18.06.16 ALL

Question 1

What is ALL?

Answer 1

ALL is a neoplasm characterised by clonal expansion of leukemic cells in the BM, lymph nodes, thymus or spleen.

ALL accounts for 75% of childhood leukaemia (85% B-cell, 15% T-cell) and 6% of adult leukaemia (75% B-cell, 25% T-cell).

Question 2

What are the features of Adult ALL?

Answer 2

ncidence 0.9-1.6/100,000 (> 60yo) and 0.4-0.6/100,000 (25-50yo). Males are more often affected than females.

Clinical features variable and depend on extent of disease; fever, fatigue, bone/joint pain, headache, weight loss, Shortness of breath, Swollen lymph nodes, particularly lymph nodes in the neck, armpit, or groin, which are usually painless, Swelling or discomfort in the abdomen, bruising, infections, lymphadenopathy, hepato/splenomegaly, and commonly haematological abnormalities including anaemia, leucocytosis, neutropenia, thrombocytopenia.

Question 3

Why is genetic testing useful at diagnosis in Adult ALL?

Answer 3

Genetic testing at diagnosis allows prognostic classification, which can help determine intensity of treatment regimen (along with other factors including age WCC at presentation, sex, immunophenotype, MRD response), in order to avoid over or under treating (risk adapted therapy). NB cytogenetics stratification is less important in T-ALL, no current protocols use this info.

Cytogenetic testing can also dictate the use of a particular drug e.g. TKI added to therapy in cases with t(9;22) and can aid diagnosis e.g. distinguish between B-ALL and Burkitt lymphoma where therapies differ.

Question 4

What are the most significant genetic prognostic factors for adult B-cell ALL?

Answer 4

t(9;22)(q34;q11) BCR-ABL1 Philadelphia chromosome.

t(4;11)(q21;q23) KMT2A (MLL)-AFF1

Complex karyotype

Hyper hyperdiploidy (51-65 chr)

Hypodiplody (<44 chromosomes) and near triploidy

TCF3 rearrangements

IGH@ rearrangements

Question 5

Describe t(9;22)(q34;q11) BCR-ABL1 Philadelphia chromosome in B-cell ALL

Answer 5

Poor prognosis (9% 5 year survival)

25-30% of adult ALL (most common rearrangement in adult ALL)

Use of TKI therapy has been shown to enhance the long-term outcomes in Ph+ve ALL (Fielding et al Blood 2014, 123:843-850) and allo BMT may not be mandatory.

Recent paper from the Leuk Res Cyto Group (Chilton et al Leukaemia 2014, 28:1511-1518) indicates that the co-existence of HeH ameliorates the adverse effect of Ph positivity (1232 patients 15-65 yo).

Common secondary abnormalities include: +Ph, -7/7q-,del(9p), +8, +X, and HeH.

~25% of Ph +ve ALLs p210 BCR-ABL1 fusion; 75% p190 (minor BCR breakpoint). p190 is only found in de novo ALL, while p210 s seen in de novo ALL and CML in lymphoid blast crisis (can determine by RT-PCR).

Recent studies have shown that the majority of Ph+ve ALLs and lymphoid BC CMLs show a deletion if IKZF1 (7p21) [significance unclear].

Question 6

Describe t(4;11)(q21;q23) KMT2A (MLL)-AFF1 in B-cell AML.

Answer 6

Poor prognosis (33% 3 year survival)

KMT2A rearrangements are seen in ~10% adult ALL [most common t(4;11)].

KMT2A rearrangements may occur in B or T-ALL.

The KMT2A gene encodes a 430 kd nuclear protein thought to be a positive regulator of gene expression in early embryonic development and haematopoiesis. HOX genes are a prominent downstream target dysregulated by the fusion protein.

> 80 partners identified so far.

Prognosis of other KMT2A rearrangements is not certain therefore important to distinguish t(4;11) (commericially available dual-fusion FISH probes).

Other KMT2A rearrangements frequently seen in ALL are t(9;11) (KMT2A-MLLAT3) and t(11;19) (KMT2A-ENL), t(10;11) (KMT2A-MLLT10).

