Tumor Cytogenetics - icg.cpmc.columbia.edu Cytogenetics Vundavalli Murty ... Short CR and prompt relapse Generally Poor t(11;19)(q23;p13.3) MLL ... NUP214/ABL1 ampliﬁcaWon ABL1 and

Tumor Cytogenetics Vundavalli Murty

Department of Pathology & Cell Biology

& Institute for Cancer Genetics

Sept 19, 2016

September 20, 2016 Page 2

Reference Material 1. Cancer Cytogenetics: Text Book: Heim S and Mitelman F, 2015

2. Recurrent Chromosome Aberrations in Cancer

Web site http://cgap.nci.nih.gov/Chromosomes/RecurrentAberrations

3. Atlas of Genetics and Cytogenetics in Oncology and Haematology

Web site: http://atlasgeneticsoncology.org//


Overview - Chromosomes in human tumors are widely abnormal:

David von Hansemann 1890: nuclear and mitotic abnormalities in tumors

Boveri 1914: Chromosome abnormalities play a central role in transformaton

- Controversy: Early 1950s

Whether the chromosome changes are primary events or merely secondary phenomena?

- Changed view:

Recurring chromosome aberrations associated with leukemia, lymphoma, sarcoma, carcinoma

Proto-oncogenes identified at the breakpoints Experimental evidence for these genes in transformation Targeted drugs that inhibit gene product: e.g. STI571 for BCR-ABL


Methods of Chromosome Preparations

• Availability of actively dividing cells can be arrested at metaphase or prometaphase stages: mitotic stimulants• Direct preparations or short-term cultures to represent in vivo conditions

• Hematologic Malignancies:

- Bone marrow, Lymph node, Peripheral blood, Other body fluids

- 12~120hr cultures with or with out mitogenic stimulation

Solid tumors: All types

Short-term cultures (days to weeks)


Chromosome preparation & Staining contd -Mitotic arrest (Colcemid: microtubule depolymerising drug)

-Hypotonic treatment & fixation-A variety of different types of methods: Giemsa-banding (best choice) -FISH(Fluorescenceinsituhybridization)-  Probes to detect translocations, inversions, deletions, amplification-Other Molecular Cytogenetic methods: "

Chromosomal CGH, SKY, array CGH, Microarray"

Methods Employed in Recognizing Chromosome Aberrations"

•  Banding methods: Trypsin-Giemsa

•  Chromosome painting:

•  FISH:

•  Comparative Genomic Hybridization CGH):

•  Spectral Karyotyping (SKY):

Ref. Rao PH et al. Molecular cytogenetic applications in analysis of the cancer genome. Methods Mol Biol. 2007;383:165-85.



Idiograms of G-band for normal Human Chromosomes Nomenclature "

p

q

Centromere

Region Band Number e.g., 1p34.1"


Symbols and Abbreviated Terms: ISCN, 2009"

Important designations to describe cancer karyotype cen centromere del deletion der derivative chromosome dic dicentric chromosome dmin double minute chromosome dup duplication hsr homogeneously staining region i isochromosome ins insertion inv inversion mar marker chromosome minus (-) loss plus (+) gain p short arm parenthesis()surround structurally altered chromosoems and breakpoints ph Philadelphia chromosome q long arm r ring chromosome semicolon(;)separates altered chromosomes t translocation

A translocation between chromosomes "9 and 22 in CML at bands 9q34 "and 22q11.2"46,XX,t(9;22)(q34;q11.2)[20]"

A deletion of long arm of chromosome 5 at band 5q31 in a leukemia patient"

46,XY,del(5)(q31)[10]/46,XY[10]"


Types & Consequences of Major chromosome changes

•  Ploidy (e.g., haploid, triploid, tetraploid): Unclear"

•  Aneuploidy (e.g.,tri-,tetra-, monosomy): Increased or decreased expression of set of genes"

•  Reciprocal or non-reciprocal translocations (inversions): ""Overexpression/fusion gene"

•  Deletions: Loss of expression"

•  Duplications and Amplifications: Increased gene expression"

•  Insertions: Loss or gain of function of genes"

Ref. Mitelman, F et al.,The impact of translocations and gene fusions on cancer causation. Nat. Rev. Cancer 7, 233–245 (2007).!


