genomics tumor board. molecular laboratory molecular pathology director: frederick nolte, ph.d....
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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
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