Question 7

What is the significance of a complex karyotype in adult B-cell ALL?

Answer 7

Poor prognosis (28% 5 year survival)

Question 8

What is the significance of high hyperdiploidy in adult B-cell ALL?

Answer 8

Good prognosis.

~7% adult ALL

Commonly gained: 4, 6, 10, 14, 17,18 , 21 (often 2 copies gained) and X.
Structural abnormalities: dup(1q), del(6q), abn 9p, abn 12p.

HeH not a mutually exclusive event. If a structural rearrangement such as t(9;22), t(4;11) or t(1;19) is also present the hyperdiploidy is thought to be a secondary event- reported to ameliorate the effects of t(9;22), see above.

Question 9

What is the difference between doubled up near haploid/low hypodipoloidy karyotype and a true hyperdiploid?

Answer 9

Important to distinguish between a doubled up near haploid/low hypodiploidy karyotype and a true hyperdiploid.

True hyperdiploid: gained chromosome tend to be trisomic (other than 21 and X)

Doubled up near haploid/low hypodiploid: usually 4 copies of gained chromosomes.

If the presence of two clones cannot be demonstrated by FISH/G-banding then flow cytometry to measure DNA index or STR zygosity testing (most duplicated chromosomes will be homozygous).

Question 10

What is the significance of Hypodiplody (<44 chromosomes) and near triploidy in adult B-cell ALL?

Answer 10

Poor prognosis

Incidence = 4% 15-29 years, 16% >60 years. 22% 5 year overall survival). In Moorman et al’s study (2007) these two entities were classified as one subgroup as these karyotypes have been shown to represent a single distinct subtype of adult ALL.

Commonly retained: 1, 4, 5, 6, 8, 10, 11, 18, 19, 21, 22 and sex chromosomes (common losses of 3 and 7).

Low hypodiploidy more common than near haploidy (converse is true for infants).

Question 11

What is the significance of TCF3 rearrangements in adult B-cell ALL?

Answer 11

Adult incidence is <5% of B-ALL. The t(1;19), which can occur in a balanced or unbalanced form [der(19)t(1;19)] is the most common rearrangement resulting in TCF3-PBX1 gene fusion. The fusion protein has an oncogenic role as a transcriptional activator. Important to characterise a TCF3 break-apart FISH result or equivocal cyto result as the t(1;19) has in intermediate prognosis and the rare t(17;19) (TCF3-HLF fusion) has a poor prognosis.

Question 12

What proportion of adult T-cell ALL have a detected cytogenetic abnormality?

Answer 12

50-70%

Question 13

What are the common rearrangements detected in adult t-ALL?

Answer 13

Approximately 35% of cases show rearrangements involving the TCR loci at 7q34 (TCRB) or 14q11 (TCRA/D).

The lack of trial data means that the prognostic significance of most T-ALL rearrangements remains uncertain; however, a complex karyotype (5 or more abnormalities) is associated with a poor prognosis in adult ALL. Numerical abnormalities are less common than in B-ALL.

TLX1-TRA/TRD t(10;14) recognised finding; prognostic significance under debate. According to data from UKALL XII/ECOG 2993 trial (Marks et al Blood 2009 114:5136-45) TLX1 rearrangements are not associated with a good prognosis as previously reported.

No FISH testing is mandatory as at present patients with T-ALL are not stratified on the basis of genetic findings.

NOTCH1 receptor activating mutations are also common in T-ALL (60%). More rarely NOTCH1 is activated by the t(7;9) translocation where is juxtaposed with TRB. Patients with mutation in NOTCH pathway may have a higher event free survival than those without.

Question 14

Some AL patients cannot be assigned to a particular cell lineage. What is a common abnormality detected in these patients?

Answer 14

t(9;22)
KMT2A rearrangements
trisomy 13

Question 15

What are the disadvantages of karyotyping of bone marrow samples?

