Transcript Genomics Tumor Board

Genomics Tumor Board
Molecular Laboratory
Molecular 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.
Normal
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
1st hit
Premalignant
Cancer
In situ
Metastatic
Cancer Microarray
FISH testing
Massively Parallel Sequencing
Current
Offerings
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
 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
Why is Copy
Number So
Important?
Copy number loss
Copy number gain
Whole gene
Partial gene
Contiguous genes
Regulatory sequences
 Targeted resequencing panel based on PCR amplification
Current
Testing:
26-Gene Solid
Tumor Cancer
Panel
 Batched once per week, start date on Monday
 Turn-around-time of 7-11 days
 Mixture of hotspot and full exon coverage
 In process of validating a 50-gene solid tumor panel
 Will include all of the genes currently covered
Future Testing
for Solid
Tumors
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
 In process of validation of myeloid panel of 49-genes by nextgeneration sequencing
Future Testing
for
Hematological
Malignancies
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 Testing
A Brief 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
Solid Tumor
Testing History
Molecular Testing Volumes for Solid Tumors
266
224
209
191
133
118
117
103
56
54
62
42
29
36
0
38
0
2012
2013
Total Cases
Lung
44
21
2014
Melanoma
Colorectal
2015
Other
2015
2014
2015 TruSight Tumor Solid Cancer Panel Analysis
By Tumor Type
2014 TruSight Tumor Solid Cancer Panel Analysis By
Tumor Type
Unable to Process
Unable to Process
15
Colon
Colon
43
Melanoma
Lung
1
2
Langerhans
1
0
20
19
HCC Trial
14
GISTs
141
Other
3
Thyroid
41
Lung
96
HCC Trial
51
Melanoma
54
Other
35
40
60
80
100
120
9
Thyroid
1
GISTs
1
Langerhans
1
0
20
40
60
80
100
120
140
• During 2015, we started creating final reports for cases with insufficient tissue
160
2015 Insufficient Samples
Tissue
11%
43%
46%
DNA
Quality Control
failure
• 43% of insufficient specimens were due to insufficient tissue prior to any
molecular analysis
2014 Positivity by Tumor Type
KIT, 2, 2.4%
MET
6
7.1%
BRAF, 18, 5.9%
APC, 24, 25.3%
BRAF
6
3.9%
EGFR,
10,
6.5%
KRAS, 21, 22.1%
KRAS, 35, 22.9%
MET
8
5.2%
NRAS
10
11.8%
PIK3CA
6
6.3%
TP53, 21, 24.7%
TP53, 30, 31.6%
TP53, 62, 40.5%
2015 Positivity by Tumor Type
BRAF
16
18.8
%
AKT1
2
3.6%
KIT
5
5.9%
NRAS
5
5.9%
TP53
9
10.6%
BRAF, 4, 4.2%
KRAS
27
28.4
%
APC
41
43.2%
PIK3C
A
9
9.5%
TP53, 41, 43.2%
BRAF, 5, 3.3%
EGFR
28
18.3
%
KRAS
46
30.1%
SKT11
16
10.5%
TP53, 86, 56.2%
2014
Frequency of Variant Classification
2015
113
127
18
60
240
Classification 1 – Clinically Actionable / May indicate specific therapeutic intervention
Classification 2 – Reported in the literature / Possible clinical relevance
Classification 3 – Variant of unknown clinical significance
276
 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
Original Intent
of the Tumor
Board
 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.
 Global review of Molecular Testing from the previous month by
Molecular laboratory
 Cases identified by clinicians for Tumor Board Discussion
Suggested
Format of the
Tumor Board
 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
PROPOSAL FOR CLINICAL PRACTICE STANDARDS DIRECTIVE
PROTOCOL
Standards
Directive for
Mutation
Testing
 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
• Simple
Chronic Myeloid Leukemia
• Complex
Most solid tumors
Diagnostic studies
Type Renal Tumor
%
Chromosome
Abnormality
Clear Cell Renal Cell ~70
Carcinoma
Loss of 3p
Papillary Renal Cell
Carcinoma
10-20
Extra copies of 7
and 17
Chromophobe
Renal Cell
Carcinoma
5
Loss of
chromosomes 1,
2, 6, 10, 13, 17,
21
Oncocytoma
<5
Normal or loss
of 1p
Microarray Result
Prognostic Significance:
Clear Cell Renal Cell Carcinoma
Clear Cell RCC with 3p-
Better prognosis
Clear Cell RCC with 3pPlus other genetic abnormalities
Worse prognosis
Aberrations Associated with Adverse
Prognosis
Clear Cell Renal Cell Carcinoma
**
**
*
*
**
*
*
*
*
*
*
*
*
Percent with abnormality
*
* P <0.5
** P <0.01
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
Karyotype
here
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 Gene
1p
NRAS
4q
TET2
7q
EZH2
9p
JAK2, CDKN2A, PAX5
11p
WT1, PAX6
11q
CBL
13q
FLT3
17p
TP53
19q
CEBPA
21q
RUNX1
cnLOH
control
Percent survival
HR 1.87
Percent relapse
Importance of LOH in Prognosis in Acute Myeloid
Leukemia and Myelodysplastic Syndromes
13q cnLOH
No 13q cnLOH
P=0.006
Relapse
HR 1.82
cnLOH
P=0.03
Percent survival
HR 6.64
Percent relapse
P=0.04
control
No 13q cnLOH
13q cnLOH
P=0.02
Survival
Min Fang, personal communication; Cancer. 2015 Sep 1;121(17):2900-8.
HR 3.45
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: Oligodendroglioma
Survival >10 years*
MA better than FISH
*Need molecular assessment of IDH1/2 gene
Gain7/Loss 10, amp 4q (PDGFRA,KIT):
High grade glioblastoma; proneural
Survival <1 year
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
Activates a tryrosine kinase that can
be targeted by imatinib
EBF1 -PDGFRB