Patricia Aoun MD, MPH

Professor and Vice-Chair for Clinical Affairs

Medical Director, Clinical Laboratories

Department of Pathology

City of Hope National Medical Center

Disclosures

• I have no disclosures and no conflicts of interest.

Diagnosis of Lymphoma in the Era of Precision Medicine

Morphological Assessment

(Pattern Recognition)

Atypical

Immunophenotyping, including for therapeutic targets

(CD20, CD30, CD38,CD138, ALK, PD-1, PDL-1, etc.)

Malignant

“Histological” Subtyping

Molecular Genetic Testing

Clonality

Classification

Therapeutic targets

Disease monitoring

Benign

The Landscape of Genetic Alterations in Lymphoma

• Immunoglobulin heavy and light chain gene rearrangements

• T-cell receptor gamma and beta chain gene rearrangements by PCR with BIOMED-2 primers

• Gene fusions (many)

• Gene mutations: single or multiple nucleotide variations, insertions, deletions, amplifications, copy number alterations (thousands…)

A Few Examples…

Non-Hodgkin Lymphoma Subtype Genetic Alterations

Diffuse Large B-cell Lymphoma Translocations: BCL2, BCL6, CMYC

Mutations: ARID1A, ARID1B, BCL6, CARD11,

CD79B, CREBPP/EP300, EZH2, KDM2,

KDM6A, KDM6B, MLL2, MLL3, MYD88,

SETD2, SGKI, SMARCA, SMARCB, SMARC,

TP53, and many more….

Chronic Lymphocytic Leukemia Aberrations: del 17p, del 11q, +12, del 13q

Mutations: ATM, DDX3X, IDH1, NOTCH1,

SF3B1, TP53, and many more….

Burkitt Lymphoma Translocations: CMYC

Mutations: DDX3X,GNA13, ID3,MKI67,

PDCD11, SMARCA4, TCF3, TP53, and many

more….

PTCL Mutations: DNMT3A, ETV6, IDH2, JAK2, JAK3,

NOTCH1, RHOA, STAT3, STAT5, TET2, and

many more….

Making Sense of the Landscape of Genetic Alterations in Lymphoma

• Cancer (lymphoma) is the result of

– Gain of function mutations in oncogenes, and

– Loss of function mutations in tumor suppressor genes.

• But, not all mutations matter.

– Driver mutations, passenger mutations, backseat driver mutations

• And, the cancer (lymphoma) genome is always changing.

– Primary and metastatic tumors have different mutations.

– Within the same tumor, there is genomic heterogeneity.

Making Sense of the Landscape of Genetic Alterations in Lymphoma

• Clinically relevant mutations are actionable:

– Diagnostic

– Prognostic

– Predictive

• On the other hand, our understanding of specific mutations is constantly changing.

2011: Case 1: 55 year-old man with acute onset of hip pain

• Imaging

– XRAY: Bony abnormality at obturator foramen

– CT: Incomplete fracture of femoral neck, c/w pathological fracture

• Normal CBC and peripheral blood smear

• Normal LDH, BUN/Creatinine

• Elevated Alkaline Phosphatase

• Normal total protein with low albumin

– Quantitative immunoglobulins: Normal

– Serum protein electrophoresis: Acute phase reaction

– Immunofixation electrophoresis: Negative

Case 1: Femoral head fragments

Case 1: Bone Marrow- No morphological,

immunophenotypic or cytogenetic abnormality.

Case 1: Femoral head fragments

• TRAP and DBA44 expression:

– Characteristic of Hairy cell leukemia (HCL) but not 100% specific

• Cyclin D1:

– Hairy cell leukemia, mantle cell lymphoma, myeloma

• CD10 expression (Jasionowski et al, AJCP 2003):

– Described in about 10% of HCL cases, all BCL-6 negative

• BCL-6 expression:

– Usually negative in HCL and therefore significance unclear.

• Next steps??

Tiacci et al, N Engl J Med 2011; 364:2305-15

• Whole exome sequencing of enriched, purified leukemic and matched normal peripheral blood cells from index patient with HCL

• Sanger sequencing of 47 additional patients with HCL

• Identified 5 missense clonal somatic mutations, including a heterozygous mutation=>

BRAF V600E variant protein

• BRAF V600E not present in 195 patients with other peripheral blood lymphomas/leukemias

Case 1: BRAF mutation analysis by pyrosequencing

2015 Case 2: 3-year old boy with skin nodules

Case 2: Bone Marrow

Case 2: Clinical course

• Failed multiple conventional therapies.