Answer 15

normal marrow outgrowing leukemic clone
apoptosis of leukemic cells
high failure rate
poor quality metaphases
cryptic/subtle rearrangements. Growth factor supplements e.g. SCF, interleukins, FLT3 ligand can be added during incubation of B-ALL to improve QA. (or PHA for T-cell cultures.

Question 16

How can multiplex RT-PCR be applied to ALL and AML patients?

Answer 16

Multiplex RT (reverse transcription)-PCR can be used as a molecular technique to identify the most common fusion transcripts in ALL or AML. This method also allows identification of the exact breakpoint so that molecular monitoring of disease can be undertaken during treatment

Other techniques are now playing an increasingly important role e.g. MLPA or arrays to detect prognostically relevant deletions such as IKAROS (IKZF1) and sequencing can be used to detect point mutations. Future trials are likely to incorporate some exome and MLPA screening. This will allow improved risk stratification.

Question 17

Microarrays and NGS have discovered new genes associated with ALL?. Give examples.

Answer 17

IKZF1 (7p21)
Other genes frequently deleted in B-ALL include PAX5, EBF1 and BTG1.
Recently mutations or deletions of CREBBP (histone acetyltransferase) have been shown to be present in 20% of cases of relapsed ALL. It is thought that these mutations may influence response of leukemic cells to chemotherapy and therefore the use of histone deacetylase inhibitors may be an important therapeutic approach in the treatment of high risk and relapsed ALL.

Question 18

What is the significance of IKZF1 in ALL?

Answer 18

a master regulator of lymphocyte development and differentiation. Alterations (deletions/mutations) are seen in ~15% of B-ALL and are associated with a poor outcome. Deletions/mutations of IKZF1 are found in 80% of Ph+ve ALL. Deletions can be detected by MLPA (or SNP array) and may be incorporated into childhood risk stratification within the next few years. Other genes frequently deleted in B-ALL include PAX5, EBF1 and BTG1.

Question 19

Why might interphase FISH be useful in ALL patients?

Answer 19

Chromosome analysis in remission is not mandatory.

Due to the difficulty of detecting a potentially low number of poor quality ALL metaphases, interphase FISH may be helpful.

MRD is best monitored by molecular means (e.g. q-PCR or PCR analysis of Immunoglobulin and TCR gene rearrangements) or flow cytometry.

At relapse cytogenetic analysis may identify karyotype evolution or secondary malignancy.

Question 20

What are the symptoms of ALL?

Answer 20

symptoms of ALL include bruising or bleeding due to thrombocytopenia, pallor and fatigue from anemia, and infection caused by neutropenia. Leukemic infiltration of the liver, spleen, lymph nodes, and mediastinum is common at diagnosis

Question 21

Describe paediatric ALL

Answer 21

It is the most common malignancy in children (25% of pediatric cancer). Incidence in childhood 3~4/100,000 per year (in adults it is <1). The peak age in children is at 2-5 years.

ALL accounts of 75% of childhood leukaemia (85% B-cell, 15% T-cell)

Question 22

What is infant ALL?

Answer 22

Infant ALL (<1year) is rare. The biological features are different from ALL in older children, with infant ALL having an immature B phenotype lacking CD10 expression, with a higher tumour load at presentation. Overall survival is considerably worse for infant ALL than for older children. 15-20% of children with ALL will relapse.

Question 23

What are the most signifiant prognostic rearrangements in paediatric infant/childhood B-ALL

Answer 23

t(9;22)(q34;q11) BCR-ABL1 rearrangement;

KMT2A (MLL) rearrangements

t(12;21) ETV6-RUNX1 rearrangement

iAMP21 (intrachromosomal amplification of chrm 21)

High Hyperdiploidy (51-65 chromosomes).

Question 24

Describe t(9;22)(q34;q11) BCR-ABL1 rearrangement; in paed B-ALL

Answer 24

Seen in 2-4% of childhood ALL, associated with poor prognosis.

The BCR-ABL1 fusion protein shows constitutive ABL1 kinase activity, conferring a growth and survival advantage.

