Prognostic significance of microRNA 17–92 cluster expression in Egyptian chronic lymphocytic leukemia patients

2. Marwa Mahmoud Khalifa
Master of Internal Medicine,
Faculty of Medicine, Alexandria University

3. INTRODUCTION

4. Chronic lymphocytic leukemia (CLL)
A malignancy characterized by clonal proliferation
and accumulation of mature, CD5 positive B
lymphocytes in the blood, bone marrow and
secondary lymphoid tissues
lymphocytosis, bone marrow infiltration,
lymphadenopathy and splenomegaly

5. Chronic lymphocytic leukemia (CLL)
 The most common leukemia in the Western world; 30-
40% of all adult leukemias.
 40%-60% of patients are asymptomatic

6. Clinical Features
 Constitutional symptoms (weakness, fatigue, night
sweats, fever)
 Lymphadenopathy bilateral, symmetrical, non-
tender and mobile often cervical and axillary lymph
nodes
 Splenomegaly, usually mild to moderate , less
frequently hepatomegaly.
 Significant anemia or thrombocytopenia.
(autoimmune/ BM involvement)

7. Diagnosis of CLL
Diagnosis requires presence of ≥ 5 x 103/dL clonal B
lymphocytes in the PB by flowcytometry sustained
for at least 3 months.

8. Immunophenotyping
 A scoring system based on five parameters:
• CD5+ (1 point)
• CD23+ (1 point)
• FMC7- (1 point)
• weak intensity of kappa/lambda chains (1 point)
• weak or negative CD22/CD79b (1 point)
 CLL score ranges between 5 (typical CLL cases) and 3 (less
typical CLL cases).
 Scores of 0–2 exclude the diagnosis of CLL.

9. Cytogenetics in CLL
Cytogenetic
aberration
Frequency Effect on
prognosis
Del 13q14 >50% Favorable Within this deleted region is the DLEU2–
miR 15-16 cluster, responsible of regulation
of expression of proteins that can inhibit
apoptosis or that are involved in cell cycle
progression.
Del 11q23 10% early stage
disease
25% advanced
disease stages
Unfavorable • Associated with alterations in ATM
• Bulky lymphadenopathy, rapid
progression, and reduced OS
Trisomy 12 10-20% Intermediate
Del 17p13 5-8% Unfavorable • Loss of TP53, tumor suppressor gene
• Resistant to standard chemotherapy

10. Somatic mutations in CLL

11. Cellular origins of CLL and
Mutational status of IGHV

12. IGHV
mutational
status
Mutated
∼60% newly
diagnosed and
asymptomatic
Unmutated
∼50%-60%
progressive
∼70%-80%
relapsed/refractory.
Unmutated IGHV
(usually defined as
≥ 98% sequence
homology to the
nearest germ line
gene)
Mutated IGHV gene
• longer TTFT
• longer PFS
• lower risk of
Richter’s
transformation
• better OS

13. Complications
of CLL
Infections
Autoimmune
Secondary
cancers
Richter’s
syndrome
Prolymphocytic
transformation
Acute
leukemia,
MDS

14. Prognostic markers of CLL cells
1- Host factors (i.e., gender and age)
2- Disease markers [i.e., lymph node (size, site)
hepatomegaly, splenomegaly, LDT, WBC, ALC, anemia,
thrombocytopenia, Rai and Binet staging]
3- Antigen expression (i.e., CD38, ZAP70, and
CD49d/VLA-4)
4- Serology (i.e., LDH, β2M, thymidine kinase and IL-8)

15. Prognostic markers of CLL cells
5- Cytogenetics (i.e., del 17p and TP53 gene mutation,
del 11q, del 13q, trisomy 12, complex karyotype)
6- Epigenetics (DNA methylation)
7- Somatic mutations(NOTCH1 mutation, ATM, SF3B1
mutation, BIRC3 mutation, BRAF mutation)
8- Immunogenetics (i.e., IGHV gene mutational status
and BCR structure)

16. Clinical staging
Low-risk (Stage 0) Lymphocytosis in the blood and/or
marrow
Intermediate-risk
(Stage I and II)
Lymphadenopathy, splenomegaly
+/- hepatomegaly
High-risk (Stage III
and IV)
Anemia, thrombocytopenia
Rai staging system

