Genomics Tumor Board

Molecular LaboratoryMolecular Pathology Director: Frederick Nolte, Ph.D.Cytogenetics and Molecular Genetics Director: Daynna Wolff, Ph.D.Medical Director: Cynthia Schandl, M.D., Ph.D.Associate Director: Julie Woolworth Hirschhorn, Ph.D.

Cancer Genetics

Tumors can be complex Genetic and genomic information can help with:

Diagnosis Prognosis Therapeutic Decisions Disease Monitoring Characterization of inherited variation contributing to cancer susceptibility

Normal

Premalignant

Cancer In situ

Metastatic

1st hit

Current Offerings

Cancer MicroarrayFISH testingMassively Parallel Sequencing

MUSC Test Directory and Specimen Collection Information

http://pathology.musc.edu/or

https://www.testmenu.com/musclabservices

Current Testing:

Cancer Microarray

How can we use microarrays in clinical cancer studies?

Diagnosis Renal cell carcinoma Glioblastoma

Prognosis/Disease monitor Chronic lymphocytic leukemia Acute myeloid leukemia/MDS Plasma cell dysplasias Renal cell carcinoma Glioblastoma

Therapy Acute lymphoblastic leukemia Acute myeloid leukemia

Genome Biology 2010, 11:R82

Why is Copy Number So Important?

Copy number variants comprise at least 3X total number SNPs

On average, 2 human differ by 4 – 24 Mb of DNA by CNV; 2.5 Mb due to SNP

Often encompass genes Important role in human disease and in drug response

Copy number loss Copy number gainWhole genePartial gene

Contiguous genesRegulatory sequences

Current Testing:

26-Gene Solid Tumor Cancer Panel

Targeted resequencing panel based on PCR amplification

Batched once per week, start date on Monday Turn-around-time of 7-11 days Mixture of hotspot and full exon coverage

Future Testing for Solid Tumors

In process of validating a 50-gene solid tumor panel Will include all of the genes currently covered

ABL1 EGFR GNAQ KRAS PTPN11

AKT1 ERBB2 GNAS MET RB1

ALK ERBB4 HNF1A MLH1 RET

APC EZH2 HRAS MPL SMAD4

ATM FBXW7 IDH1 NOTCH1 SMARCB1

BRAF FGFR1 IDH2 NPM1 SMO

CDH1 FGFR2 JAK2 NRAS SRC

CDKN2A FGFR3 JAK3 PDGFRA STK11

CSF1R FLT3 KDR PIK3CA TP53

CTNNB1 GNA11 KIT PTEN VHL

Future Testing for Hematological Malignancies

In process of validation of myeloid panel of 49-genes by next-generation sequencing

ASXL1 BCOR BCOR1 BRAF CALR CBL CBLB

CEBPA CSF3R DNMT3A ETV6 EZH2 FLT3 GATA1

GATA2 GNAS HRAS IDH1 IDH2 JAK1 JAK2

JAK3 KDM6A KIT KMT2A/ MLL-PTD KRAS MEK1 MPL

MYD88 NOTCH1 NPM1 NRAS PHF6 PML PTEN

PTPN11 RAD21 RUNX1 SETBP1 SF3B1 SMC1A SMC3

SRSF2 STAG2 TET2 TP53 U2AF1 WT1 ZRSR2

Solid Tumor TestingA Brief History

Solid Tumor Testing History

Molecular Laboratory performed targeted real-time PCR analysis of the EGFR, KRAS, and BRAF genes from late June of 2011 til January of 2014