~25% of Ph +ve ALLs p210 BCR-ABL1 fusion; 75% p190 (minor BCR breakpoint). p190 is only found in de novo ALL, while p210 s seen in de novo ALL and CML in lymphoid blast crisis (can determine by RT-PCR).

The prognosis of Ph+ve ALL was dire until the advent of TKIs. Initially imatinib was combined with intensive chemotherapy. 3 year EFS rates as high as 80% in children and 60% in adults The introduction of 2nd generation TKIs has raised the possibility that less intensive chemo may be used and allo BMT may not be mandatory.

Recent paper from the Leuk Res Cyto Group (Chilton et al Leukaemia 2014, 28:1511-1518) indicates that the co-existence of HeH ameliorates the adverse effect of Ph positivity independent of age; however, study only included patients 15-65 yo therefore effect on children <15 not clear. Currently, in these patients poor prognosis of the 9;22 overrules the favourable prognosis of the hyperdiploidy.

Recent studies have shown that the majority of Ph+ve ALLs and lymphoid BC CMLs show a deletion if IKZF1 (7p21) [significance unclear].

Question 25

Describe KMT2A (MLL) rearrangements in paed B-ALL

Answer 25

The age specific incidence of KMT2A translocations is highly skewed.

Most common abnormality in infants <1yr - Incidence in infant ALL= 60-80% infants harbouring a MLL translocation with ~50% being t(4;11).

Incidence in childhood ALL = 3~8% but the incidence of t(4;11) increases with age among adults.

The KMT2A gene encodes a 430kd nuclear protein though to be a positive regulator of gene expression in early embryonic development and haematopoiesis. HOX genes are a prominent downstream target dysregulated by the fusion protein.

Gene fusions juxtapose 5’KMT2A and 3’ partner gene (>60 partner genes identified so far and 71 recurrent translocations).

KMT2A translocations may occur in utero (has been identified in neonatal blood spots of infants who go on to develop leukaemia).

Question 26

What are the common KMT2A rearrangements in B-cell ALL. What is the prognostic significance?

Answer 26

Common rearrangements;

t(4;11)(q21;q23) KMT2A-AFF1 (40~50% infant ALL, i7q common secondary abnormality- poor prognosis)

t(9;11)(q22;q23) KMT2A-MLLT3 (10% infant ALL)

t(11;19)(q23;p13.3) KMT2A-MLLT1 (10% infant ALL)

t(1;11)(p32;q23) KMT2A-EPS15

There is some conflicting data regarding the prognostic significance of translocations other than t(4;11); in general infant ALL <6months age with an KMT2A rearrangement has a poor prognosis.

Question 27

Describe t(12;21) ETV6-RUNX1 rearrangement in childhood B-ALL

Answer 27

Common translocation in childhood B-ALL (25% cases); typically 3-6yo.

Not seen in infants and frequency decreases in older children (rare in adulthood).

Cryptic translocation which can be detected by FISH or RT-PCR (although the latter may miss some rare variant breakpoints); very few cases have been reported cytogenetically visible.

The translocation results in a fusion protein that acts in a dominant negative fashion to interfere with the normal function of the transcription factor RUNX1. The translocation alone may not be sufficient to cause ALL. The frequency of detection of the fusion gene in Guthrie cards (dried blood spots) is x100 than the frequency of paediatric ALL patients.

Commonly loss of the other functional ETV6 (seen in 75% by FISH).

Common secondary abnormalities (e.g CDKN2A/B, PAX5 and IKZF1 deletions do occur in this subgroup)

This translocation has a good prognosis (>90% cure rate). Relapses occur later than in other ALL. This may be due to the persistence of a ‘preleukaemic’ clone harbouring the translocation which undergoes additional genetic events after the elimination of the first leukaemic clone.

Question 28

Describe iAMP21 in paediatric B-ALL

Answer 28

The incidence of iamp21 in pediatric B-ALL is 1.5-2% (median age 9 years).

