U nusually Aggressive BCLU - PowerPoint Presentation

Slide1

Unusually Aggressive BCLU with Double-Hit Genetics by Chromothripsis Identified on Chromosomal Microarray

EAHP16-LYWS-281

Charles

Van

Slambrouck

MD, Kai

Lee Yap PhD

, Sandeep

Gurbuxani

MBBS PhD,

Joo

Song MD,

Sonali

M Smith MD,

Madina

Sukhanova

PhD, Girish

Venkataraman

MD

University of Chicago

Medicine

Slide2

52 year-old maleDeveloped left shoulder pain, initially thought to be a musculoskeletal injury, but symptoms progressively worsened.Was admitted to an outside institution for evaluationClinical exam/imaging

: Mass in the left chest wall/shoulder.

Core biopsy: High-grade B-cell lymphoma (C-MYC focal, BCL2 negative, Ki67 >90% by IHC) – Path not available for reviewStaging bone marrow: Negative for lymphoma.PET scan: Disease localized to the left chest.Received 4 cycles of R-CHOP

Clinical History

Slide3

Clinical HistoryDuring treatment with R-CHOP,

the patient’s disease progressively worsened with:

Overlying skin ulcers/nodulesNew lymphadenopathyEnlargement of the primary massA new left distal femur lesionThe patient transferred care to University of Chicago and underwent debridement of the enlarged left shoulder/chest wall mass.

Chest CT after 4 cycles of R-CHOP showing enlargement of primary mass

Slide4

H&E, 0.5x

Fibroadipose and skeletal muscle tissue extensively involved by a diffuse, sheet-like proliferation of tumor cells

Large areas of necrosis are present (~40% of tissue)

Slide5

H&E, 2x

H&E, 20x

Slide6

H&E, 40x

Monomorphic medium-sized tumor cells with blastoid features.

Numerous mitoses and apoptotic debris.

Slide7

CD20:

POS

Pax5:

POS

TdT:

NEG

Ki67:

POS in ~100% of tumor nuclei

Slide8

BCL-6:

POS

CD10:

POS

Mum1:

NEG

CD30:

NEG

Slide9

BCL-2 (SP66):

POS

BCL-2 (clone 124):

NEG

CD5:

NEG

C-MYC:

POS

Slide10

Immunophenotype: SummaryPositive

: CD20, Pax5, CD10, BCL-6,

C-MYC, BCL-2 (SP66).Negative: TdT, Mum1, CD30, CD5, BCL-2 (clone 124)

Ki67

is expressed in essentially 100% of the tumor

cells.

Slide11

Cytogenetic FindingsKaryotype analysisUnsuccessful

due to an insufficient number of metaphase

cellsLikely due to the extensive tumor necrosis in specimen.

Slide12

LSI BCL2 Dual Color, Break Apart Rearrangement Probe

BCL2 rearrangement,

5’BCL2 loss, 3’BCL2 gain

1F2G

(74% nuclei)

BCL2 translocation,

5’BCL2 gain

1F3R1G (18% nuclei)

Result

:

Slide13

LSI MYC Dual Color Break Apart Rearrangement Probe

Result

:

Normal signal pattern

Slide14

To resolve

this discrepancy, the case

was analyzed using

chromosomal microarray analysis (CMA)

CMA - Methodology

High resolution method which detects submicroscopic

deletions

and

duplications

.

Limitations include an inability to detect balanced translocations or low level mosaicism (<20%).

MYC Discrepancy (

IHC+

,

FISH-

)

IHC:

POS

FISH:

NEG

Slide15

CMA Results

Array Karyogram

Numerous complex chromosomal abnormalities were detected involving multiple chromosomes

Slide16

CMA ResultsChromosome 11

No copy number variations

Helped exclude this case being a high-grade B-cell lymphoma with recurrent abnormality in 11q [reference]

Salaverria

I,

et al;

A

recurrent 11q aberration pattern characterizes a subset of MYC-negative high-grade B-cell lymphomas resembling Burkitt lymphoma

.

Blood

. 2014 Feb 20;123(8):

1187-98.

Slide17

CMA Results

Chromosome 8

Numerous copy number losses and gains consistent with a

chromothripsis

event

p-arm

q-arm

Slide18

Chromothripsis“Chromo”

=

Chromosome + “Thrypsis” = shattering into piecesSingle catastrophic event shatters a chromosome into tens/hundreds of fragments.

DNA-repair machinery reassembles some of the fragments incorrectly leading to multiple rearrangements and lost genetic material

.

Tubio

JM,

Estivill

X. Cancer:

When catastrophe strikes a cell

.

Nature

.

2011. Feb

24;470(7335):476-7

.

Stephens

PJ,

et al.