Chromosomal Translocations in Leukemia and Lymphoma

Consequences: Altered Expression and/or Fusion Proteins"

Gain of function; BCR/ABL

Over a 420 genes associated with translocations have been identified in human cancer.

Ref. Rowley JD. Chromosome translocations: dangerous liaisons revisited. Nature Reviews Cancer 1, 245-250, 2001.

- Tumor cells exhibit non-random chromosome abnormalities

- Recurrent in specific types of malignancies

- Have been used as diagnostic and prognostic markers

- Have lead to the understanding of biology of cancer

Cancer is a Genetic (Chromosomal) Disease


Chronic Myelogenous Leukemia (CML)

t(9;22)(q34;q11.2)

BCR/ABL fusion

Ref. Goldman JM1, Melo JV. Chronic myeloid leukemia--advances in biology and new approaches to treatment.

N Engl J Med. 2003 Oct 9;349(15):1451-64.

05-1363

06-174

02-15

t(9;22) partial karyotype BCR/ABL FISH

t(9;22)(q34;q11.2)

t(9;22)(q34;q11.2)

t(9;22)(q22;q11.2)

CML: Karyotyping and FISH

Chromosome changes in CML progression

A: Karyotype: 47,XX,t(9;22)(q34;q11.2),+8,i(17)(q10) B: FISH--BCR/ABL C: FISH--CEP 8 (green) and D20S108 (red) D: FISH– TP53 (red) and ATM (green)

Chromosomal changes in Myelodysplatic Syndrome (MDS): Clinical significance

5q- syndrome Single defect; favorable with low risk of transformation (distinct morphologic entity; high prevalence in elderly females; RPS 14, CTNNA1 as target genes

del(5q)/- 5 Favorable as sole abnormality Additional abnormalities, therapy related--poor prognosis

del(7q)/-7 Unfavorable outcome

Trisomy 8 Intermediate

del(20q) Good prognosis (as sole change) Intermediate/unfavorable (with other abnormalities)

• Heterogenous group"• Primary and secondary MDS"• Cytogenetic changes have prognostic implications"The classification of MDS is based on clinical data (previous history, age) and biologic characteristics (morphology, cytochemistry, immunophenotype, cytogenetic and molecular alterations)


Acute Myeloid Leukemia (AML)

>85% karyotypic abnormalities

Translocations, deletions, and inversions

Diagnostic and classification of risk groups

The classification of acute myeloid leukemia (AML) similar to myelodysplasic syndromes (MDS) is based on clinical data (previous history, age) and biologic characteristics (morphology, cytochemistry, immunophenotype, cytogenetic and molecular alterations)


Recurring Chromosome Abnormalities in AML Morphologic Subset Chromosome change Genes Prognosis

M2 t(8;21)(q22;q22) ETO-AML1/RUNX1 (attenuates spindle checkpoint)

Favorable

(No adverse for additional changes)

M3 t(15;17)(q22;q12-q21) PML-RARA Variants: PLZF, NPM, NUMA

Favorable

M4Eo inv(16)(p13q22)"

t(16;16)(p13;q22)"

del(16)(q22)"

CBFß/MYH11 (core binding factor/myosin heavy chain)

Favorable

M1,M2, M4, M5a, therapy-related (Topoisomerase inhibitors)

11q23 abnormalities" MLL (KMT2A) Poor, generally

Variable, translocation-based

Biphenotypic leukemia (BAL)

t(9;22)(q34;q11), 11q23 abnormalities"

BCR/ABL, MLL Worse

Many other categorized, Uncategorized, and Treatment-related

Inversion 16 in AML: Good prognostic AML"

9/20/16 Page 19

t(15;17) is a charcteristic chromosome change in APML

Therapy Related MDS and AML

An AML patient with complex karytoype and MLL amplification predicts poor prognosis

MLL gene amplified"

Diagnosis: "AML"

Age: " "74 years (Female) ""

Karyotype: 45,XX,-4,der(5)t(5;17)(q11.2-q21;q11.2),+6,+8, der(11)dup(11)(q13q23)ins(11;4)(q23;q22q28)hsr(11)(?23),-17,-18[cp35]

Amplification: MLL, ATM: Deletion: EGR1, TP53 "

Treatment: "Mylotarg, ATRA "

Survival: "5 days after diagnosis"

A patient of P. vera transformed to AML with complex karytoype

Diagnosis: "tAML"

