MRD in rarer chronic lymphoid leukemias

Tadeusz Robak

Department of of HematologyMedical University of Lodz, Copernicus Memorial

Hospital, Lodz, Poland

Hematology 2020 – To be or not to be MRD negative – this is the question…

Tel Aviv, Israel; February 27th 2020

Professor Aaron Polliack

Rarer chronic lymphoid leukemias

1. Hairy cell leukemia (HCL)

2. T-cell prolymphocytic leukemia (T-PLL)

3. Large granular lymphocyte leukemia (LGLL)

Diagnostic work-up of classical HCL

•

Hairy cells characteristic: medium in

size with moderately abundant pale

blue cytoplasm, a round or kidney-

shaped nucleus, open chromatin,

absent nucleoli, and a characteristic

serrated cytoplasmic border

Full blood cell counts, peripheral

blood film morphology [I, C]

Bone marrow aspiration (often “dry

tap”), and trephine biopsy – H& E

stain, reticulin stain, and

immunohistochemistry for CD20,

Annexin-1, DBA-44 [I, C]

Immunophenotypic analysis by flow

cytometry: CD19+, CD20+, CD11c+,

CD25+, CD103+, CD123+

BRAF V600E mutation

Tartrate-resistant acid phosphatase

(TRAP).

Grever et al. Blood. 2017;129:553-560; Robak et al. Ann Oncol 2015;26 (Supplement 5): v100v107;

Cladribine as initial treatment of clasic HCL

• CR rate of 85–91% after a single course of therapy

• Median time to relapse -16 years (disease-free and relapse-free survival curves have not reached a plateau, most patients who live long enough will eventually relapse)

• A high percentage of patients in CR have minimal residual disease (MRD;) even if assayed after 16 years

• Second course (with or without rituximab) should be considered ifPR after the first course at least 6 months after the end of the first course to achieve a CR

Gidron etTallman MS. . Leuk Lymphoma 2006;47:2301–7; Else et al.. J Haematol 2009;145:733–40S Sigal et al. Blood 2010;115:1893–6.. Robak et al. Ann Oncol 2015;26 (Supplement 5): v100v107, 2015

Cladribine vs. pentostatin: relapse-free survival

0 2 4 6 8 10 12 14 16 18 20

Years from start of treatment

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Pentostatin (n=188)

Cladribine (n=54)

Else et al, Br J Haematol. 2009;145:733.

0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Years from start of treatment

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Complete response

Partial response

p<0.0001

Relapse-free survival in HCL after pentostatin or cladribine

Dearden et al, Leuk Lymphoma, 2011;52(S2): 21.

Therapies active in R/R HCL• Rituximab alone, obinutuzumab

• Cladribine + rituximab

• Fludarabine 40 mg/m2/day (p.o.) x 5d + rituximab 375 mg/m2 day 1 every 28 days (4 cycles)

• Bendamustine 70-90 mg/m2 day 1 -2 + rituximab375 mg/m2 day 1

• Moxetumomab Pasudotox

• BRAF inhibitors: Vemurafenib, Dabrafenib

• BTK inhibitors: Ibrutinib

Robak T et al. Expert Opin Investig Drugs. 2015;24(11):1419-31;. Burotto M et al. Clin Cancer Res 2013; 19:6313. Robak et al. Annals of Oncology 2015;26 (Supplement 5): v100-v107

Methods for MRD in HCL• Characteristic immunophenotype useful for detection of MRD

(CD19, CD20, CD22, CD25, CD79a, CD11c, CD103, surface immunoglobulin ):

- Flow cytometry - sensitivity usually 1 cell in 10 000 (1x10 -4 )

- Immunohistochemistry

• Molecular - Unique immunoglobulin heavy chain (IgH) gene rearrangements

- amplification using consensus V primers (usually for the framework 1-3

regions of IgH, [cpPCR] (sensitivity ranging 1x10 -4 – 1x 10-5 ;

- more sensitive PCR techniques with clone specificity (sensitivity 1 x 10-6 )

• AS-PCR for BRAF-V600E mutation in PB and BM aspirate.