• Underwent hematopoietic stem cell transplantation.

• Relapsed with high burden of disease.

• Further therapeutic options?

COH Clinical Molecular Diagnostics Laboratory Comprehensive tumor testing by Next Generation Sequencing

• Onco Gene Mutation Panel

– Targets >2800 mutations in 49 key cancer genes

ABL1 AKT1 ALK APC ATM BRAF

CDH1 CDKN2A CSF1R CTNNB1 EGFR ERBB2

ERBB4 EZH2 FBXW7 FGFR1 FGFR2 FGFR3

GNA11 GNAQ GNAS HNF1A HRAS IDH1

IDH2 JAK2 JAK3 KDR KIT KRAS

MET MLH1 MPL NOTCH1 NPM1 NRAS

PDGFRA PIK3CA PTEN PTPN11 RB1 RET

SMAD4 SSMARCB1 SMO SRC STK11 TP53

VHL

Case 2: BRAF mutation testing of skin biopsy

• BRAF gene analysis by Next Generation Sequencing (NGS) – 77 mutations within exons 11 and 15 – Genomic alteration identified in skin biopsy:

• c1799T>A; p. V600E

EGFR Signaling pathway:

KRAS, NRAS, BRAF, PIK3CA, PTEN

BRAF mutations are driver mutations

Vemurafenib

“Molecular diagnostics enable precision medicine”

Clayton Christensen, Harvard Business School 2008

2011National Research Council Task Force:

Toward Precision Medicine

• Is it possible to

develop a new

taxonomy of human

disease by

classifying diseases

based on molecular

information instead

of by groups of

symptoms or organ

sites?

“Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations” Hyman et al, N Engl J Med 2015;373:726-36

• Histology-independent early phase 2 ”basket” trial • 122 patients from 23 centers in specific cancer cohorts

– Langerhans Cell Histiocytosis/Erdman-Chester Disease – Non-small Cell Lung Carcinoma – Colorectal Carcinoma

• Vemurafenib monotherapy • Vemurafenib + Cetuximab (anti-EGFR agent)

– Cholangiocarcinoma – Anaplastic Thyroid Carcinoma – Others: Primary Brain Tumors, Multiple Myeloma, Ovarian,

Breast, Salivary Duct, Pancreatic, Clear Cell Sarcoma, Unknown Primary

• Excluded: Melanoma, Papillary Thyroid Carcinoma, Leukemia, Lymphoma

• Primary Endpoint: Response at week 8

Vemurafenib in Multiple Nonmelanoma Cancers with BRAF V600 Mutations Hyman et al, N Engl J Med 2015; 373:726-36

Response Complete

Response

Partial

Response

Stable

Disease

Progressive

Disease

LCH/ECD

(N=14)

7% 36% 57% 0%

NSCLC

(N=19)

0% 42% 42% 11%

CRC MonoRx

(N=10)

0% 0% 50% 50%

CRC ComboRx

(N=26)

0% 4% 69% 27%

Cholangio CA

(N=8)

0% 12% 50% 38%

Anaplastic Thyroid

(N=7)

14% 14% 0% 57%

Precision Medicine: Towards a New Molecular Taxonomy of Cancer?

• Not all BRAF-mutated tumor types respond to BRAF-targeted therapy.

• Histology still matters.

• In 2016, histology with molecular subtypes matters most.

• But, the medical community has no practice standards for which genes to test in order to assign molecular subtypes.

Molecular

Diagnostics

Lab

Results Specimens

What Test Do I Order ?????