In Feb of 2014, we began offering a 26-gene solid tumor cancer panel

2012 2013 2014 2015

191209

224

266

118 117103

133

54 564236

38 44

62

0 0 2129

Molecular Testing Volumes for Solid Tumors

Total Cases Lung Melanoma Colorectal Other

2014

Langerhans

GISTs

Thyroid

HCC Trial

Other

Lung

Melanoma

Colon

Unable to Process

0 20 40 60 80 100 120

1

2

1

14

3

96

54

43

15

2014 TruSight Tumor Solid Cancer Panel Analysis By Tumor Type

2015

Langerhans

GISTs

Thyroid

HCC Trial

Other

Lung

Melanoma

Colon

Unable to Process

0 20 40 60 80 100 120 140 160

1

1

1

9

19

141

41

51

35

2015 TruSight Tumor Solid Cancer Panel Analysis By Tumor Type

• During 2015, we started creating final reports for cases with insufficient tissue

43%

46%

11%

2015 Insufficient Samples

TissueDNAQuality Control failure

• 43% of insufficient specimens were due to insufficient tissue prior to any molecular analysis

2014 Positivity by Tumor Type

BRAF, 18, 5.9%

APC, 24, 25.3%

KRAS, 35, 22.9%

KRAS, 21, 22.1%

EGFR,10,

6.5%

NRAS10

11.8%

PIK3CA6

6.3%

TP53, 21, 24.7%

TP53, 30, 31.6%

TP53, 62, 40.5%BRAF

63.9%

KIT, 2, 2.4%

MET6

7.1%

MET8

5.2%

2015 Positivity by Tumor Type

TP53, 86, 56.2%

TP53, 41, 43.2%

TP53 9

10.6%

BRAF1618.8

%

KIT5

5.9%

KRAS27

28.4%

KRAS46

30.1%

APC41

43.2%

EGFR28

18.3%

NRAS5

5.9%

PIK3CA9

9.5%

AKT12

3.6%

BRAF, 4, 4.2%

SKT1116

10.5%

BRAF, 5, 3.3%

Frequency of Variant Classification

Classification 1 – Clinically Actionable / May indicate specific therapeutic interventionClassification 2 – Reported in the literature / Possible clinical relevanceClassification 3 – Variant of unknown clinical significance

2014 2015127

60

276240

18

113

Original Intent of the Tumor Board

Meeting on the Second Monday of each month at 4 pm in HCC 120

Focus on clinical cases with molecular genetic analyses

Global review of molecular cases analyzed in the previous month

In-depth case discussions (up to 4 cases each month) For example, cases may be selected because an

actionable mutation that was unexpected in a particular tumor type was identified or there was informative referral lab testing information available.

This meeting will also provide a forum to discuss advanced diagnostic tests for cancer and any unmet local needs. 

Suggested Format of the Tumor Board

Global review of Molecular Testing from the previous month by Molecular laboratory

Cases identified by clinicians for Tumor Board Discussion

Cases sent for tumor board agenda Cases should be worked up by fellow(s) , with

assistance from oncologist and pathologist/laboratory director

Case presented by fellow(s) Summaries of these cases, including age, diagnosis,

tumor site, pathology, molecular pathology test results, and prior therapy will be distributed before the meeting

Additional items included on agenda might be future testing, new testing on the market

Standards Directive for Mutation Testing

PROPOSAL FOR CLINICAL PRACTICE STANDARDS DIRECTIVE PROTOCOL

This is a document that will allow mutation analysis to be ordered directly by the diagnosing pathologist if the given criteria are met.

This type of protocol must be accepted by the ordering physicians at the Hollings Cancer Center.

Example: All metastatic or unresectable melanoma; diagnostic specimen: Mutation status

should be assessed to allocate appropriate therapy. Mutation analysis must include BRAF V600E variant testing and KIT activating mutation testing as indicated by NCCN guidelines. Analysis of progressive, newly metastatic (after mutation analysis of the primary site), recurrent disease, and suspected acquired resistance testing will require physician order.

MUSC assay*: Cancer Panel Next Generation Sequencing Analysis Note: Once diagnosis is made, request for testing may be initiated by the resident /

fellow / attending pathologist. The block should accompany the stained slide to molecular pathology for testing. An ideal area for DNA extraction should be circled by the referring pathologist with an associated percent tumor indicated. If insufficient tissue is available on the block, the pathologist should determine whether another specimen (e.g. fine needle aspirate smear, metastatic site biopsy) is available for testing.