CD103 Fitc

CD

11

c A

PC

100

101

102

103

104

100

101

102

103

104

CD103 FITC

CD

11

c V

45

0

10 2 10 3 10 4 10 5

102

103

104

105

CD103 Fitc

CD

11

c A

PC

100

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CD103 Fitc

CD

11

c A

PC

100

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103

104

No CD103+, CD11c bright Hairy cells

Pre-Cycle 1

Pre-Cycle 3

CD

11

c A

PC

CD

11

c A

PC

Definitions of CR and minimal residual disease (MRD)

BMBx H/E BMBx IHC Blood Flow BMA Flow(3 cells in 104) (most sensitive)

Courtesy of Dr R Kreitman , Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019 Rome, Italy

VH DH JH

R Q

Clone-specific PCR for HCL - real-time quantitative PCR - Sensitivity: 10-6 cells

(Sequence-specific primer)

sequence-specific (clonogenic) primer

((clonogenic)

Reverse consensus primer

Forward consensus primers

VH DH JH

Deep sequencing

Design Primers & Probe

Deep sequencing after treatment

. HCL cells express unique Immunoglobulin rearrangements. To improve the sensitivity of detection of MRD, consensus primers are used to clone them, using CD11c sorting if needed to enrich for the malignant band. After cloning and sequencing each patient a consensus 5’ primer, a clonogenic 3’ primer, and a clonogenic probe containing a reporter and a quencher are- designed . This method, called real-time quantitative PCR, is able to detect 1 HCL cell in 106 normal

Courtesy of Dr R Kreitman, Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019 Rome, Italy

Comparisson of flow cytometry with immunohistochemistry for MRD in HCL

• Comparison of mmunohistochemistry (IHC) (CD20, DBA.44) and flow cytometry (FC)

(CD20, CD22, CD25, SIg, CD11c, CD103) to detect MRD after therapy with 2-CdA.

• The definition for positive MRD

- IHC: 1–10% CD20/DBA44 scattered or clustered cells with tricoleukocyte morphology

- FC: any expression of CD11c/ CD25/CD103 in the BM or PB.

• The MRD positivity rate for IHC - 46% compared with 64% for FC, suggesting that

FC is more sensitive technique than IHC.

Bengio et al. Haematologica 2000;85:1227–1229; Sausville et al. Am J Clin Pathol 2003;119:213

Comparisson of FC with PCR for the detection of MRD

• Comparison of FC immunophenotyping with clonal analysis using

- consensus primer PCR (cpPCR) for the heavy chain gene in previously treated HCL

- PB multi-parameter FC (CD19, CD22, CD103, FMC7, CD23, CD19, CD20,

CD11c, CD25)

• FC more sensitive: 31% of the cases positive by FC

were negative by cpPCR and only 1% of cases positive by cpPCR were

negative by FC.

Sausville et al. Am J Clin Pathol 2003;119:213–217.

MRD assessed by clone specific polymerase chain reaction -Splinkerette PCR (spPCR) seems to be most sensitive

• MRD positivity after therapy with the recombinant immunotoxin BL22 assessed by FC and consensus primer (cpPCR) for the heavy chain gene, and splinkerette PCR (spPCR)

• The MRD positivity rates – FC 74%, cpPCR 55%, and spPCR 98%, respectively. Quantitative levels of spPCR correlated with disease status.

• FC superior to cpPCR for the heavy chain gene for the detection of MRD but inferior to spPCR

• The use of spPCR may be most useful once negativity for MRD by FC has been established.

Arons et al. Clin Cancer Res 2006;12:2804–2811.

MRD after therapy with deoxycoformycin

• MRD by FC in patients with HCL (n = 23) in CR after treatment

with deoxycoformycin (DCF).

• PB and BM samples collected at variable time points (median of 10 months) assessed for expression of CD3, CD22, CD11c, CD25, HC2, and CD103.

• The incidence of detectable MRD either in PB , BM or both sites - 43%

• No significant correlation between the presence of MRD and subsequent relapse

Matutes et al. Br J Haematol 1997;98:375–383

Timing of marrow assessments for MRD status in HCL treated with nucleoside analogs

• Serial asesmentof MRD in BM by IHC staining with DBA.44 at 2–4, 6, 12–18, and 24 months after one course of standard 2-CdA therapy.