Age: " "70 years (Male) ""

Karyotype: 61-63,<3N>,XY,-2,-3,-4,-5,-7,der(8)t(2;8)(?;p21),-9,+der(9)(?)x6,-10,-12,-13,-14,-15,-17,�-18,+der(18)t(11;18)(q13;p11.2),+21[25]

Treatment: "No data"

Survival: "No data"

Myeloid neoplasms with complex karyotypes predict poor prognosis"

No Diagnosis (age)

Treatment Karyotype Amplification

Increased copies

Deletion Survival

1 AML (74) Mylotarg, ATRA Complex MLL, ATM - EGR1, TP53 5 days

2 MDS/AML (65)

Doxorubicin, AraC HU " Complex MLL TP53 EGR1, AML1, TEL

8 months

3 MDS (74) NA Complex MLL ATM EGR1, TP53 NA

4 AML (87) No RX Complex MLL, ATM - EGR1, TP53 <5 days

5 MDS/AML (69)

Ida/Ara C refractory disease failed induction "

Complex MLL ATM EGR1, TEL 1 month

6 MDS/AML (69)

Topotecan/Ara C G-CSF persistent leukemia "

Complex MLL, ATM - EGR1, TEL 8 months

7 p.veraàAML NA Complex MLL ATM EGR1, TEL NA

8 AML (49) Ara C/Ida R " Complex - - EGR1, TP53 1 month

9 CML (69) HU, Ida/Ara " Complex MLL ATM EGR1, TP53 1 month

10 AML (65) No RX Complex MLL, ATM - EGR1, TEL 4 days

11 MDS/AML (75)

No RX Complex MLL - EGR1, TEL 1 month

Probabilities of 5-year survival according to cytogenetic stratifications in children with advanced primary or secondary MDS

Gohring G et al., Blood 116: 3766, 2010


Acute Lymphoblastic Leukemia (ALL): Cytogenetics Diagnostic and prognostic significance"

Chromosome change Genes involved Subtype Diagnosis/Prognosis High hyperdiploidy

(>55 chromosomes)

Not known

(+4,+6,+8,+10,+14,+21)

Pre-B Good

(>90% cure in children)

Near haploidy (26-28) Not known Pre-B Poor (short complete remission)

Normal -- -- Poor

t(9;22)(q34;q11.2) BCR/ABL (p210 or p190)

B-cell (stem cell or progenitor)

Poor

t(4;11)(q21;q23) AF4/MLL B-cell (biphenotypc)

(also AML M5)

Short CR and prompt relapse

Generally Poor

t(11;19)(q23;p13.3) MLL (KMT2A)/MLLT1 B-cell (biphenotypc)

Very Poor

t(12;21)(p12;q22) ETV6(TEL)/RUNX1(AML1) B-cell Excellent

t(8;14)(q24;q32) t(2;8)(p12;q24) t(8;22)(q24;q11)

MYC/IGH MYC/IGK MYC/IGL

B-cell ALL(L3) /NHL(BL)

Diagnostic and Adverse

t(1;19)(q23;p13) PBX/E2A ALL Adverse

Accumulation of malignant and immature lyphoid cells in marrow


Chromosomal abnormalities in ALL: T-Cell �Diagnostic and prognostic significance

Chromosome change Genes

involved

Subtype Diagnosis/

Prognosis

t(10;14)(q24;q11) HOX11/TCRA T-cell Favorable(Adult)

t(7;10)(q34;q24) TCRB/HOX11 T-cell Notknown

t(11;14)(p13;q11) LMO2/TCRA T-cell Notknown

t(8;14)(q24;q11) MYC/TCRA T-cell Poor

t(7;9)(q34;q34) TCRB/NOTCH1 T-cell Notknown

inv(14)(q11q32.1)

t(14;14)(q11;q32.1)

TCRA-TCRD/TCL1 T-PLL

AT

T-cellleukemia

/lymphoma

Aggressivecourse

NUP214/ABL1amplificaWon ABL1andNUP214(CAN)

T-cell SensiWvetoTyrosineKinaseinhibitorGleevac

Cytogenetic Subsets in Pediatric ALL"

