This document discusses various techniques used to analyze hematologic malignancies including morphology, immunohistochemistry, flow cytometry, cytogenetics, and molecular studies. It provides details on karyotyping and fluorescent in situ hybridization (FISH), including examples of normal results and common abnormalities. The document also reviews molecular marker testing for various leukemias and lymphomas, highlighting frequently mutated genes and how to report testing results on disease forms. Case studies are presented to demonstrate how to interpret and report cytogenetic and molecular testing results.

1. TRAINING & DEVELOPMENT | 1 .
Reporting Karyotype, FISH & Molecular Results
Janet Brunner-Grady, PA-C
February 2019

2. TRAINING & DEVELOPMENT | 2 .
Hematologic Malignancy Evaluation
• Histology/ Morphology (Least sensitive)
– What the cells look like
• Immunohistochemistry (IHC)
– Staining the cells to identify specific markers
• Flow cytometry
– Looks at individual cells based on staining for specific markers
• Cytogenetics
– Karyotype
- FISH analysis
• Molecular studies (Most sensitive)
– Identifying abnormal genes and/or gene products

3. TRAINING & DEVELOPMENT | 3 .
Types of Cytogenetic Analysis
• Karyotype vs. FISH
Karyotype- is the characterization of structural and numerical changes in
chromosomes while cells are dividing. They are displayed as a systematized
arrangement of chromosome pairs in descending order of size.
FISH (Fluorescent in situ hybridization)-
• A molecular cytogenetic technique using fluorescent probes that bind to a
specific part of a chromosome
• Used to detect the presence or absence of specific DNA sequences on
chromosomes

4. TRAINING & DEVELOPMENT | 4 .
Normal Karyotype

5. TRAINING & DEVELOPMENT | 5 .
Chromosomal Banding
Giemsa staining (or G-banding)
of chromosomes reveals
characteristic patterns of
horizontal bands like bar codes.

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Chromosome Definitions

7. TRAINING & DEVELOPMENT | 7 .
Chromosome Structure

8. TRAINING & DEVELOPMENT | 8 .
Definitions of a Clone
• Two or more cells with gain of a specific chromosome (e.g.,
trisomy 8 or +8)
• Two or more cells with the same structural chromosomal
abnormality (e.g., t(9;22))
• Three or more cells with loss of a specific chromosome (e.g.,
monosomy 7 or -7)

9. TRAINING & DEVELOPMENT | 9 .
Types of FISH probes
• Centromere enumeration probes (CEP)
– Monitors number of a specific chromosome in a cell (e.g., trisomy 8
or +8)
• Locus specific probes
– Used to rule out deletions, gains or rearrangements of specific loci
(e.g., del(7q) or del(7)(q22q31))

10. TRAINING & DEVELOPMENT | 10 .
Types of FISH probes (continued)
• Dual fusion probes
– Used to confirm presence of a translocation (e.g., t(9;22))
– Fusion signal on each partner chromosome
– Highly specific (very low false positives)
• Break-apart probes
– Used to confirm rearrangements of genes
– 3’ portion of gene or region in one color, 5’ in another
– If rearranged, colors are separated

11. TRAINING & DEVELOPMENT | 11 .
Examples of karyotype & FISH abnormalities
• Trisomy 8 (or +8) using CEP
• Translocation of chromosome 9 & 22 (t(9;22)) using dual-
fusion probe

12. TRAINING & DEVELOPMENT | 12 .
Karyotype with trisomy 8
• 47,XY,+8

13. TRAINING & DEVELOPMENT | 13 .
FISH for trisomy 8

14. TRAINING & DEVELOPMENT | 14 .
Karyotype findings in CML

15. TRAINING & DEVELOPMENT | 15 .
Dual-fusion probes

16. TRAINING & DEVELOPMENT | 16 .
t(9;22) by FISH

17. TRAINING & DEVELOPMENT | 17 .
Case Study #1
• The following karyotype is from a 55 y/o WM with newly
diagnosed AML.
• Cytogenetic report-
20 metaphases analyzed
43XY, add(4)(q21), add(5)(q11.2), add 7(q11.2),
-11, add(12)(p11.2), add(15)(p11.2), -16, -18, add(19)(q13.3),
add(22)(p11.2)[4]
46,XY[16]