Developing Practice Standards for Genomic Testing

• Number of genes/panel: 6-408

• Total number of genes across all panels: 611

• Number of genes present in EVERY panel: 0

• 54 genes were listed in 3 panels

• 43 genes were listed in 4 panels

• 393 genes were listed in only 1 or 2 panels

Foundation Heme ™

• Entire coding sequence of 405 genes

– Base substitutions, indels, copy number alterations

• Selected introns of 31 genes involved in rearrangements (gene fusions)

• RNA sequencing of 265 genes involved in hematological malignancies, sarcoma, and pediatric cancers

• List Price = $7200

2016 Development Pipeline: Hematological Malignancies NGS 74 gene mutation panel

ASXL1 CCND1 EP300 IDH1 KMT2D NRAS SF3B1 WHSC1L1

ATM CCND3 ETV6 IDH2 KRAS PAX5 SPI1 WT1

BCOR CD79A EZH2 IL7R MEF2B PDGFRA SRSF2 ZRSR2

BIRC3 CD79B FBXW7 JAK1 MIR142 PHF6 STAG2

BRAF CDKN2A FLT3 JAK2 MPL PIK3CA SUZ12

BTK CEBPA GATA1 JAK3 MYC PTEN TCF3

CALR CHEK2 GATA2 KDM6A MYD88 PTPN1 TET2

CARD11 CREBBP HRAS KIT NOTCH1 PTPN11 TP53

CBL CSF3R ID3 KMT2A NOTCH2 RUNX1 U2AF1

CBLB DNMT3A IKZF1 KMT2C NPM1 SETBP1 WHSC1

2016 Development Pipeline: Hematological Malignancies Fusion Panel by NGS

ABL1 ABL2 ALK BCR CBFB

CRLF2 CSFIR FGFR1 JAK2 KMT2A

MECOM MKL1 NOTCH1 NUP214 PDGFRA

PDGFRB PICALM RARA RBM15 RUNX1

RUNX1T1 TAL1 TCF3

Developing Practice Standards for Genomic Testing

• Remember, clinically relevant mutations are actionable: – Diagnostic, Prognostic, Predictive

• Single gene tests: – Still useful for diagnosis, prognosis, or prediction of therapeutic

response when applied in the context of morphological findings. • Targeted gene panels:

– Focus on clinically relevant mutations. – Allow for greater depth of sequencing to detect mutations found

at low frequencies. • Large gene panels:

– Rare tumors for which the driver mutations are unknown. – Common tumors that have been shown not to have the usual

mutations. – Relapsed tumors not responsive to standard of care therapies

Leveraging Tumor Mutation Profiles to Monitor

Patients During/After Therapy: “Liquid Biopsies”

• Peripheral blood:

– Quantitative allele –specific,

real-time polymerase chain

reaction test for the BRAF

V600E mutation

– Lower limit of detection

0.02% in whole blood

• Urine

– Qualitative droplet digital

PCR assay for cell free

DNA

– Lower limit of detection

0.03%

Leveraging Tumor Mutation Profiles to Monitor Patients During and After Therapy

• Next Generation Sequencing of B and T receptor genes in lymphoma and leukemia – Identification of multiple clones when present – Subsequent testing of peripheral blood for circulating tumor DNA to

detect minimal residual disease or early relapse – Lower limit of detection 0.0001% (1 in a million)

• Studied in – Diffuse large B-cell lymphoma – Mantle cell lymphoma – Chronic lymphocytic leukemia – Acute lymphoblastic leukemia – Multiple myeloma – Cutaneous T-cell lymphoma – Classical Hodgkin Lymphoma

• Commercially available as clonoSEQ ID and clonoSEQ MRD assays for circulating tumor DNA

Leveraging Tumor Mutation Profiles to Monitor Patients During and After Therapy

• The medical community has few practice standards for monitoring patients with “liquid biopsies.”

• Which test and how often?

• Outside of a Clinical Trial?

• How much does it cost?

– Peripheral blood BRAF quantitative PCR assay: $890

– Urine cf DNA BRAF assay: $1495

– Initial tumor profiling, clonoSEQ ID $1200

– Peripheral blood monitoring, clonoSEQ MRD $1300

Molecular Testing in the Era of Precision Medicine Lymphoma and Other Malignancies

• Understand and acknowledge the goal of performing each test for each patient each time. – Clinical utility (drive patient care) – Discovery (research) – Both

• Decide up front when, why, and how often testing will be performed. – Consider the cost and the “return on the investment.”

• Select tests and reference labs thoughtfully. – The rate of genomic discovery and test design is much

faster than the rate of clinical utility. – Molecular testing for hematological malignancies (and all

cancer) is about balancing discovery with clinical utility and cost.

COH Clinical Molecular Diagnostics Laboratory