17. Stage A Up to 2 lymphoid areas involved, no anemia
or thrombocytopenia
Stage B ≥3 lymphoid areas involved, no anemia or
thrombocytopenia
Stage C Hb <10 g/dL and/or platelet count <100x109/L.
Binet staging system
Areas of involvement involved in staging include: head and neck, axillae, groins,
splenomegaly and hepatomegaly

18. International prognostic index for
CLL (CLL-IPI)
Del 17p on FISH or TP53 mutation (4 points)
Unmutated IGHV genes (2 points)
Serum β2 microglobulin >3.5 mg/L (2 points)
 Binet B-C /Rai stage I–IV (1 point)
Age >65 years (1 point)

19. International prognostic index for
CLL (CLL-IPI)
CLL-IPI category
Number
of points
OS at 5
years
Potential clinical consequence
Low risk 0-1 93.2% Do not treat
Intermediate risk 2-3 79.3%
Do not treat except if the disease is really
symptomatic
High risk 4-6 63.3%
Treatment indicated except if the disease is
asymptomatic
Very high risk 7-10 23.3%
If you need to treat do not use chemotherapy
but rather novel agents or treatment in
clinical trials.

20. Indications For Treatment CLL
1. Progressive marrow failure (development / worsening
of anemia and/or thrombocytopenia).
2. Massive or progressive or symptomatic splenomegaly.
3. Massive (≥10 cm in longest diameter) or progressive or
symptomatic lymphadenopathy.
4. Progressive lymphocytosis (an increase of ≥50% over
2-month period), or LDT < 6 months.

21. Indications For Treatment CLL
5. Autoimmune complications (AIHA/ITP) refractory to
corticosteroids.
6. Symptomatic or functional extranodal involvement (e.g.,
skin, kidney, lung, spine).
7. Disease-related B symptoms:
a. Unintentional weight loss ≥10% within the last 6 months.
b. Significant fatigue
c. Fevers ≥ 38.0°C for ≥2 weeks without evidence of infection.
d. Night sweats for ≥1 month without evidence of infection.

22. Drug Classes
Chemo-
immuno-
therapy
• FCR
• BR
• ClbO
BTKi
• Ibrutinib
• Acalabrutinib
• Zanubrutinib
PI3Ki
• Idelalisib
• Duvelisib
BCL-2
inhibitor
• Venetoclax
Monoclonal
Ab
• Anti CD20
Rituximab
Ofatumumab
Obinutuzumab
• Anti CD52
Alemtuzumab

23. Treatment of CLL
The following parameters should be taken in
consideration :
1. The clinical stage of the disease.
2. The patient's symptoms.
3. The fitness and comorbidities, particularly potential
organ toxicity of the newer targeted agents.
4. The genetic risk.
5. The treatment situation.

24. Treatment algorithm as first line according to ESMO
recommendations.
Stage Del 17p/
TP53
mutation
IGHV Fitness Therapy
Symptomatic
early stage
or
advanced stage
No Mutated Fit CIT: FCR (BR if age ≥65 y)
Ibrutinib
Unfit Venetoclax + Obinutuzumab
CIT: CLBO
Ibrutinib or Acalabrutinib
No Unmutated Fit Ibrutinib
CIT: FCR (BR if age ≥65 y)
Unfit Venetoclax + Obinutuzumab
Ibrutinib or Acalabrutinib
CIT: CLBO
Yes Irrelevant Any Ibrutinib or Acalabrutinib
Venetoclax + Obinutuzumab
Venetoclax
Idelalisib + Rituximab

25. Treatment algorithm in relapse setting according
to ESMO recommendations.
TP53 mutation/ del 17p Ibrutinib or acalabrutinib
Venetoclax + Rituximab
Venetoclax alone
Idelalisib + Rituximab
Discuss Allogeneic SCT
Short remission duration
(<36 months)
Ibrutinib or acalabrutinib
Venetoclax + Rituximab
Venetoclax alone
Idelalisib + Rituximab
Long remission duration
(>36 months)
Repetition of 1st line is possible
Ibrutinib or acalabrutinib
Venetoclax + Rituximab
Idelalisib + Rituximab
CIT

26. Allogeneic Transplant in CLL
 Treatment of choice and the only curative approach for
high risk CLL patients.
 Have a role in patients who fail, intolerant or do not
have access to the novel agents

27. Chimeric antigen receptor bearing
T cell (CAR T) in CLL
 Relapsed/refractory CLL who have run out possible
effective therapeutic options.
 Tisagenlecleucel is an anti-CD19 CAR that has a
costimulatory domain.
 Ibrutinib pretreatment before T cell collection for
CART cell therapy can reverse the dysfunction of T
cells and induce the variation of CART cell phenotypes
with low CRS severity and high response rates.

