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Chronic Myelogenous Leukemia :

Making Sense of Special Tes1ng

Andrew Hansen, MD UTHSC San Antonio

11/25/2013

ObjecHves

•  Summarize the clinicopathologic features of chronic myelogenous leukemia (CML)

•  Describe cytogeneHc and molecular diagnosHc tests for the diagnosis and management of CML

•  Explain tyrosine kinase inhibitor (TKI) therapy and resistance to it

Chronic Myelogenous Leukemia (CML)

•  Prototype for integraHng cytogeneHc and molecular tests to: – Classify and diagnose – Predict response to treatment – Monitor response to treatment –  IdenHfy disease progression – Design novel drugs

Case Study – Mr. Roadrunner

•  63 year old man with HTN, DM, HLP: – Presents for rouHne annual exam – CBC shows WBC count 38k/μl

•  Automated differenHal: –  Absolute neutrophilia –  Absolute basophilia

•  Peripheral smear is prepared

Mr. Roadrunner – peripheral blood

Mr. Roadrunner – bone marrow


•  MyeloproliferaHve neoplasm – Granulocyte hyperplasia with maturaHon

•  Annual incidence: 1-‐2 / 100,000 •  Median age: 65 years •  Clinical presentaHon:

– FaHgue, weight loss, fever, splenomegaly – AsymptomaHc (incidentally found)

CML -‐ Morphology (chronic phase)

Jaffe. Hematopathology. Saunders/Elsevier, 2011.

Peripheral blood (PB):

•  hypercellular •  granulocyte hyperplasia

(↑M:E ratio) •  <10% blasts

•  granulocytosis •  myelocytes, segs

•  absolute basophilia •  <10% blasts

Bone marrow (BM):

CML -‐ Morphology •  Accelerated phase:

–  Persistent ↑ or increasing WBC count or splenomegaly –  Persistent thrombocytosis or thrombocytopenia –  Clonal cytogeneHc evoluHon –  ≥ 20% basophils in PB –  10% -‐ 19% blasts in PB or BM

•  Blast phase: –  ≥ 20% blasts in PB or BM –  Extramedullary blast proliferaHon

CML -‐ Morphology (blast phase)

CML – Disease Course

•  Starts in chronic phase •  Progresses to:

– Accelerated phase, then blast phase – Blast phase, directly

•  Median survival without treatment: 3 years

Discovery of the “Philadelphia Chromosome” -‐ 1960

“Philadelphia Chromosome” = t(9;22)(q34;q11.2)


BCR-‐ABL fusion protein

Kumar, Abbas. Robbins and Cotran Pathologic Basis of Disease,8th ed. Saunders/Elsevier, 2011.

CML -‐ diagnosis

•  BCR-‐ABL1 required for diagnosis – WHO 2008: CML, BCR-‐ABL1 posiHve

•  How is it detected? – ConvenHonal cytogeneHcs

Image courtesy of Dr. Velagaleti

CML -‐ diagnosis


•  How is it detected? – ConvenHonal cytogeneHcs – FISH

der 9 Ph

22 9

Figure and Images courtesy of Dr. Velagaleti

CML -‐ diagnosis


•  How is it detected? – ConvenHonal cytogeneHcs – FISH – RT-‐PCR

•  QualitaHve •  QuanHtaHve

–  qRT-‐PCR

QuanHtaHve Reverse Transcriptase PCR (qRT-‐PCR)

A. B.

Image courtesy of Dr. Fan

CML -‐ diagnosis

•  QuanHtaHve PCR – InternaHonal scale (IS): – Useful for residual disease monitoring – Proposed in 2005 at the NIH in Bethesda –  Individual labs convert results to internaHonal scale (IS)

•  Reported as percentage of IS – Allows for inter-‐laboratory comparison – Recommended every 3-‐6 months (NCCN, 2013)

CML -‐ diagnosis

Is there sHll role for cytogeneHcs? •  YES!

–  Complex/variant translocaHons correlate with prognosis, response to treatment (Dr. VelagaleH’s poster in hallway)

–  Clonal cytogeneHc evoluHon = progression to accelerated phase

•  Need to know original karyotyope –  NCCN (2013) -‐ recommends cytogeneHcs:

•  At diagnosis •  If not responsive to treatment •  If progression or relapse is suspected

Mr. Roadrunner

•  RT-‐PCR shows BCR-‐ABL1 p210 – 46% (IS)

Treatment

•  We have a diagnosis, now what? •  Treatment opHons (unHl 2001):

–  Interferon + cytarabine •  Limited efficacy, not curaHve •  Median survival: 6 years

– Allogeneic stem cell transplant •  75% 5-‐year survival (chronic phase)

“Oncogene AddicHon”

•  CML: signaling via BCR-‐ABL gene required for tumor to persist, despite accumulaHon of other mutaHons –  inhibiHon of its acHvity is effecHve therapy Can we inhibit tyrosine kinase acHvity of BCR-‐ABL?

Kumar, Abbas. Robbins and Cotran Pathologic Basis of Disease,8th ed. Saunders/Elsevier, 2011.