• MRD negativity defined as <5% staining with DBA.44

• MRD negativity improved from 57% at the 2-month assessment to 77% at the 6-month assessment, without significant changes thereafter

• Correlation between positive DBA.44 immunostaining on the 6-month marrow assessment and an increased incidence of relapse.

• 6 months reccommended time for the full effects of the nucleoside analog therapy

Bastie et al. Leuk Lymphoma 1999;35:555–565

MRD may predict bone marrow relapse in HCL patients treated with 2-CDA or DCF

• Patients : 2-CdA (N =39) vs DCF (N=27)

• MRD detectable by IHC:

- 2-CDA at 3 months and then yearlyafter therapy

- DCF every 6 months

• Relapse was defined as detection ofHCL via H & E microscopy of BM

• The 4-year relapse-free survival ratewas significantly lower if MRD wasdetected

(55% versus 88%, p=0.0025).

Tallman et al. Clin Cancer Res 1999;5:1665–1670. Wheaton S et al. Blood 1996;87:1556–1560.

Is MRD detection clinically important in PNA treated HCL patients ?

• Persisting disease detected by immunohistochemistry using anti-CD45RO, anti-CD20, and DBA.44 in paraffin-embedded bone marrow sections after initial therapy with nucleoside analogs associated with a shorter relapse-free survival 1

• Long-term relapse-free survival possible in patients harboring HCL cells 2.

Scrips Clinic experience - BM samples from 19 asymptomatic patients in continuouscomplete hematological response after initial therapy with a course of 2-CDA with a median relapsed time from therapy of 16 years – presence of MRD or morphological disease in 10 of 19 (53%) patients

• Proliferative capacity and the kinetics of relapse in HCL are important factors and may argue against the need for MRD monitoring

1Wheaton et al. Blood. 1996; 87(4):1556-1560. 2Sigal DS, et al. Blood. 2010;115(10):1893-1896.

Purine analogs and rituximab may allow the eradication of MRD in most HCL patients

• 10 patients received 4 cycles of Rituximab after Cladribrine

• Before Rituximab, 2 pts in CR, 6 in PR and 2 NR. Median time from the last 2‐CdA infusion was 5.7 months.

• Two months after the end of Rituximab, all patients in CR

• Rituximab increased percentage of molecular remission up to 100% 1yr after the end of treatment. All but one, showed MRD levels lower than before Rituximab

Cervetti et al. Eur J Haematol 2004;73:412–7.

MRD in HCL patients treated with Cladribine followed by rituximab

• Untreated HCL (N = 59), relapsed HCL (N = 14) and HCL variant (HCLv, N = 7).

• Cladribine 5·6 mg/m2 IV) daily for 5 d followed 1 month later with rituximab 375 mg/m2 IV weekly for 8 weeks

• Response assessed at day 28 (+/4 d) in bone marrow specimens colectedprior to rituximab and after completion of rituximab.

• Median duration of response to 2-CDA + R longer than the first‐line 2-CDA only (72 months vs not reached, P = 0·004).

Ravandi et al., Blood, 2011;118:3818 ; Chihara et al., Br J Haematol. 2016 Sep;174(5):760-766

MRD in HCL patients treated with Cladribinefollowed by rituximab

• MRD was assed in BM by multiparameter FC with the limit of detection 0.002%.

• Eradication of MRD priorior to rituximab - 76% of untreated patients with classic HCL and 64% of patients with relapsed disease

• MRD negativity after the treatment with rituximab in PB flow monitoring - 94%

achieved at median time 2.9 months, (range 0.8-18.9)

• Only 4 patients had MRD recurrence

• Positive MRD during the follow up did not result in clinically relevant relapse.

• Cladribine followed by rituximab is highly effective even in patients with relapsed disease and HCLv, and can achieve durable remission.

TRavandi et al., Blood, 2011;118:3818 ; Chihara et al., Br J Haematol. 2016 Sep;174(5): 760-766

9.