28. Evaluating response to treatment
Complete
remission
Partial remission
Progressive
disease
Stable disease
Lymph nodes
None /
≤1.5 cm
Decrease ≥50% from
baseline
Increase ≥50% from
baseline / response
Change of -49 to
+49%
Liver and/or
spleen size
Spleen size ≤13 cm;
liver size normal
Decrease ≥50% from
baseline
Increase ≥50% from
baseline /Response
Change of -49 to
+49%
Constitutional
Symptoms
None Any Any Any
Circulating
Lymphocyte count
Normal
Decrease ≥50% from
baseline
Increase ≥50% over
baseline
Change of -49 to
+49%
Platelet count ≥100 x 109/L
≥100 x 109/L or increase
≥50% over baseline
Decrease of ≥50%
from baseline
Change of -49 to
+49%
Hemoglobin ≥11 g/dL
≥11 g/dL or increase ≥50%
over baseline
Decrease of ≥2 g/dL
from baseline
Increase <11 g/dL or
<50% over baseline, or
decrease <2 g/dL
Marrow
Normocellular, no
CLL cells, no B
lymphoid nodules
Presence of CLL cells, or
of B-lymphoid nodules
Increase of CLL cells
by ≥50% on successive
biopsies
No change in
marrow infiltrate

29. Eradicating MRD
 MRD assessment
1- Six-color flow cytometry
2- allele-specific oligonucleotide PCR
3- high throughput sequencing
 Reliably sensitive down to a level of <1 CLL cell in 10.000
leukocytes.
 Useful to decide when to stop treatment
 Increase/reappearance of MRD could guide the
rechallenge of therapy after treatment discontinuation.

30. MicroRNAs
Biology of MicroRNAs
 Short noncoding RNAs, with a length of 17–25
nucleotides
 Regulate gene expression by promoting degradation or
repressing translation of target mRNAs.
 Implicated in the pathogenesis of hematological
malignancies (NHL, CLL, MM, CML, AML, ALL) and
solid tumors.

31. Biosynthesis of miRNA

32. Role of microRNAs in CLL
CLL was one of the first human diseases found to be
associated with alterations in miRNA.
Several microRNAs affect the key pathways implicated
in the pathogenesis and prognosis of CLL
Examples: miR15a/16-1 , miR34a, miR155, miR29,
miR181b, miR221, miR150

33. Micro RNA 17-92
 Polycistronic miRNA cluster
 Located in non-protein-coding gene at 13q31.
 The precursor transcript yield the six mature miRNAs:
miR17, miR18a, miR19a, miR20a, miR19b-1, and
miR92a-1
 Strong oncogene
 Regulates multiple cellular processes that favor
malignant transformation, promoting cell survival,
rapid cell proliferation, and increased angiogenesis.

34. Targets of microRNA 17-92 cluster

35. AIM OF THE WORK

36.  To study the expression of miRNA17-92 cluster as a
prognostic factor in Egyptian CLL patients.
 To assess the relation between miRNA17-92 cluster
with the known prognostic markers in CLL including
17p deletion / P53 mutation, CD38 and ZAP70
expression, serum LDH, serum β2 microglobulin and
the stage of the disease at diagnosis.
To assess the relation between miRNA17-92 cluster
with time to start treatment in patients with early
disease stages and with response to 3-4 cycles of either
FCR or ibrutinib therapy.

37. PATIENTS

38. 40 treatment naïve CLL Egyptian patients either newly
diagnosed or in the “watch and wait” phase (22 males
& 18 females) and 20 healthy age and sex matched
controls.
Attending the hematology clinics at Alexandria Main
University Hospital and Damanhour Oncology Center
From May 2019- December 2020.
A written informed consent.
Approval of research ethics committee of Alexandria
Faculty of Medicine.