ImaHnib (Gleevec) ABL kinase domain

•  1st generation tyrosine kinase inhibitor (TKI)

imatinib

i.e. ≤0.1% of IS

Baccarani, et al. Haematologica, 2008.

Response to imaHnib (5 years)

InternaHonal Randomized Interferon and STI571 (IRIS) Study •  STI571 = imaHnib •  Complete cytogeneHc response (18

mos): –  ImaHnib: 76% –  Interferon + cytarabine: 14.5%

Druker, et al. NEJM, 2006.

Mr. Roadrunner

•  Started on imaHnib •  RQ-‐PCR BCR-‐ABL1 p210 (IS):

– 3 months: 1.2% – 6 months: 0.2% – 12 months: 0.07% = major molecular response – 18 months: 0.6% – 21 months: 1.8% -‐ uh-‐oh!

Resistance to imaHnib

•  Primary resistance: no response from beginning – ~20-‐25% of newly diagnosed CML paHents

•  Secondary resistance: iniHal response occurs, followed by relapse – ~20-‐25% of paHents with iniHal response

Mechanisms of resistance to imaHnib

Apperley. Lancet Oncol, 2007.

50% of resistance


Missense MutaHons in ABL kinase domain

Threonine at position 315 forms crucial hydrogen bond with imatinib •  substitution with bulky isoleucine (T315I) results in steric hindrance

MutaHons IdenHfied (>70)


When to test for mutaHon?

•  NCCN guidelines: CML (Nov 2013) – Chronic phase:

•  Inadequate iniHal response: –  At 3 months -‐ failure to achieve parHal cytogeneHc response or BCR/ABL >10% (IS)

–  At 12 months – failure to achieve complete cytogeneHc response

•  Any loss of response: –  Hematologic or cytogeneHc relapse –  1 log increase in BCR/ABL and loss of MMR

– Progression to accelerated or blast phase

When to test for mutaHons?

•  Why not at diagnosis? – Some cases, even without prior exposure to TKI, have low numbers of mutated BCR-‐ABL1

•  Not associated with to response to imaHnib, event-‐free survival, or overall survival

•  Detected by allele-‐specific PCR

•  So, when is it clinically significant? – ~5%?

Willis, et al. Blood, 2005.

What tests are available?

•  ARUP: – BCR-‐ABL1 Kinase Domain MutaHon Analysis

•  Direct (Sanger) sequencing •  Detects 90% of mutaHons (clinical sensiHvity)

– BCR-‐ABL1 T315I mutaHon •  Pyrosequencing of codon 315 •  QuanHtaHve •  IndicaHon?

Direct (Sanger) Sequencing

Direct (Sanger) Sequencing

c.764A>T E255V glutamic acid (E) to valine (V)

Pyrosequencing

http://www.qiagen.com/products/pyromarkq96md.aspx#Tabs=t1

“sequencing by synthesis”

Pyrosequencing

ATT (isoleucine) c.944C>T

ACT (threonine)

943 944 945

943 944 945

Comparing Methods

Jones, et al. J Mol Diagn, 2009.

Why do pyrosequencing of codon 315? •  T315I is most common and most clinically significant mutation •  if < 20%, may not be detected by sequencing

Ø  but what percentage is clinically significant?

Next GeneraHon Sequencing

•  High throughput •  Can detect compound/polyclonal mutaHons

Khorashad, et al. Blood. 2013

Next GeneraHon Sequencing •  Basic principles:

–  Create “DNA library” by fragmenHng •  Targeted region amplified by PCR •  Genomic DNA (whole genome extracted from nuclei)

–  Sequence the small fragments individually –  Align sequenced fragments to reference sequence

Mr. Roadrunner

•  T315I mutaHon detected by sequencing

What next?

•  We found a point mutaHon accounHng for resistance to imaHnib – what can we do? –  Increase dose of imaHnib

•  Generally not effecHve – Switch to newer generaHon TKI

Second-‐GeneraHon TKI’s

•  NiloHnib (2007), dasaHnib (2006): –  useful for many imaHnib-‐resistant mutaHons

–  Beuer than imaHnib as first-‐line drugs

•  BosuHnib (Sept 2012): –  Useful for resistant or refractory cases

nilotinib

Choice of drug depends on mutaHon

Branford, et al. Blood, 2009.

PonaHnib (Dec 2012) “3rd generaHon”

•  Triple bond overcomes steric hindrance by isoleucine (T315I)

Cortes, et al. NEJM, 2013.

PonaHnib (Dec 2012) “3rd generaHon”

Risk of life-threatening blood clots and severe narrowing of blood vessels: •  heart (12%) •  brain (6%) •  extremities (8%)

http://www.fda.gov/Drugs/DrugSafety/ucm373040.htm

Looking ahead •  Will ponaHnib prove to be beuer first-‐line drug than imaHnib (like dasaHnib and niloHnib)? –  If so, why ever use imaHnib?