Mullighan CG. J. Clin Invest. 122; 3407, 2012

Favorable outcome

Poor outcome

Excellent prognosis

Poor prognosis

Poor outcome

Karyotype showing near haploid chromosomes in an ALL patient

Karyotype: 27<1N>,XY,+14,+18,+21

t(4;11)(q21;q23) in ALL

A: Karyotype--46,XY,t(2;19)(q23;q13.4),t(4;11)(q21;q23)

B: FISH: MLL break apart probe


Prognostic Impact of Diagnostic Karyotypes in Pediatric ALL"

Karyotype

Event Free Survival (%)

2 yrs 5 yrs >50 chromosomes 90 80

47-50 chromosomes 70 60

Normal karyotype 70 60

t(1;19)(q23;p13) 50 40

t(9;22)(q34;q11.2) 40 30

t(8;14)(q24;q32) 40 30

11q23 abnormalities 30 20

Hypodiploid (<46) 30 20

t(4;11)(q21;q23) 20 20

Heim and Mitelman, 1995

Cytogenetics: Mature B- and T-Cell Lympoma


Chromosome Translocations: Lymphocyte Development • Translocations are considered to arise as errors during intragenic physiologic rearrangements that assemble productive copies of IG and TCR genes during normal B- and T-cell development

• Translocations tend to be specific for breakpoints and show a high degree of association with histologic subsets

• Three Main Subsets of NHLs:

-- B-Cell

-- T-Cell/Natural Killer (NK) Cell

-- Hodgkin’s Lymphoma (HL)

• First description of karyotype in BL in 1963 (Jacobs et al., 1963)

• Identified as t(8;14)(q24;q32) in 1972 (Manolov and Manolova, 1972)

• t(8;14) was shown that MYC rearranges with IGH (Dalla-Favera et al., 1987)


Burkitt’s Lymphoma"

Primary Chromosome change:

•  --t(8;14)(q24;q32) MYC/IgH •  Variant translocations:

•  -- t(8;22)(q24;q11) (MYC/IgL)

•  -- t(2;8)(p11;q24) (IgK/MYC)

• Duplication 1q: progression


Follicular Lymphoma

Primary Chromosome Change: t(14;18)(q32;q21) (IgH/BCL2)(>75%)

variant translocations involving 18q21 break point (BCL2)

»  3q27 rearrangements (BCL6) »  6q21 deletions

t/der(1q), +7, del(6q), del(17p): Progression/transformation

t(14;18) negative tumors do exist, which exhibit complex and heterogeneous breakpoints (3q27; 8q24, ect)


Diffuse Large B-Cell Lymphoma (DLBCL)

Generally complex, hyperdiploid

t(14;18)(q32;q21) (IgH/BCL2) followed by 3q27 rearrangements (BCL6)(many partners) t(8;14)(q24;q32) (MYC/IgH) Correlations with chromosome changes contradictory

-- der(1)(q21), +7, del(6q), del(17p) Associate with progression/transformation; predict adverse outcomes

--Chromosome amplifications frequent


t(11;14)(q13;q32): The cytogenetic hallmark found in 70% of cases (CCND1/IgH)

•  Monosomy 13/del(13q)

Mantle Cell Lymphoma (MCL)

del (17p13) (TP53) (v)"

del(11q22-q23) (ATM) (iv)"

Trisomy 12 (ii)"

Normal (iii)"

del(13q14.3)(sole) (i)"

"

Adverse

Progression "

Intermediate"

Intermediate"

Favorable"

30 "

80 "

115 "

110"

135

Chronic lymphocytic leukemia (CLL/SLL) A chronic lymphoproliferative disorder of mature B-lymphocytes

Tumor cells are inert with only a small proliferative compartment50-60% abnormal by karyotype (>80% by FISH)

Chromosome change Prognosis OS (mo)

FISH panel targeting all the above changes

Absence of IGHV mutation: Aggressive course

Prognosis of deletions in CLL"

Dal Bo M et al. GENES, CHROMOSOMES & CANCER (2011)


Multiple Myeloma (MM)

Cytogenetic change" Genes" Prognosis"

Hyperdiploidy (3, 5, 9, 11, 15, and 19)"

Not known" Good"

1p deletions" Not known" Poor"

14q32 rearrangements"

(at least 5 partners)"

IGH"

(partners: FGFR3, cMAF)"

Poor"

11q13, t(11;14)" IGH/CCND1" Favorable outcome"

6q deletions" MYB" --"