This is defined as 5 or more copies of RUNX1 (21q22), corresponding to 3 or more extra copies of the gene on one chromosome 21 (not truly iAMP21 if a total of 5 chromosome 21s in the context of a hyperdiploid karyotype). In interphase the signals may cluster.

If there is uncertainty between iAMP21 and high hyperdiploidy further metaphase FISH or subtelomeric FISH may be employed to clarify the number of copies of chromosome 21.

Childhood prognosis is poor (but may be improved by intensive treatment protocols).

Question 29

Describe High Hyperdiploidy (51-65 chromosomes) in paed B-cell ALL

Answer 29

Incidence in childhood B-ALL is 25-30% and is associated with a good prognosis.

Commonly gained chromosome are 4, 6, 10, 14, 17, 18 , 21 (often 2 copies gained) and X. Structural abnormalities commonly seen are dup(1q), del(6q), abn 9p, abn 12p.

The children’s oncology group (COG) currently use the presence of the triple trisomy (simultaneous gain of chromosomes 4, 10 and 17) to identify a subgroup of HeH with low risk of relapse. However, Moorman (2012) found that trisomy 18 and NOT triple trisomy to be the better indicator of improved survival.

It is also important to distinguish between a doubles up near haploid/low hypodiploidy karyotype and a true hyperdiploid. In genuine hyperdiploid the gained chromosome tend to be trisomic (other than 21 and X) whereas in doubled up near haploid/low hypodiploid cases there are usually 4 copies of gained chromosomes. If the presence of two clones cannot be demonstrated by FISH/G-banding then flow cytometry to measure DNA index or STR zygosity testing (most duplicated chromosomes will be homozygous).

Question 30

Describe Near haploidy (23-29 chromosome) /low hypodiploidy (30-39 chromosomes)/ high hypodiploidy (40-44 chromosomes)) in paed B-cell ALL

Answer 30

This is a rare finding which confers a poor prognosis (although prognosis of 44-45 group is better).

In a large MRC study, excluding patients with an established structural abnormality 5% had hypodiploidy, of which 1% has fewer than 45 chromosomes.

Near haploidy appears to be confined to childhood.

Diagnosis of near haploid/low hypodiploid B-ALL may be missed by G-banding as the clone may undergo doubling up (endoreduplication). Chromosomes 1, 11 and 17 are usually disomic in haploid clone and tetrasomic in doubled up clone. Chromosomes 3 and 7 are typically monosomic/disomic.

Question 31

Describe TCF3 rearrangements in B-cell ALL

Answer 31

Childhood incidence is 5-6% of B-ALL.

The t(1;19), which can occur in a balanced or unbalanced from is the most common rearrangement resulting in TCF3-PBX1 gene fusion. The fusion protein has an oncogenic role as a transcriptional activator. The t(1;19) has in intermediate prognosis.

The rare t(17;19) (TCF3-HLF fusion) on the other hand has a poor prognosis.

If a TCF3 rearrangement is detected by FISH and karyotyping is unsuccessful then additional testing must be undertaken to distinguish between the two rearrangements.

Question 32

Describe IGH@ rearrangements in B-cell ALL

Answer 32

IGH@ rearrangements – emerging as significant subgroup in childhood ALL-(8% childhood B-ALL predominantly adolescents. Preliminary data suggests a poor prognosis)

These juxtapose IGH@ transcriptional enhancers to genes on partner chromosomes and result in de-

regulated expression. A number of partner genes have been described:

Question 33

What other chromosomal abnormalities are seen in paed B-cell ALL?

Answer 33

dic(9;12) (PAX5-ETV6)- Childhood incidence <1%. Secondary abnormalities include +8, +21. Good prognosis according to a number of studies but coded as intermediate in childhood B-ALL according to Moorman et al (2010).

dic(9;20) 2% childhood ALL (may be under diagnosed due to subtlety). Variable breakpoints. Intermediate prognosis (according to Moorman et al (2010)), but may be unfavourable according to some reports. Secondary abnormalities include +X, +21.