Randomized trial CDAR vs CDA

| | | | | | | | | | | | | | | |

| | | | | | | |

CdA 0.15 mg/Kg SQ qd x5 (d1-5) Rituximab 375 mg/m2 qw x8

CdA + delayed Rituximab

CdA + concurrent Rituximab vs

> 6 mo after CdAwhen blood MRD+

| | | | | | | |

> 6 mo after RTXwhen blood MRD+

Goals: • Allow CDA + R to work synergistically• Determine how well CDA alone eradicates MRD• 1o endpoint: Determine if MRD different at 6 months• 2o endpoint: Determine benefit of delayed rituximab

• Potential advantages of delayed: Avoid rituximab, target isolated HCL cells rather than tumor

collections, recovery of B-cells after >6 months, avoid steroids for rituximab, avoid additional

risk of infections.

• Trigger for delayed rituximab was blood MRD by flow

Courtesy of Dr R Kreitman Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019, Rome, Italy

MRD in HCL patients treated with Cladribine followed by rituximab: Results at 6 months

• CR rates of 100% with CDAR and 88% with cladribine are expected results.

• MRD-free CR 6 months after concurrent CDAR 97%, vs 24% with CDA alone. Eventually increased to 32%.

• .

Characteristics Concurrent (CDAR) Delayed (CDA) p-value

N 34 34

Overall response rate (ORR) 34 (100%) 34 (100%) 1.0

Complete response rate (CR) 34 (100%) 30 (88%) 0.11

Peripheral blood flow cytometry neg 34 (100%) 17 (50%) < 0.0001

Bone marrow IHC neg 34 (100%) 27 (79%) 0.011

Bone marrow flow cytometry neg 33 (97%) 8 (24%) < 0.0001

All three tests negative 33 (97%) 8 (24%) < 0.0001

Courtesy of Dr R Kreitman Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019, Rome, Italy

Very high rate of MRD-negativity in R/R HCL treated withbendamustine and rituximab.

• 12 pts with HCL ≥2 prior therapies

• Treatment : Rituximab 375 mg/m2 days 1 and 15 + bendamustine 70 or 90 mg/m2, days 1 and 2, for six cycles at 4-week intervals.

• OR rate 100%, 7 (CR 58%).

• MRD in PB and BM absent in 67% (in all with CRs) as detectable by flow cytometry

Burotto et al. Clin Cancer Res. 2013;19(22):6313-6321.1896.

Moxe

Moxe

Kreitman et al., CCR, 17:6398, 2011

•Moxetumomab pasudotox is composed of a light chain variable domain and a heavy chain variable domain of a anti-CD22 monoclonal antibody genetically fused to a truncated form of Pseudomomas exotoxin PE38

•Moxetumomab have been shown to induce immunogenic cell death, eliminating tumor cells and promoting tumor recognition by the immune system

Moxetumomab pasudotox -recombinant , first in class Pseudomonas-based immunotoxins

• 8-color multiparametric approach on a 3-laser FACSCanto II (BD Biosciences, San Jose, CA)

• 1-1.5 million cells acquired for each cocktail.

• Cells coexpressing CD19, CD11c, CD25, CD103 and CD123 were identified and quantitated.

• The validated limit of detection of minimal residual hairy cell leukemia is 0.002% of cells, similar to the 10-4 limit reported for CLL

Kreitman RJ et al. . Blood. 2018;131(21):2331–2334.

Importance of MRD-free CR from Phase 1 Moxe study

•N= 33 patients - treatment 50 μg/kg:- 12 previously reported patients- 21- extension cohort

• OR - 88% ; CR – 20( 64%)

• 20 CRs evaluated for MRD by BM aspirate: - 9 MRD-positive: median CR duration 13.5 (4.9-42.4) m- vs 11 MRD-negative: median CR duration 42.1 (24.0-69.2) m (P < .001).

Kreitman et al. Blood, 131:2331, 2018

CR duration is shown for patients at 50 mg/kg by consolidation cycles received

Tiacci E, et al. ASH 2017. Abstract 409.