Massive genomic rearrangement acquired in a single catastrophic event during cancer development

.

Cell

. 2011 Jan 7;144(1):

27-40

.

Slide19

CMA results

MYC region (8q24.2) of chromosome 8

~180

kb two copy number gain (tetrasomy) at 8q24.21 associated with the MYC locus.

This duplication occurred as the result of the

chromothripsis

event and likely

caused deregulation of MYC gene

expression

Slide20

BCL2 region (18q21.22)Duplication of the 18q21.22 region associated with the BCL2 locus

A finding likely associated with the BCL2 rearrangement confirmed by FISH analysis.

CMA Results

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Cytogenetic Findings: SummaryKaryotype

Unsuccessful (extensive necrosis)

FISHMYC Dual Color Break Apart ProbesNo MYC rearrangement (normal signal pattern)BCL2 Dual Color Break Apart ProbesBCL2 translocation with gain of 5’BCL2 (74% cells)BCL2 rearrangement with gain of 3’ BCL2 and loss of

5’BCL2

(18% cells)

CMA

Chromothripsis event on chromosome 8

 ~180 kb two copy number gain (tetrasomy) at 8q24.21 associated with the MYC locus.Duplication of the 18q21.22 region associated with the BCL2 locusGain of chromosome 12Copy neutral loss of heterozygosity affecting the whole chromosome 13

23Mb

copy number loss (deletion) at 17p13.3p11.1 resulting in loss of TP53

gene

Slide22

Interesting Features of the CaseThis is a

case

of an unusually aggressive B-cell lymphoma with significant disease progression through chemotherapy.While MYC protein was overexpressed by IHC, FISH analysis was negative for a MYC rearrangement. Additional CMA analysis showed MYC gene over-activation as the result of a chromothripsis event on chromosome 8.BCL2 was initially negative

by IHC (Dako clone 124

), but positive using the SP66 clone. FISH

analysis

and CMA confirmed

BCL2 over-activation as the result of both increased copy number and translocation.

Overall, the combined results of IHC, FISH, and CMA provide evidence for this case having “double-hit” genetics, helping to explain the unusually aggressive clinical course.Conventional karyotyping and FISH analysis may not always reveal significant aberrations of MYC and BCL2.CMA can be useful in identifying key genetic alterations in unusual cases when conventional methods fail.Chromothripsis is a novel mechanism of gene alterations which is increasingly being recognized in lymphoma.

Chromothripsis

has been reported mechanism in 1 case of CLL [

1],

1 case of Burkitt lymphoma [2], plasma cell myeloma [3], DLBCL [4], and in 3 classical Hodgkin lymphoma cell lines [3,5].

Still,

chromothripsis

in lymphoma has not been significantly investigated, and more cases

need to be

examined to determine if a proportion of highly aggressive lymphomas exist with submicroscopic alterations in MYC, BCL2 or BCL6.

Stephens

PJ, et al. Massive genomic rearrangement acquired in a single catastrophic event during cancer development. Cell. 2011 Jan 7;144(1):27-40.

Sarova

I, et al. Jumping-like translocation-a rare chromosomal rearrangement in a patient with Burkitt lymphoma/leukemia. Cancer Genet. 2014 May;207(5):221-5.

Nagel

S, et al.

Chromothripsis

in Hodgkin lymphoma. Genes Chromosomes Cancer.

2013 Aug;52(8

):741-7.

Morin

RD, et al. Mutational and structural analysis of diffuse large B-cell lymphoma using whole-genome sequencing. Blood. 2013 Aug 15;122(7):1256-65.

Krem

MM, Horwitz MS. Mitotic errors, aneuploidy and micronuclei in Hodgkin lymphoma pathogenesis.

Commun Integr Biol. 2013 May 1;6(3):e23544.

Slide23

Proposed DiagnosisB-cell lymphoma, unclassifiable with features intermediate between diffuse large B-cell lymphoma and

Burkitt lymphoma

(BCLU-DLBCL/BL) with evidence of double-hit genetic alterations in MYC and BCL2

Slide24

Category for WorkshopDe novo large B-cell lymphomas with molecular features suggesting more aggressive behavior

Slide25

Clinical Follow-UpReceived 1 cycle of RICE salvage

without an objective

response.Switched to HYPER-CVAD with clinical improvement and continued with MTX/AraC every 3 weeks the goal of proceeding to hematopoietic stem cell transplant.However, after 2 cycles of MTX/

AraC

, the patient developed pelvic pain and was found to have new masses in the left

pelvis.

The patient is receiving radiotherapy and is being considered for clinical trials versus palliative management with lenalidomide/rituximab.

PET/CT scan after RICE, HYPER-CVAD, and MTX/

AraC