Abn/del 9p; Visible deletion in ~10% childhood ALL. CDKN2A deletions detected (by SNP array analysis in 21-34% of childhood B-ALL. CDKN2A inactivation found in 20% of B and 50% of T lineage childhood ALL. Deletions may be mono or biallelic. Prognostic significance unclear in childhood ALL (intermediate according to Moorman et al (2010)).

Abn 17p. Poor risk in pediatric ALL according to Moorman et al (2010) (but only in patients without ETV6-RUNX1 or high hyperdiploidy)

13q- Poor risk in pediatric ALL according to Moorman at al (2010).

Rare Chromosomal Translocations (Moorman et al 2012):

t(9;10)(q34;q22.3)/ZMIZ1-ABL1

Previously only reported as a single case, Moorman 2012 collected an additional 5 cases representing an incidence of 0.05%. Interestingly, 5 out of 6 cases were young girls aged between 1 and 3 years old. None of the patients had a high WCC or other high risk clinical feature and all are currently alive and well 8 or more years after diagnosis.

t(7;12)(q36;p13)/ETV6-MNX1

This translocation is associated with infant AML but has occasionally been reported in ALL. Moorman et al 2012 has seen just two ALL cases, both infants, representing an overall incidence of 0.02% but

1% of infants. Both were females and unfortunately both died.

Question 34

Give an overview of childhood T-ALL (15% of childhood ALL).

Answer 34

T-ALL is characterised by recurring rearrangements that commonly juxtapose regulatory elements of the TCR loci with transcription factors or homeobox genes. More common in adolescents than younger children. TCR gene rearrangements are common (35%); TCR alpha/delta (14q11.2), TCR beta (7q35) and TCR gamma at 7p14-15 with a variety of partner genes. Juxtaposition of one of the TCR loci leads to target gene over expression. Currently due to the lack of trial data the prognostic significance of most T-ALL rearrangements is unclear and there are no mandatory FISH tests for T-ALL. Optional testing could include TRA/D, TLX3, TLX1, SIL-TAL1, LMO2 and LMO

Question 35

What is the utility of gene expression profiling in ALL

Answer 35

T-ALL is characterised by recurring rearrangements that commonly juxtapose regulatory elements of the TCR loci with transcription factors or homeobox genes. More common in adolescents than younger children. TCR gene rearrangements are common (35%); TCR alpha/delta (14q11.2), TCR beta (7q35) and TCR gamma at 7p14-15 with a variety of partner genes. Juxtaposition of one of the TCR loci leads to target gene over expression. Currently due to the lack of trial data the prognostic significance of most T-ALL rearrangements is unclear and there are no mandatory FISH tests for T-ALL. Optional testing could include TRA/D, TLX3, TLX1, SIL-TAL1, LMO2 and LMO

Question 36

How can NGS be used in ALL?

Answer 36

NGS has identified novel lesions in ALL improving our understanding of its pathogenesis. It has also helped to discover key biomarkers of diagnostic and prognostic importance.

Interpretation is challenging due to disease heterogeneity

Question 37

Where have novel somatic mutations been identified in B-cell ALL?

Answer 37

B-cell differentiation and development

RAS signalling

JAK/STAT signalling

cell cycle regulation and tumor suppression

Question 38

Where have novel somatic mutations been identified in T-cell ALL?

Answer 38

NOTCH signaling pathway

transcription factors

RAS signalling

JAK/STAT signalling

PI3K/AKT/mTOR signalling

Wnt/β-catenin signaling pathway

chromatin structure modifiers and epigenetic regulators

ribosomal processes

DNA repair complex

Question 39

What is the utility of NGS in ALL?

Answer 39

Some novel genes identified can aid prognosis. For example LEF1 mutations are associated with a favorable outcome and IKZF1 alterations (mutation/deletion) are associated with disease relapse.

NGS use can also be used for residual disease monitoring as an alternative to flow cytometery. The increased sensitivity may allow peripheral blood testing and better identify those patients who will benefit from transplantation. All though standardisation of the workflow need to be advanced it may be that in time NGS panel analysis will become the gold standard for MRD monitoring.