Specialized Diagnostics Unit

ImmunohistochemistryMolecular Diagnostics

Lymphoma Pathogenesis

Mark Raffeld, MD

Primary Research Area(s)

• What are your expertise? • Immunohistochemisty • Molecular Diagnostics, FFPE approaches, clonality assays, cancer gene

mutations• Lymphoma pathogenesis• Lung cancer pathogenesis• Melanoma pathogenesis• Glioblastoma pathogenesis

• What technologies / approaches / methodologies do you utilize in your research area?• Immunohistochemistry – test panels• PCR/CE bases antigen receptor rearrangement studies, insertions,

deletions• RT-PCR for fusion transcripts and qRT-PCR (ABI, Fluidigm)• Enrichment for minor molecular populations, COLD PCR, clamped PCR,

LCM • Tumor signatures on Nanostring platform• Sequencing- pyrosequencing• Promoter methylation analysis• Next Generation Sequencing, amplicon resequencing, genomic

bioinformatic approaches

Research Implications• How does your research fit in the broader context?

– How does it fit into LP and broader areas?

• The technologies being developed and implemented in the laboratory are broadly applicable to translational research into the pathogenesis of disease

• My work in lymphoma pathogenesis both supports and complements work and expertise of the Hematopathology section of LP

– How does your research translate into potential clinical applications?

• Molecular test development and antibody assay development goals are designed to support NCI/NIH clinical protocols

• Identification of molecular and phenotypic abnormalities in lymphomas lead to better diagnostics and potentially new treatment modalities

Example: Molecular Lab Development

NGS Nanostring panels

LCM

Amplificationmaximizing

• tumor enrichment

• allele enrichment• ciculating t-DNA detection• nanoreactions

Discovery

Translational research

Clinical laboratory testing

Molecular Lab Core Space

Regulation

Sample maximizing Multiplexing technologies • Sequenom• Snapshot• Pyroseq

Example: Molecular Lab Development

ION TORRENT PGM

316 Chip, 6,348,236 wells

ABL1 EZH2 JAK3 PTENAKT1 FBXW& IDH2 PTPN11ALK FGFR1 KDR RB1APC FGFR2 KIT RETATM FGFR3 KRAS SMAD4BRAF FLT3 MET SMARCB1CDH1 GNA11 MLH1 SMOCDKN2A GNAS MPL SRCCSF1R GNAQ NOTCH1 STK11CTNNB! HNF1A NPM1 TP53EGFR HRAS NRAS VHLERBB2 IDH1 PDGFRA  ERBB4 JAK2 PIK3CA  