Three protocols have been submitted for approval by HCC –colorectal, NSCLC, and melanoma. Myeloid panel may also be submitted once available in-house

Genetic Complexity

• SimpleChronic Myeloid Leukemia

• ComplexMost solid tumors

Type Renal Tumor

% Chromosome Abnormality

Microarray Result

Clear Cell Renal Cell Carcinoma

~70 Loss of 3p

Papillary Renal Cell Carcinoma

10-20 Extra copies of 7 and 17

ChromophobeRenal Cell Carcinoma

5 Loss of chromosomes 1, 2, 6, 10, 13, 17, 21

Oncocytoma <5 Normal or loss of 1p

Diag

nost

ic st

udie

s

Prognostic Significance: Clear Cell Renal Cell Carcinoma

Clear Cell RCC with 3p-Clear Cell RCC with 3p- Plus other genetic abnormalities

Better prognosis Worse prognosis

Aberrations Associated with Adverse Prognosis Clear Cell Renal Cell Carcinoma

Percent with abnormality

** **

***

*

*

*

*

**

*

*

**

* P <0.5** P <0.01

Karyotype here

Prognosis/Therapy for Chronic Lymphocytic Leukemia (CLL)79 yr old male with new dx CLL; Flow 71% WBC; Cyto: deletion 11q21, +12[1/20]

60% del 13q14, 50% +12, 40% del 11q, 60% LOH 17q

Clonal Evolution: Patient more likely to need therapy or on therapy

Acute Myeloid Leukemia Prognosis

Standard Cytogenetic Testing 25% Good prognosis: balanced rearrangements

[t(8;21),t(15;17),inv(16)] 50% Intermediate prognosis: +8 (10%), NORMAL cytogenetics

(40%) 25% Unfavorable prognosis: deletions 5q, 7q, 17p, KMT2A

(MLL) rearrangement, complex karyotypes (>4 abn)

Microarray Analysis Provides exact breakpoints for known cytogenetic aberrations Reveals cryptic abnormalities Copy number neutral loss of heterozygosity (10-20% of cases of

normal cytogenetics cases; like LOH for 7q) LOH regions often harbor genes with homozygous mutations

LOH is associated with gene mutations

Region of LOH Associated Gene1p NRAS4q TET27q EZH29p JAK2, CDKN2A, PAX511p WT1, PAX611q CBL13q FLT317p TP5319q CEBPA21q RUNX1

Perc

ent r

elap

sePe

rcen

t rel

apse

Perc

ent s

urvi

val

Perc

ent s

urvi

val

cnLOH

cnLOH

control

control

No 13q cnLOH

No 13q cnLOH

13q cnLOH

13q cnLOH

P=0.02P=0.006

P=0.03P=0.04

HR 1.87

HR 3.45HR 6.64

HR 1.82

Relapse Survival

Importance of LOH in Prognosis in Acute Myeloid Leukemia and Myelodysplastic Syndromes

Min Fang, personal communication; Cancer. 2015 Sep 1;121(17):2900-8.

Among FLT3-ITD patients, 13qLOH associated with poor prognosis

P=0.01 P=0.01 n.s.

Min Fang, personal communication; Cancer. 2015 Sep 1;121(17):2900-8.

Diagnosis/Prognosis for Glioma

Loss 1p/19q: OligodendrogliomaSurvival >10 years*MA better than FISH

Gain7/Loss 10, amp 4q (PDGFRA,KIT):High grade glioblastoma; proneuralSurvival <1 year

*Need molecular assessment of IDH1/2 gene

Implication for therapy: 23 year old male with Philadelphia-like B-cell Acute Lymphoblastic Leukemia; Cytogenetics and FISH testing negative

8.6 Mb deletion of 5q32q33.3

Molecular Mechanism

EBF1 -PDGFRBActivates a tryrosine kinase that can be targeted by imatinib