegaly common.
o CNS involvement is more common than in T-
LBL.
o Involvement of extranodal sites.
o Skin, tonsil, spleen, liver, CNS, and testis may
be involved.

51. Prognosis:
T-ALL:
Childhood:
• Higher risk compared to B-ALL.
• Increased risk of induction failure and early relapse.
• Increased risk of isolated CNS relapse.
Adult:
• Better prognosis than B-ALL likely due to low rate of adverse cytogenetic
abnormalities in adult T-ALL.

52. T-LBL:
Childhood:
• Inferior event-free survival with BM &/or CNS involvement compared to patients with stage
I-III disease.
Adult:
• Longer survival, complete remission (CR) rate, and CR duration in patients with:
oAge < 40 years.
oLDH level < 2x upper limits of normal.
oAbsent or single extranodal site of disease.
• Short survival with failure to achieve CR in patients with:
oAge > 40 years.
oLDH level > 2x upper limits of normal.
oHemoglobin level < 10 g/dL.

53. MICROSCOPIC PATHOLOGY:
T-ALL:
Peripheral blood:
• Leukocytosis with circulating lymphoblasts.
• T lymphoblasts are morphologically indistinguishable from B lymphoblasts.
Bone marrow:
• Lymphoblasts in BM aspirate show morphologic features similar to those found in
blood.
• Extensive marrow involvement on core biopsy and clot sections.
oSheets of blasts with immature nuclear chromatin.
oHigher mitotic figures compared to B-ALL.
• In contrast to B-lineage ALL, normal trilineage hematopoiesis relatively preserved.

54. T-LBL:
Peripheral blood:
• Absent to minimal involvement of peripheral blood.
Bone marrow:
• BM involved in < 20% of cases at diagnosis.
• By definition, blasts account for < 25% of nucleated BM cells in cases with
marrow involvement.

56. 1. Flow cytometric immunophenotyping:
• T lymphoblasts usually express TdT.
• T lymphoblasts variably express CD1a, CD2, CD3 (surface or cytoplasmic), CD4,
CD5, CD7, and CD8.
oCD7 and cytoplasmic CD3 are most often positive.
oOnly CD3 is considered lineage specific.
• T lymphoblasts frequently coexpress CD4 and CD8 (double positive) but could
express only former/latter (single positive) or neither (double negative).
• T lymphoblasts may also coexpress CD10.
• Subset of T-ALL/T-LBL cases also express CD34.
oIn addition to CD34 and TdT, expression of CD1a and CD99 help to determine
precursor nature of T lymphoblasts.
ANCILLARY TESTS:

58. Stage
Immunophenotype
CD1a CD2 cCD3 sCD3 CD4/CD8 CD5 CD7 TdT CD34
Pro-T - - + - Double - - + + +/-
Pre-T - + + - +/- + + + +/-
Cortical T + + + - Double + + + + -
Medullary T - + + + Single + + + - -
T-ALL Stages of Intrathymic Differentiation:

59. 2. Genetic testing:
a). Cytogenetic analysis:
i. Recurrent cytogenetic abnormalities:
• Structural chromosomal abnormalities detected in 60% of T-ALL/T-LBL.
• Translocations involving T-cell receptor genes are the most common recurrent
cytogenetic abnormality.
oFound in about 40% of T-ALL cases.
oTRA and TRD at 14q11.2, TRB at 7q34, and TRG at 7p14 are involved.
• Transcription factor genes commonly juxtaposed to T-cell receptors regulatory
regions:
oTLX1 (HOX11) in t(7;10)(q34;q24) and t(10;14)(q24;q11.2)
oHOXA in inv(7)(p15q34), t(7;7)
oTAL1 in t(1;14)(p32;q11.2) and t(1;7)(p32;q34)

60. ii. Cryptic deletions leading to loss of tumor suppressor genes:
• Deletion of CDKN2A at 9p21, is the most frequent cryptic deletion.
oPresent in > 50% of T-ALL.
iii. ABL1 rearrangements in T-ALL:
• NUP214-ABL1 gene fusion in t(9;9), strictly associated with T-ALL, 3-6%
oCryptic translocation not detectable by cytogenetics.
• ETV6-ABL1 gene fusion in t(9;12), < 1%
• BCR-ABL1 gene fusion in t(9;22): very rare

61. b). PCR:
Antigen receptor gene rearrangements:
• Clonal rearrangements of T-cell receptor genes detected in virtually all cases.

62. DIFFERENTIAL DIAGNOSIS:
1. Precursor B-Lymphoblastic Leukemia/Lymphoma:
• B lymphoblasts are cytomorphologically similar to T lymphoblasts.
• Flow cytometry or immunohistochemistry can easily separate T lymphoblasts from B
lymphoblasts.
2. Mature T-Cell Lymphoma:
• Subset of T lymphoblasts with mature-appearing morphology may mimic mature T-
cell lymphoma.
• Angioimmunoblastic T-cell lymphoma may have overlapping immunophenotype
including expression of CD3 and CD10.
• Expression of immature markers in T lymphoblasts are useful to separate T
lymphoblasts from mature T-cell lymphoma.

63. EARLY T-CELL PRECURSOR (ETP) ALL:
• Provisional entity in revised 2016 WHO.
• Diagnosed when case of T-ALL meets all following criteria:
1. Absence of expression of CD1a and CD8.
2. Absence or weak expression of CD5 in < 75% of blasts.
3. Expression of 1 or more of following myeloid or stem cell associated antigens in
≥ 25% of blasts:
oCD117.
oCD34.
oHLA-DR.
oCD13.
oCD33.
oCD11b &/or CD65.