AMPLISEQ CANCER PANEL V.2

Example: Molecular Lab Development

10

100

1000

10000

CHP2_PIK3CA

_5CH

P2_RB1_9CH

P2_ATM

_11CH

P2_JAK3_1

CHP2_CD

KN2A

_2CH

P2_APC_7

CHP2_SM

AD

4_7CH

P2_ATM

_2CH

P2_FGFR3_3

CHP2_JA

K3_3CH

P2_SMA

D4_2

CHP2_G

NA

S_1CH

P2_NO

TCH1_1

CHP2_RB1_2

CHP2_TP53_2

CHP2_H

RAS_1

CHP2_STK11_4

CHP2_FG

FR3_2CH

P2_FGFR3_5

CHP2_RB1_1

CHP2_G

NA

11_1CH

P2_ATM

_15CH

P2_RB1_7CH

P2_ATM

_1CH

P2_VH

L_1CH

P2_ERBB4_1CH

P2_STK11_3CH

P2_EGFR_6

CHP2_PIK3CA

_2CH

P2_ATM

_9CH

P2_HRA

S_2CH

P2_STK11_5CH

P2_NO

TCH1_3

CHP2_TP53_7

CHP2_FLT3_4

CHP2_FG

FR1_1CH

P2_FGFR3_1

CHP2_FG

FR3_4CH

P2_ATM

_13CH

P2_TP53_8CH

P2_RET_5CH

P2_TP53_4CH

P2_RB1_8CH

P2_STK11_2CH

P2_SMA

RCB1_4CH

P2_PTEN_2

CHP2_TP53_5

CHP2_SM

O_3

CHP2_A

LK_2CH

P2_CDKN

2A_1

CHP2_PIK3CA

_8CH

P2_SMO

_4CH

P2_KIT_5CH

P2_FBXW7_2

CHP2_A

TM_14

CHP2_A

LK_1CH

P2_RB1_6CH

P2_SMO

_5CH

P2_TP53_3CH

P2_FLT3_2CH

P2_STK11_1CH

P2_IDH

2_1CH

P2_RB1_4CH

P2_KIT_6CH

P2_BRAF_2

CHP2_FG

FR2_1CH

P2_ERBB2_2CH

P2_CDH

1_3CH

P2_PIK3CA_10

CHP2_V

HL_3

CHP2_RET_4

CHP2_TP53_1

CHP2_KRA

S_3CH

P2_PIK3CA_4

CHP2_SM

ARCB1_2

CHP2_RB1_10

CHP2_FLT3_3

CHP2_RET_3

CHP2_KD

R_1CH

P2_HN

F1A_1

CHP2_N

OTCH

1_2CH

P2_FGFR1_2

CHP2_N

RAS_1

CHP2_A

TM_3

CHP2_RET_1

CHP2_SM

ARCB1_3

CHP2_M

PL_1CH

P2_AKT1_1

CHP2_ERBB2_3

CHP2_SRC_1

CHP2_RET_2

CHP2_PTEN

_6CH

P2_ERBB4_8CH

P2_MET_5

CHP2_A

TM_5

CHP2_ERBB4_5

CHP2_TP53_6

CHP2_A

BL1_3CH

P2_CTNN

B1_1CH

P2_HN

F1A_2

CHP2_M

ET_2CH

P2_KDR_9

CHP2_SM

ARCB1_1

CHP2_N

PM1_1

CHP2_FG

FR2_4CH

P2_ATM

_10CH

P2_KDR_2

CHP2_A

BL1_4CH

P2_RB1_5CH

P2_CSF1R_1CH

P2_GN

AQ

_1CH

P2_PTEN_8

CHP2_PIK3CA

_7CH

P2_EGFR_8

CHP2_PTPN

11_2CH

P2_KDR_8

CHP2_PIK3CA

_1CH

P2_RB1_3CH

P2_ABL1_2

CHP2_ERBB4_4

CHP2_PD

GFRA

_1CH

P2_KRAS_1

CHP2_A

TM_17

CHP2_V

HL_2

CHP2_N

RAS_3

CHP2_ERBB2_1

CHP2_SM

AD

4_4CH

P2_MLH

1_1CH

P2_BRAF_1

CHP2_SM

O_1

CHP2_FLT3_1

CHP2_A

PC_3CH

P2_CSF1R_2CH

P2_ATM

_6CH

P2_PTEN_4

CHP2_A

KT1_2CH

P2_ABL1_1

CHP2_PD

GFRA

_2CH

P2_PTEN_3

CHP2_JA

K2_1CH

P2_FBXW7_1

CHP2_EG

FR_4CH

P2_KIT_2CH

P2_ATM

_12CH

P2_GN

AS_2

CHP2_PTEN

_1CH

P2_SMA

D4_1

CHP2_PTEN

_5CH

P2_KDR_3

CHP2_SM

O_2

CHP2_SM

AD

4_5CH

P2_PDG

FRA_3

CHP2_EZH

2_1CH

P2_APC_5

CHP2_PIK3CA

_3CH

P2_FGFR2_2

CHP2_SM

AD

4_9CH

P2_APC_6

CHP2_PIK3CA

_6CH

P2_CDH

1_2CH

P2_SMA

D4_3

CHP2_ERBB4_2

CHP2_JA

K3_2CH

P2_SMA

D4_6

CHP2_CD

H1_1

CHP2_FG

FR2_3CH

P2_EGFR_2

CHP2_PTPN

11_1CH

P2_ATM

_8CH

P2_FBXW7_3

CHP2_KD

R_4CH

P2_SMA

D4_8

CHP2_M

ET_3CH

P2_ERBB4_3CH

P2_PDG

FRA_4

CHP2_A

PC_1CH

P2_PTEN_7

CHP2_KD

R_6CH

P2_ATM

_7CH

P2_APC_4

CHP2_N

RAS_2

CHP2_FBXW

7_5CH

P2_ATM

_4CH

P2_MET_6

CHP2_EG

FR_3CH

P2_EGFR_5

CHP2_KIT_9

CHP2_EG

FR_7CH

P2_KIT_8CH

P2_ERBB4_7CH

P2_KIT_1CH

P2_MET_1

CHP2_ERBB4_6

CHP2_KD

R_5CH

P2_KDR_7

CHP2_KIT_4

CHP2_EG

FR_1CH

P2_ATM

_16CH

P2_MET_4

CHP2_ID

H1_1

CHP2_KRA

S_2CH

P2_KIT_3CH

P2_APC_2

CHP2_PIK3CA

_11CH

P2_KIT_7CH

P2_PIK3CA_9

CHP2_FBXW

7_4

Cove

rage

AmpliSeq CHPv2 Amplicons

Q1

MIN Cov.

MEDIAN Cov.

MAX Cov.

Q3

Box-plot of Coverages of 207 AmpliSeq CHPv2 Amplicons in 34 FFPE DNA SamplesAMPLICON COVERAGE ON 316 CHIP

INTER AND INTRA LAB REPRODUCIBILITY

R² = 0.8712

0

10

20

30

40

50

60

70

80

90

100

0 10 20 30 40 50 60 70 80 90 100

Alle

le Q

uanti

ficati

on -

Pyro

sequ

ence

(%)

Variant Frequency - Ion variantCaller (%)

ACCURACY OF NGS VARIANT QUANTITATION

Future Direction • Where do you see your research going in the next 5-10

years?– Continued development of automated solutions in specialized diagnostics– Miniaturization of assays, provide more answers from less tissue– Signatures for diagnosis, prediction, prognosis– Circulating tumor specific nucleic acid markers– Development of IHC panels to functional and immunogenic protein

targets to assist in identifying personalized therapies. – Development of highly multiplexed IHC technologies– Application of technologies to lymphoma pathogenesis and diagnosis

• What are the current challenges & obstacles? – Inconsistent involvement of pathology in clinical protocol development – Institutional focus on small PI dependent clinical protocols, rather than a

programmatic commitment to specific cancer areas– Expectations to develop protocol specific assays without necessary

resource allocations.– Fragmentation and duplication of institutional resources in molecular

pathology– Inadequate protected research time

CollaboratorsWho do you consider your primary collaborators & partnerships?

•Drs. Pittaluga and Jaffe- Hematopathology•Rosenberg- Surgery Branch•Rajan, Hassan- Thoracic oncology group•Buck, Tangrea, Hipp- LCM•Kreitman- HCL•Kreisl- Neuro-oncology •Other LP: Barr, Bachelor, Pack, Stetler- Stevenson, Merino, Filie, Roth, Hewitt, Miettinen, •Other NCI/NIH: Wilson, Staudt, Apolo, Klion, Holland, Meltzer, Kraemer