18. TRAINING & DEVELOPMENT | 18 .
Case Study #1
• What to report on the Pre-TED Disease form (F2402)….
• Cytogenetic report
43XY, add(4)(q21), add(5)(q11.2), add(7)(q11.2),
-11, add(12)(p11.2), add(15)(p11.2), -16, -18, add(19)(q13.3),
add(22)(p11.2)

19. TRAINING & DEVELOPMENT | 19 .
Case Study #1
• F2402 r3 AML disease section Q3-89
Cytogenetics tested via karyotyping:
• There are three monosomy chromosomes listed in the report: -11, -16,
-18
• Q18 option ‘-18’ would be checked, while the -11 & -16 would be
reported in Q19 ‘Specify other abnormality’

20. TRAINING & DEVELOPMENT | 20 .
Case Study #1
• How to report the rest of the abnormalities-
add(4q), add(5q), add(7q), add(12p), add(15p), add(19q) & add(22p)
Q18 Specify abnormalities
• All the abnormalities listed above would be reported in Q19 or
simply write “See attached cytogenetic report”.

21. TRAINING & DEVELOPMENT | 21 .
CIBMTR Request 
• Please submit a copy of the chromosome reports (karyotype
and /or FISH)
• It will decrease the number of queries sent related to
chromosome abnormalities!

22. TRAINING & DEVELOPMENT | 22 .
Case Study #2
• The following karyotype is from a 10 y/o AAF with relapsed
ALL.
• Cytogenetic report-
20 metaphases analyzed
28, X, +X, +8, +10, +18, +21[7]; 46, XX[13]
Interpretation: near haploid clone detected
• The report could have been written this way- 28,XX,-1,-2,-3,
-4,-5,-6,-7,-9,-11,-12,-13,-14,-15,-16,-17,-19,-20,-22[7];
46,XX[13]

23. TRAINING & DEVELOPMENT | 23 .
Case Study #2
• How to report the following karyotype on the Pre-TED
Disease form (F2402 r3)…..
28, X, +X, +8, +10, +18, +21[7]; 46, XX[13]
• How would you answer Q104?
A) Check options +8 & +21
B) None of the abnormalities should be reported here

24. TRAINING & DEVELOPMENT | 24 .
Case Study #2
• Keep in mind this karyotype was a “near haploid clone”.
What does that even mean?
– It means most of the chromosomes only had one copy instead of
the normal two.
– In this case, indicating +8, +10, +18 & +21 means there are two
copies and not three copies as in the case of a trisomy finding.
28, X, +X, +8, +10, +18, +21[7]; 46, XX[13]

25. TRAINING & DEVELOPMENT | 25 .
Case Study #2
• How to report the rest of the abnormalities…
-1,-2,-3,-4,-5,-6,-7,-9,-11,-12,-13,-14,-15,-16,-17,-19,-20,-22

26. TRAINING & DEVELOPMENT | 26 .
Case Study #2
• How to report the rest of the abnormalities…
-1,-2,-3,-4,-5,-6,-9,-11,-12,-13,-14,-15,-16,-17,-19,-20,-22
Q104
• ‘Hypodiploid’ and ‘Other abnormality’ would be checked
• Report all the remaining abnormalities in Q105 or write “See
attached cytogenetic report”.

27. TRAINING & DEVELOPMENT | 27 .
CIBMTR Request 
• Please submit a copy of the chromosome reports (karyotype
and /or FISH)
• Again, it will decrease the number of queries sent related to
chromosome abnormalities!

28. TRAINING & DEVELOPMENT | 28 .
Case Study #3
• Interpreting FISH results can be a challenge….
– The CEP 8 (8 centromere) and D20S108 (20q12) dual-color probe set
were used for enumeration analysis. Of 200 cells examined, 15 (7.5%)
demonstrated three signals for the 8 centromere and two signals for
20q12.
– The RUNX1T1 (8q21.3) / RUNX1 (21q22.1) dual-color, dual fusion probe
set was used for detection of RUNX1T1/RUNX1 fusion associated with
t(8;21). Of 200 cells examined, 14 (7%) demonstrated three signals for
RUNX1T1 and two signals for RUNX1. There was no evidence of
fusion.