A. CR duration for patients at 50 mg/kg by consolidation cyclesB. CR duration for Pts evaluable for MRD C. Durations of MRD-negative CRD. MRD negativity in blood for patientsachieving CR or PR

Conclusions:1. Moxetumomab pasudotox can

eliminate MRD in a significant percentage of HCL patients

2. Elimination of MRD was significantlyassociated with prolonged CR.

Kreitman RJ et al. . Blood. 2018;131(21):2331–2334

Key Inclusion Criteria: Histologically confirmed HCL and ≥1 of the following:

– Neutrophils <1.0 × 109/L

– Platelets <100 × 109/L

– Hemoglobin <10 g/dL, or

– Symptomatic splenomegaly

≥2 prior systemic therapies

• Option to discontinue with <6 cycles if patient achieved MRD-negative CR (investigator assessed, by flow cytometry)

ECOG PS, Eastern Cooperative Oncology Group performance status; HCL, hairy cell leukemia; PNA, purine nucleoside analog.

Moxetumomab Pasudotox in Heavily Pretreated Patients with R /R HCL : Results of a Pivotal International Study

29

ParameterBICR Investigator-assessed

n (%) 95% CI† n (%) 95% CI†

Durable CR 24 (30%) 20, 41 38 (48%) 36, 59

Best overall response

CR 33 (41%) 30, 53 41 (51%) 40, 63

CR, MRD-negative 27 (34%) 24, 45 26 (33%) 22, 44

PR 27 (34%) 22 (28%)

SD 12 (15%) 9 (11%)

PD 2 (3%) 3 (4%)

NE 6 (8%) 5 (6%)

ORR (CR or PR) 60 (75%) 64, 84 63 (79%) 68, 87

*By IHC. †Two-sided confidence interval was calculated using the exact probability method based on the binomial distribution. EHA 2018 - Presented by Francis Giles, MD – Study Principal InvestigatorKreitman RJ, et al. Leukemia. 2018 ;32(8):1768-1777.

30

Moxetumomab Pasudotox in Heavily Pretreated Patients with Relapsed/Refractory Hairy Cell Leukemia - Results of a Pivotal International Study

Response and MRD Status*

Moxe 1053 study: Moxetumomab pasudotox in R/R HCL: Final analysis

Kreitman RJ, et al. 2020 . Manuscript in preparation

MRD negativity is associated with high rates of durable complete response

• Moxetumomab pasudotox induces deep durable responses and eradication of MRD in a substantial proportion of extensively pretreated patients with R/R HCL

• Moxetumomab pasudotox has an acceptable tolerability profile - Low rates of treatment-related AEs leading to discontinuation

• Moxetumomab pasudotox is a non-chemotherapeutic agent that may become a standard of care for patients with relapsed/refractory HCL – FDA Approval in 2018

• Long-term outcome may be improved by clearing MRD in patients with advanced HCL

CLS, capillary leak syndrome; HCL, hairy cell leukemia; HUS, hemolytic uremic syndrome; MRD, minimal residual disease. 32

Moxe 1053 study: Moxetumomab pasudotox in R/R HCL

Conclusions

RAF-MEK-ERK signaling pathway in hairy cell leukemia.

Roider T, Falini B and Dietrich S. Recent advances in understanding and managing hairy cell leukemia [version 1]. F1000Research 2018, 7:509 (doi: 10.12688/f1000research.13265.1)

Vemurafenib in relapsed or refractory HCL

Vemurafenib in relapsed/refactory HCL previously treated with purine analogs - Twophase 2 studies – in Italy and USA Dose 960 mg x 2 daily

ITALY: Median treatment duration 16 weeks; Median follow-up 23 m ; OR- 96%, CR 35% (9/26) ; , Median treatment free survival 25m for CR i 18 m for PR

USA: Median treatment duration 18 weeks; OR 100%,CR 42% (10/24), 1 yr PFS –73%

Updated results:

OR rate in refractory/relapsed HCL 100%, with 35% - 40 CRs

Median relapse free-survival was about 19 months in patients who had achieved CR and 6 months in those who had obtained a partial response

Tiacci et al. N Engl J Med 2015; 373:1733.; Fallini et al. Blood. 2016 Oct 13;128(15):1918

Vemurafenib + Rituximab in relapsed or refractory HCL HCL-PG03: Study Design

• Single-center, single-arm phase II trial

Courtesy of Dr Tiacci E, et al. ASH 2017. Abstract 4; Tiacic et al. Rome - October 24-25, 2019

09.