39. Inclusion criteria:
• CLL patients diagnosed according to the modified
International Workshop on CLL (iwCLL) 2018 criteria
• ECOG performance status: ≤ 2
Exclusion criteria:
• Prior treatment.
• Heart failure or coronary artery disease.
• Significant comorbidities e.g.: renal, hepatic,
respiratory or collagen disorders.
• Concomitant or previous hematologic or non-
hematologic malignancies.

40. METHODS

41. All patients in this study were
subjected to:
Clinical
examination
History
taking
Investigations

42. Investigations:
 CBC , reticulocyte count , routine
 Serum LDH level , Serum β2 microglobulin
 Flowcytometry for CD38 and ZAP 70 expression.
 Fluorescence in situ hybridization (FISH) for 17p
deletion.
 Micro RNA 17-92 cluster expression by qRT-PCR
(miR17, miR18a, miR19a, miR19b-1,
miR20a and miR92a-1)

43. RESULTS

44. The frequency of clinical
findings in patients group
0
10
20
30
40
50
60
70
percentage %

45. Classification of patients according
to modified Rai staging system
Low-risk (Stage
0)
40%
Intermediate-
risk (Stage I
and II)
37%
High-risk
(Stage III and
IV)
23%

46. Classification of patients according
to Binet staging system
Stage A
45%
Stage B
32%
Stage C
23%
CLL patients (n=40)

47. Treatment eligibility & response to
treatment
Treatment
eligibility
(n=40)
wait and see
strategy (n=23)
Follow up
2 patients
progressed
Indicated for
treatment
(n=17)
Standard
chemotherapy
(n=13)
Ibrutinib
(n=4)

48. Response
to
treatment
Standard
chemotherapy
(n=13)
Objective
response (n=5)
No response
(n=6)
Died / lost
follow up (n=2)
Ibrutinib (n=4)
Objective
response (n=2)
No response
(n=1)
Died / lost
follow up (n=1)
Objective response =
CR + PR
No response = stable
disease+ progressive
disease

49. Comparison between the patient &
control groups
10
10.5
11
11.5
12
12.5
13
13.5
Patients Control
Hb (g/dl)
0
50
100
150
200
250
300
Patients Control
Platelets (x 109/L)
(p<0.001)
(p= 0.001)

50. Comparison between the patient & control
groups
0
10
20
30
40
50
60
70
80
90
100
Patients Control
WBCs (x 109/L)
0
10
20
30
40
50
60
70
80
90
Patients Control
ALC (x 109/L)
(p<0.001)
(p<0.001)

51. Laboratory tests of some
prognostic factors in CLL patients
77%
23%
β2 microglobulin (mg/L )
Elevated Normal
42%
58%
LDH (U/L)
Elevated Normal
Min. – Max. Mean ± SD Median
LDH (U/L) 166 – 1481 508.12 ± 276.41 438.5
β2M (mg/L) 1.7 - 10.5 4.8±2.5 4.05

52. Laboratory tests of some
prognostic factors in CLL patients
32%
68%
CD38
Positive Negative
37%
63%
ZAP70
Positive Negative

53. Laboratory tests of some
prognostic factors in CLL patients
7%
30%
25%
38%
Combined CD38/ZAP70
expression
(CD38+/ZAP+) (CD38-/ZAP+)
(CD38+/ ZAP-) (CD38-/ZAP-)
10%
90%
17p Del
Positive
Negative

54. Micro RNA 17-92 cluster expression
 Overexpression of all members of miRNA17-92 cluster
was detected in CLL patients compared to controls

55. MiR17
ROC curve for
miR17 to
discriminate
patients (n = 40)
from control cases
(n = 20)

56. MiR18a
ROC curve for
miR18a to
discriminate
patients (n = 40)
from control cases
(n = 20)

57. MiR19a
ROC curve for
miR19a to
discriminate
patients (n = 40)
from control
cases (n = 20)

58. MiR19b-1
ROC curve for
miR19b-1 to
discriminate
patients (n = 40)
from control cases
(n = 20)

59. MiR20a
ROC curve for
miR20a to
discriminate
patients (n = 40)
from control cases
(n = 20)

60. MiR92a-1
ROC curve for
miR92a to
discriminate
patients (n = 40)
from control cases
(n = 20)

61. Positive correlation between miR17 expression and hemoglobin
concentration in CLL patients. (r = 0.324, p = 0.041)

62. Negative correlation between miR19b-1 expression and hemoglobin
concentration in CLL patients. (r = -0.376, p = 0.017)