29. TRAINING & DEVELOPMENT | 29 .
Case Study #3
• What do the FISH results tell us?
• 15 of 200 cells (7.5%) demonstrated three signals for the 8 centromere
and two signals for 20q12.
 The three signals indicate the presence of 3 copies of chromosome 8 (i.e.,
trisomy 8 or +8) and the two signals for 20q12 indicate a normal copy number of
chromosome 20.
• 14 of 200 cells (7%) demonstrated three signals for RUNX1T1
(associated with chromosome 8q21.3) and 2 signals for RUNX1
(21q22.1).
 The three signals indicate the presence of 3 copies of chromosome 8 (i.e.,
trisomy 8 or +8) and the 2 signals for RUNX1 (21q22.1) indicate a normal copy
number of chromosome 21. There’s no evidence of t(8;21).

30. TRAINING & DEVELOPMENT | 30 .
Molecular Marker Testing
• Purpose
Diagnostic accuracy
Prognostic markers to guide therapy options and estimate outcomes
Monitor for minimal residual disease

31. TRAINING & DEVELOPMENT | 31 .
Types of Molecular Testing
• Polymerase Chain Reaction (PCR)
– Amplifies DNA fragments
• Gene Expression Profiling (microarray technology)
– Identifies a molecular signature of a tumor
• Proteomics (microarray technology)
– Identifies protein expression profiles of tissue/cell type
• Single nucleotide polymorphism (SNP) &
Comparative genomic hybridization (CGH)
• Next Generation Sequencing (NGS)

32. TRAINING & DEVELOPMENT | 32 .
Mutations in AML
• CEBPA
• FLT3- D835 point mutation
• FLT3- ITD mutation
• IDH1
• IDH2
• KIT
• NPM1

33. TRAINING & DEVELOPMENT | 33 .
AML: Example
• AML/MDS Gene Mutation Panel

34. TRAINING & DEVELOPMENT | 34 .
AML: F2402 r3 Q26-27
• FLT3- ITD allelic ratio
– Refers to the # of ITD-mutant alleles compared to the number of
wild-type alleles (normal alleles)
– In the example, the mutant allele burden is 70%, therefore the wild-
type alleles (normal) would be 30%. The ratio = 2.3 (70:30)

35. TRAINING & DEVELOPMENT | 35 .
AML: F2402 r3 Q3-89
2.3

36. TRAINING & DEVELOPMENT | 36 .
FLT3-ITD allelic ratio
• 2017 European Leukemia Net (ELN) guidelines defined 0.5
as the cutoff between low & high allelic ratios
– Low allelic ratio < 0.5 (FLT3-ITDlow)
– High allelic ratio > 0.5 (FLT3-ITDhigh)
• A low FLT3-ITD allelic ratio is a favorable prognostic factor

37. TRAINING & DEVELOPMENT | 37 .
Mutations in MDS/MPN
• ASXL1
• JAK2 (MPN only)
• ETV6
• EZH2
• P53
• RUNX1

38. TRAINING & DEVELOPMENT | 38 .
Case Report-
Patient with Primary Myelofibrosis
Panel reports data on 21 genes
for myeloid malignancies

39. Case Report Continued

40. TRAINING & DEVELOPMENT | 40 .
MDS/MPN: F2014 r3 Q29-38
CALR

41. TRAINING & DEVELOPMENT | 41 .
Mutations in ALL
• BCR / ABL
• TEL-AML / AML1
ALL: F2402 r3 Q107-111

42. TRAINING & DEVELOPMENT | 42 .
CML: F2012 r3 Q56-82

43. TRAINING & DEVELOPMENT | 43 .
BCR/ABL
• Fusion protein that
results in increased
activity of a tyrosine
kinase
• Present in CML,
ALL (30 -35% adult
B-cell), and some
AML
• Can be followed quantitatively with a Major Molecular
Response (MMR) determined as ≤ 0.1% BCR-ABL (ratio of
BCR-ABL/BCR

44. TRAINING & DEVELOPMENT | 44 .
Mutations in CLL
• Immunoglobulin heavy chain variable (IGHV) mutation
• NOTCH 1
• TP53 (or P53)
• SF3B1

45. TRAINING & DEVELOPMENT | 45 .
CLL: F2013 r3 Q41-50

46. TRAINING & DEVELOPMENT | 46 .
Mutations in Lymphomas
• BCL-1/Cyclin D1; t(11;14)
• BCL2; t(14;18)
• BCL-6
• B-cell, Immunoglobulin Heavy (IgH) chain rearrangement
• T-cell receptor (TCR) gene rearrangement

47. TRAINING & DEVELOPMENT | 47 .
Questions