Del(13q)/-13" D13S319" Adverse outcome; shorter survival"

Karyotype detects only 30-50% cases with abnormalities

Overall survival according to chromosome abnormality in multiple myeloma

Hervé Avet-Loiseau et al., J Clin Oncol: 30 (16): 1949-52, 2012

Mature T-cell Neoplasms

Entity"

Chromosome change"

Anaplastic large cell lymphoma

t(2;5)(p23;q35) (NPM/ALK) Other partners exists; favorable prognosis

T-cell prolymphocytic leukemia (T-PLL)"

inv(14)(q11q32), t(14;14)(q11;q32)"(TCRa/d-TCL1)"7q34-36 translocation (TCRB)"

Angioimmunoblastic T-cell lymphoma"(EBV+ve in >75%)"

Trisomy 3 or i(3q), +5, +X, del(6q)"

Chromosome change in Anaplastic Large Cell Lymphoma (ALCL)

Ref. Amin HM, Lai R. Pathobiology of ALK+ anaplastic large-cell lymphoma. Blood. 110(7):2259-67, 2007.

Genetic hallmark of T-PLL is inv(14)(q11.2q32) or t(14;14)(q11.2;q32) TCL1 gene at 14q32 juxtapose next to TCRA/D locus at 14q11.2 resulting in up regulation of the TCL1 expression. The TCR-associated translocations are regarded as the primary oncogenic events in T-PLL. T-PLL are generally very aggressive tumors among T-cell lymphomas and poorly responsive to chemotherapy.


Karyotype Changes in NHL: �diagnostic and prognostic significance

Type Diagnostic Change Progression/ Transformation

Poor outcome Good outcome

B-CLL/SLL - +12,del(11q23),t(14q32)

+12, del(11q) del(13q)

MM/LPL t(9;14)(p13;q32) - -13/del(13q) -

MALT t(11;18)(q21;q21) - - -

FL t(14;18)(q32;q21) +7,del(q),t(8;14) t(8;14) -

MCL t(11;14)(q13;q32) - del(17p) -

DLBCL - der(1q21),+7,del(6q),del(17p)

der(1q21),del(6q),del(17p

BL t(8;14)(q24;q32) dup(1q) - -

ALCL

(anaplastic)

t(2;5)(p23;q35) t(2;5)(p23;q35)

Chromosome abnormalities of 1, 6, and 17 are virtually seen in all lineage NHLs:Associated with poor clinical outcome

Solid Tumor Cytogenetics


Ewing sarcoma/Primitive neuroectodermal tumor"• Heterogeneous group of distinct histologic types

• 90% cases with t(11;22)(q24;q12); in all histologic types

• Translocation results in the fusion of the EWS with FLI1, forming a chimeric protein

• variant translocations: t(21;22)(q12;q12)[EWS-ERG] and t(7;22)(p22;q12) [EWS-ETV1 in 5% cases

• EWSR1 is also rarely rearranged in other soft tissue tumors (Myxoid chondrosarcoma, Desmoplastic small round cell tumor, malignant melanoma of soft parts)

Ref. Crompton BD. et al. The genomic landscape of

pediatric Ewing sarcoma. Cancer Discov. 4(11):1326-41, 2014.

Page 48

Other soft tissue tumors- Karyotypic Changes

Tumor Chromosome translocation

Genes Comments

Myxoid liposarcoma" t(12;16)(q13;p11)"

Variant: t(12;22)"

DDIT3 (CHOP)-FUS (TLS)"

DDIT3/EWS"

Fusion protein; oncogenic"

Alveolar rhabdomyosarcoma (A-RMS)"

t(2;13)(q35;q14) (~80%)"

t(1;13)(p36;q14) (~15%)"

Variants"

PAX3 - FOXO1 (FKHR)"

PAX7-FKHR"

Poorer survival than ERMS"

ARMS and ERMS are two distinct genetic entities"

Synovial sarcoma" t(X;18)(p11.2;q11.2)"

Variants"

SSX1, SSX2, SSX4-SYT (SS18)"

Hybrid protein"

Poor prognosis"

Page 49

Neuroblastoma Sympathetic neuronal precursor: Embryonic origin

Common Extracranial pediatric tumor of infancy

Classified as low, intermediate, and high-risk groups

"

Two major types of Cytogenetic abnormalities:

Segmental chromsome aberrations 1p and 11q deletion (LOH)—High-risk

Amplification of NMYC is the most robust prognostic factor in NB

Landscape of genetic changes in neuroblastoma"

Page 50

Pugh TJ et al., Nat Genet 45: 279, 2013


Gene Amplification: Double Minute Chromosomes

Tumors of Nervous System Gliomas

Oligodendrogliomas

Page 52

•  1p36 and 19q13 co-deletion occur in over 75% both grade II and III oligodendrogliomas •  Correlate with response to chemotherapeutic drugs and radiation

1p36/1q25" 19q13/19p13" 1p36/1q25" 19q13/19p13"

Page 53

ALK break apart Probe for Lung Cancer

• In non-small cell lung cancer (NSCLC) development rearrangements of ALK gene are implicated"

• Rearrangement of ALK with echinoderm microtubule-associated protein-like 4 gene (EML4) results in in-frame fusions of EML4-ALK genes."

• ALK also forms fusions partners with TFE and KIF5B genes."

• Patients with ALK rearrangement positive NSCLC are eligible for treatment with crizotinib and achieves high-rate of tumor response in first 8 weeks.

Case History of a Patient with ALK+ve

•  A 61 yr. old male, former smoker with stage IV lung cancer, with multiple rounds of chemo, metastatic to liver and bone.

•  After 4 years of chemotherapy, no response was seen.

•  The patient was about to enter hospice, but

•  Patient sought 2nd opinion at CUMC and among other tests ALK was tested and found positive for rearrangement.

•  The patient was treated with Xalkori (trade name for Crizotinib) and achieved complete remission in 10 weeks.

Apparently responded to targeted therapy


Renal Epithelial Neoplasms: Origin and Genes Involved� -- Morphologically and behaviorally heterogeneous group, epithelial

Origin (Brit Hogg Dubé

Hereditary form (Fumarate hydratase)


Clear Cell RCC

• Characteristic feature: Loss of 3p due to deletions or unbalanced translocations

• Number of common regions of deletions identified: 3p14, 3p21, and 3p25 (3q21 is essential)

• Trisomy 5q21-qter (partial trisomy): Predictive of good prognosis

• +12, +20

• Germ line mutation in VHL gene at 3p25 in familial cases

• No consensus on tumor suppressor on 3q21(Candidates: RASSF1A, FHIT, DUTT1, TTRC1, NRC1)


Papillary RCC • Tri- or Tetrasomy of 17, 7, 16, 20, 12 • Loss of Y in men • Loss of Y, +7, +17 may correspond to papillary adenomas • Additional changes diagnostic papillary RCC • Inherited form of pRCC--Missense mutations in MET oncogene


•  Chromosome modal number 38-39"•  Loss of chromosomes 1, 2, 6, 10, 13, 17, and 21"

•  Difficulties in differential diagnosis from Oncocytoma"

•  Karyotype (FISH) is useful in differential diagnosis

Chromophobe RCC


CCND1 rearrangement of 11q13 in Oncocytoma


Testicular Germ Cell Tumors

• Seminoma and Non-seminoma

• Over representation of 12p --85% with one or more copies of i(12p)

--tandem duplications of 12p (10%)

--Rarely, 12p amplification


Her-2/neu (ERRB) Amplification in Breast Cancer

• ERBB2 gene is amplified and overexpressed in 20-25% of breast tumors

• Tumors showing ERBB2 amplification have predominantly lost estrogen receptor expression (ER-)

• Worsened course of the disease

• Target for therapeutically approaches using engineered anti-ERBB2 antibodies


Utilities of Tumor Cytogenetics • To Establish Malignant Clone (normal karyotype doesn’t rule out)

• To Clarity/Establish Diagnosis

• To Indicate Prognosis (some changes associated with poor response)

• To Assist in Choice of Treatment Strategy

• To Monitor Response to Treatment

• To Establish Engraftment Status and Monitor

Number of targeted therapies already exists against specific genetic change that patient’s tumor uniquely possesses. "

"

Documents

Tumor Cytogenetics - icg.cpmc.columbia.edu Cytogenetics Vundavalli Murty ... Short CR and prompt relapse Generally Poor t(11;19)(q23;p13.3) MLL ... NUP214/ABL1 ampliﬁcaWon ABL1 and