•  $$ -‐ generic imaHnib in 2015

•  With new drugs like ponaHnib, why even do mutaHon analysis? –  Why not just start with ponaHnib? A: New/compound mutaHons may confer resistance to new drugs – idenHficaHon may allow for design of new drugs


•  Prototype for integraHng cytogeneHc and molecular tests to: – Classify and diagnose – Predict response to treatment – Monitor response to treatment –  IdenHfy disease progression – Design novel drugs

References 1.  Jaffe, Elaine Sarkin. Hematopathology. Philadelphia, PA: Saunders/Elsevier, 2011. 2.  Kumar, Vinay, Abul K. Abbas, Nelson Fausto, Stanley L. Robbins, and Ramzi S. Cotran.Robbins and Cotran Pathologic Basis of

Disease. 8th ed. Philadelphia: Elsevier Saunders, 2009. 3.  Swerdlow SH, Campo E, Harris NL et al. WHO Classifica1on of Tumours of Haematopoie1c and Lymphoid Tissues. Lyon: IARC; 2008 4.  Nowell PC, Hungerford DA. Chromosome studies on normal and leukemic human leukocytes. J Natl Cancer Inst. 1960 Jul;25:85-‐109. 5.  NaHonal Comprehensive Cancer Network. Clinical PracHce Guidelines in Oncology: Chronic Myelogenous Leukemia. Version

01.2014. 9/9/2013. 6.  Baccarani M, Pane F, Saglio G. Monitoring treatment of chronic myeloid leukemia. Haematologica. 2008 Feb;93(2):161-‐9. doi:

10.3324/haematol.12588. Review. 7.  Druker BJ, Guilhot F, O’Brien SG, et al: Five-‐year follow-‐up of paHents receiving imaHnib for chronic myeloid leukemia, N Engl J

Med 355:2408, 2006. 8.  Apperley JF. Part I: mechanisms of resistance to imaHnib in chronic myeloid leukaemia. Lancet Oncol. 2007 Nov;8(11):1018-‐29.

Review. 9.  Qiagen: pyrosequencing. hup://www.qiagen.com/products/pyromarkq96md.aspx#Tabs=t1. Accessed 01/2013. 10.  Branford S, Melo JV, Hughes TP. SelecHng opHmal second-‐line tyrosine kinase inhibitor therapy for chronic myeloid leukemia

paHents ayer imaHnib failure: does the BCR-‐ABL mutaHon status really mauer? Blood. 2009 Dec 24;114(27):5426-‐35. 11.  Willis SG, Lange T, Demehri S, Ouo S, Crossman L, Niederwieser D, Stoffregen EP, McWeeney S, Kovacs I, Park B, Druker BJ, Deininger

MW. High-‐sensiHvity detecHon of BCR-‐ABL kinase domain mutaHons in imaHnib-‐naive paHents: correlaHon with clonal cytogeneHc evoluHon but not response to therapy. Blood. 2005 Sep 15;106(6):2128-‐37.

12.  Jones D, Kamel-‐Reid S, Bahler D, Dong H, Elenitoba-‐Johnson K, Press R, Quigley N, Rothberg P, Sabath D, Viswanatha D, Weck K, Zehnder J. Laboratory pracHce guidelines for detecHng and reporHng BCR-‐ABL drug resistance mutaHons in chronic myelogenous leukemia and acute lymphoblasHc leukemia: a report of the AssociaHon for Molecular Pathology. J Mol Diagn. 2009 Jan;11(1):4-‐11.

13.  Khorashad JS, Kelley TW, Szankasi P, Mason CC, Soverini S, Adrian LT, Eide CA, Zabriskie MS, Lange T, Estrada JC, Pomicter AD, Eiring AM, Kray IL, Anderson DJ, Gu Z, Alikian M, Reid AG, Foroni L, Marin D, Druker BJ, O'Hare T, Deininger MW. BCR-‐ABL1 compound mutaHons in tyrosine kinase inhibitor-‐resistant CML: frequency and clonal relaHonships. Blood. 2013 Jan 17;121(3):489-‐98.

14.  Cortes JE, Kim DW, Pinilla-‐Ibarz J, le Coutre P, Paqueue R, Chuah C, Nicolini FE, Apperley JF, Khoury HJ, Talpaz M, DiPersio J, DeAngelo DJ, Abruzzese E, Rea D, Baccarani M, Müller MC, GambacorH-‐Passerini C, Wong S, Lustgarten S, Rivera VM, Clackson T, Turner CD, Haluska FG, Guilhot F, Deininger MW, Hochhaus A, Hughes T, Goldman JM, Shah NP, Kantarjian H; PACE InvesHgators. A phase 2 trial of ponaHnib in Philadelphia chromosome-‐posiHve leukemias. N Engl J Med. 2013 Nov 7;369(19):1783-‐96.

15.  FDA Drug Safety CommunicaHon: FDA asks manufacturer of the leukemia drug Iclusig (ponaHnib) to suspend markeHng and sales. hup://www.fda.gov/Drugs/DrugSafety/ucm373040.htm. Accessed 11/25/2013.

Questions? Comments?

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