63. Positive correlation between miR19b-1 expression and WBCs in CLL
patients. (r = 0.359, p = 0.023)

64. Positive correlation between miR19b-1 expression and ALC in CLL
patients. (r = 0.362, p = 0.022)

65. Positive correlation between miR18a expression and serum LDH in
CLL patients (r = 0.458, p = 0.003)

66. Positive correlation between miR92a-1 and serum β2M in CLL
patients. (r = 0.438, p = 0.005)

67. Relation between miRs and CD38
0
2
4
6
8
10
12
14
16
18
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a
Poitive CD38
Negative CD38
P=
0.034
P=
0.749
P=
0.333
P=
0.851
P=
0.073
P=
0.762

68. Relation between miRs and ZAP70
P=
0.061
P=
0.091
P=
0.41
P=
0.655
P=
0.459
P=
0.162
0
2
4
6
8
10
12
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a
Poitive ZAP70
Negative ZAP70

69. Relation between miRs and 17p del
P=
0.26
P=
0.652
P=
0.718
P=
0.652
P=
0.875
P=
0.787
0
5
10
15
20
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a
Poitive del 17p
Negative del 17p

70. Relation between miRs and
modified RAI stages
P=
0.626
P=
0.439
P=
0.547
P=
0.061
P=
0.667
P=
0.113
0
5
10
15
20
25
30
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a-1
Low risk
Intermediate risk
High risk

71. Relation between miRs and Binet
stages
P=
0.413
P=
0.486
P=
0.603
P=
0.05
P=
0.977
P=
0.141
0
5
10
15
20
25
30
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a-1
Stage A
Stage B
Stage C

72. Relation between miRs and
treatment plan
P=
0.619
P=
0.4
P=
0.934
P=
0.05
P=
0.314
P=
0.089
0
2
4
6
8
10
12
14
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a-1
Eligible for
treatment
Wait and see

73. Relation between miRs and
response to treatment
P=
0.535
P=
0.073
P=
0.620
P=
0.209
P=
0.259
P=
0.165
0
5
10
15
20
25
30
MiR17 MiR18a MiR19a MiR19b-1 MiR20a MiR92a-1
Objective
response
No response

74. Survival Results
• Overall survival
of CLL patients
90.2% after 1
year from the
time of
diagnosis
• reached 67.6%
by the end of
May 2021

75.  High expression
of miR19a above
the median is
associated with
better OS
(p=0.04).

76. No significant difference between low and high levels of
expression of the other miRs regarding OS

77. CONCLUSIONS

78. 1- All members of the miR17-92 cluster are overexpressed
in Egyptian CLL patients.
2- Significant positive correlation between Hb and miR17
and statistically significant negative correlation between
Hb and miR19b-1.
3- Significant positive correlations between miR19b-1
expression and both WBCs and ALC.
4- Significant relation between miR19b-1 expression and
advanced stage of Binet system.

79. 5- Significant positive correlation between miR18a and
LDH.
6- Significant positive correlations between miR92a-1
and β2 microglobulin.
7- Significant relation between miR17 and negative
CD38.
8- Patients who express miR19b-1 were more likely
indicated for treatment and this was statistically
significant.

80. 9- MiR18a, miR19b-1, and miR92a-1 have an adverse
prognostic value while miR17 can be considered as a
good prognostic marker in Egyptian CLL patients.
10- High expression of miR19a is associated with better
overall survival.

81. RECOMMENDATIONS

82.  More studies using larger prospective patient
cohorts together with cytogenetic studies are still
needed
 to exactly define the prognostic relevance of these
miRNAs in patients with CLL.
 to precisely detect the role of micro RNA 17-92 cluster
in CLL progression, response to treatment, prediction
of time to treatment and clinical outcome of CLL.
 to clarify if alterations in microRNA expression are the
initiating events in CLL pathogenesis, or they occur
later during the progression of the disease.

83.  Clinical trials on the use of micro RNA antagonists as a
novel therapeutic approach to the treatment of CLL
are warranted.