Pts with HCL that

relapsed following or

refractory to purine

analogues (N = 31)

CR*: Follow-upVemurafenib 960

mg PO BID on

Days 1-28 +

Rituximab 375

mg/m2 IV Q2W on

Days 1, 15

Rituximab 375

mg/m2 IV Q2W

for 4 doses

Cycles 1-2 Consolidation

No CR*:

Repeat 1 cycle of

vemurafenib + rituximab

followed by consolidation

and response re-

evaluation

*CR defined as: normal blood counts (neutrophil count ≥ 1500/mm3,

platelet count ≥ 100,000/mm3, Hb ≥ 11 g/dL), no hairy cells observed in

BM and blood smear by morphology, no splenomegaly.

Endpoints: response, time to response, depth of response, safety

Response evaluation

4 wks post

consolidation

Response

evaluation

Day 42

HCL-PG03: Vemurafenib + Rituximab in Relapsed/Refractory HCL

RELAPSE-free* survival (RFS; n=26 responding pts.)

• Median follow-up: 26 months (range: 13-43)

• 84% of pts. relapse-free

• All relapses (n=4) in MRD+ pts.; 3/4 had received a prior monotherapy with a BRAF inhibitor (BRAFi)

• RFS longer in pts. MRD- (n=17) vs MRD+ (n=9),and in pts. naive (n=19) vs exposed (n=7) to a BRAFi

*N ≥1,500/mm3 and PLT ≥100,000/mm3 and Hb ≥11g/dl

MRD -

MRD +

BRAFi-naive

BRAFi-exposed

p=0.003

p=0.009

Courtesy of Dr E Tiaci

HCL-PG03: Vemurafenib + Rituximab in R/R HCL

MRD-free survival (n=17 pts.)

• Median follow-up: 24 months

(range: 12-36)

• 16/17 (94%) of pts. remain MRD- negative

• The only pt. who lost MRD-negativityis still in CR at 40 months

Courtesy of Dr E Tiaci., Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019 Rome, Italy

Vemurafenib in HCL: Conclusions• Vemurafenib (BRAF inhibitor) is associated with high response rate in

patients with R/R HCL

• Vemurafenib is more efective in combination with rituximab

• Deep and durable response in refractory and heavily pre-treated pts.

• Fixed and short duration - 8 weeks of vemurafenib + 8 rituximab doses

• Clearly superior to historical results with either agent alone

• Direct combination therapy (Vemurafenib +8 rituximab ) seems better than Brafi-monotherapy followed by Vemurafenib +8 rituximab at relapse

Courtesy of Dr Tiaci, Hairy Cell Leukemia Foundation Annual Conference – October 24-25, 2019 Rome, Italy

HCL casewith permission of the Patient

Mazowsze - Polish Folk Song and Dance Ensemble

Clinical History (1)General description

• 34-year-old female first seen in in May, 2013 - pancytopenia .• Physical examination: splenomegaly 2 cm below the costal margin, no

lymphadenopathy• Hb -84 g/l, WBC – 1.75x109/l, Neutrophils - 0.31x109/l and PLT 59x109/l. • BM aspiration – 20% ‘hairy’ cells• BM Trephine biopsy - 80% hairy cells• Diagnosis: Hairy cell leukemia

Case 1• The 1st line treatment:

- 2 cycles of cladribine (2-CdA) 0.12 mg/kg) in 2-hour iv infusion – the 1st once a week over 6weeks (18.06.2013 – 1.08.2013) – PR, the 2nd one – 5 consecutive days (13-17.01.2014) – PR

BM biopsy (16.09.2014) – 80% HCL infiltration

• The 2nd line treatment:

- Interpheron IFN alpha 3 mln U x 3 per week (29.09.2014 - 4.12.2014) – NR

• The 3rd line treatment:

- cladribine (19-23.01.2015) in combination with 8 doses of rituximab (17.02.2015 – 7.04.2015) –PR

- BM biopsy (28.05.2015) – 50% HCL infiltration

• CBC (8.09.2015): Hb 8,7 WBC 0,73 PLT 129• BM biopsy (9.09.2015) 100% HCL infiltration; PB immunophenotyping: CD19+, CD20+, CD11c+,

CD25+, CD103+, CD123+ • BRAF V600E mutation present

Case 1

The 4th line treatment:

- moxetumomab pasudotox 6 cycles (28.09.2015 – 4.03.2016)

- a pivotal multicenter,open-label, single-arm trial of moxetumomab pasudotox in relapsed/refractoryhairy cell leukemia; moxetumomab pasudotox 40µg/kg IV on days 1, 3 and 5 of each 28 day cycle ; no AEor SAE was observed; hematologic remission was obtained; BM biopsy (date) – 80% HCL infiltration

• November 2016 – recurrent infections, transfusion – dependent anemia, progressive pancytopenia

• BM biopsy (12.2016) – 75% HCL infiltration - NR

• The 5th line treatment:

- vemurafenib 960mg BID oral (3.01.2017 – 10.04.2017) and rituximab 375mg/m2/d/iv scheduled for 8doses given every 2 weeks (3.01.2017 – 20.05.2017); due to vemurafenib intolerance (photosensivity,arthralgia, musculoskeletal pain, rash) – dose reduction to 240mg BID;

CR MRD (-) the response assessment was established by trephine biopsy (no HCL cells), BM+PB flowcytometry (no HCL cells), BM+PB AS-PCR for BRAF- V600E mutation (negative)

Case 1• 9.01.2019 admitted to the emergancy room because of fever and fatigue with CBC: WBC

0,91 Neu 0,54 Hb 8,1, PLT 135

- BM biopsy: 95% HCL infiltration

• The 6th line of treatment:

- retreatment with vemurafenib 2x240mg (11.02.2019 – 3.06.2019) BID and rituximab375mg/m2/d/iv every 2 weeks, 8 doses in total (11.02.2019 – 20.05.2019)

- CBC 9.01.2019 : Hb 12,7; WBC 6,06; ANC 4,23; PLT 204

- 29.10.2019 5-10% residual HCL (MRD+ CR) (BM+PB AS-PCR for BRAF- V600E mutationpositive)

- CBC 19.02.2020: Hb 12,2; WBC 3,72 ANC 2,10; PLT 212;

MRD in HCL- Conclusions

• The prognostic implications of detectable MRD after therapy for HCL are

influenced by variability in sensitivity of the various techniques used.

• FC appears to be the most informative and practical method

• Nonuniformity of the criteria used to define MRD and variability in timing

of MRD assessments after therapy

• The prognostic relevance of MRD in HCL can only be determined in the context

of uniform definitions, methodology, and therapy.

MRD in HCL- Conclusions (2)

• Proliferative capacity and the kinetics of relapse in HCL are important factorsand may argue against the need for MRD monitoring

•Interventions to eradicate MRD may improve outcome but need further

investigation

• Clinical trials further exploring the potential for MoAbs, immunotoxins and BRAF inhibitors to eradicate MRD and improve disease outcomes areunderway.

• Clone-specific rearrangements of the T-cell receptor (TCR) genes are present

in virtually all T-PLL cases.

• Identification and quantification of clonal T cells by PCR can be a valuable tool

to monitor MRD in T-PLL and other T-cell malignancies

T-prolymphocytic leukemia (T-PLL).

• Cuantitative MRD monitoring performed by clone-specific real-time PCR of TCR rearrangements (n=7), and TCR repertoire diversity assessment by next-generation sequencing (NGS; n=3)

• Significant reduction (>1 log) of MRD levels in 7 of 10 occasions

• Durable MRD clearance observed in two patients

• In all three patients analyzed by TCR-NGS, MRD responses were reproducibly associated with a shift from a clonal, T-PLL-driven profile to a polyclonal signature.

Sellner et al. Bone Marrow Transplant. 2017;52(4):544-551..

T-prolymphocytic leukemia (T-PLL).

• The MRD responses to immune modulation provide first molecular evidence for GvL activity in T-PLL which may be often only transient and reliant on a poly-/oligoclonal rather than a monoclonal T-cell response.

• The results suggest that TCR-based MRD quantification is possible.

• GvL is effective in T-PLL but often only limited or transient, and is driven by poly- or oligoclonal T-cell responses rather than single dominant T-cell clones.

Sellner et al. Bone Marrow Transplant. 2017;52(4):544-551..

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