renal biopsy

1
IPAM 2 Diagnostic Applications in Cancer
Timothy Triche, MD, PhD Department of
Pathology Childrens Hospital Los Angeles
2
Goals for Microarrays in Cancer Studies

• Can Gene Expression Profiles detect differences
  in tumor types (eg, confirm diagnosis)?
• Can they detect atypical variants of these
  typical entities?
• Can they detect biologic/genetic variants
  otherwise undetectable (eg, do they add anything
  new)?
• Can they go beyond diagnosis?

3
RMS Histology
Embryonal Alveolar
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RMS Undifferentiated
5
RMS Muscle Protein Expression(Desmin)
6
RMS Minimal Criteria for Dx
Uncommitted Mesenchymal Cell
Myogenic Transcription Factors
1) Myf5 or MyoD
2) Myogenin
Committed Myoblast
3) MRF4
Transforming Event
Malignant Myoblast
muscle proteins (myosin, desmin)
7
RMS Gene Expression

• All RMSs express one or more myogenic TFs
• Some RMSs express only some myogenic TFs
• Non-RMS STSs express no myogenic TFs
• Therefore, minimum Dx of RMS is expression of
  even a single myogenic TF

Non-RMS ERMS ARMS
MYOD MYF5 MYOG MIC2
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MyoD Expression in RMS
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Clinical Classification ofChildhood Cancer

• Historical Morphologic diagnosis clinical data
  gt risk group, protocol eligibility, treatment
  (eg, group-based treatment)
• Current Combined (morphology, immunophenotype,
  genomic defect) gt patient-specific group-based
  treatment (eg, NMYC amplified NB patients)
• Future Patient-specific therapy, based on
  multi-genic phenotype?

10
Clinical Questions in Diagnosis

• What is it? (eg, Diagnosis)
• How will it behave? (eg, Prognosis)
• localized vs. disseminated
• High grade vs. indolent
• Treatment responsive or not
• What is the appropriate therapy for this patient?
  (eg, Protocol)

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Alveolar RMS Chromosomal Translocation
chr 2
chr 13

• t(213)gt Pax3/FKHR (55)
• t(113) gt Pax7/FKHR (22)
• 23 No known translocation!

n d
n d
n d
13q14
2q35
Data 78 IRS IV cases (Sorensen et al)
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Age Incidence of Rhabdomyosarcoma
Pax7/FKHR
Pax3/FKHR
13
ARMS Survival by Translocation Status
IRS IV Patients, all metastatic ARMS with tissue
available
1.0
0.9
Log Rank Test p0.0026
0.8
PAX7
0.7
0.6
Proportion
0.5
Negative
0.4
0.3
PAX3
0.2
0.1
0.0
0
1
2
3
4
5
6
7
8
9
10
Overall survival (years)
Data Jim Lynch, IRSG
14
ARMS Survival by Translocation Status
IRS IV Patients, all ARMS with tissue available
1.0
0.9
Log Rank Test p0.17
0.8
0.7
0.6
PAX7
Proportion
0.5
PAX3
0.4
0.3
0.2
Negative
0.1
0.0
0
1
2
3
4
5
6
7
8
9
10
Overall survival (years)
Data Jim Lynch, IRSG
15
Study Population
Patients diagnosed and treated at CHLA
between 1988 and 2000 and with frozen tumor
tissue available for analysis

38 separate samples from 34 separate tumors
(primary and metastatic) from 29 different
patients
4 separate samples from 4 separate tumors
from 2 different patients, UBC

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38 Tumors
24 Primary tumors
14 Metastatic tumors
9 ARMS
10 ERMS
5 other 1 c/w ERMS 2 ARMS/ERMS 2 RMSdiver
12 ARMS 5 post rx 5 untrtd 2 unk
2 ERMS post rx
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Demographic Characteristics
ERMS (n10) 7 male, 3 female 3mo - 12yr
mean 3.7yr Primary ARMS (n9) 2 male, 5
female, 2 unk 8mo - 14yr 4 lt 3yr, 3 gt
8yr Metastatic ARMS (n9 12) 4 male, 3
female, 2 unk 13mo - 16yr



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Primary Tumors - Site
Other, n5 trunk, n3 extremity paratesticular
ERMS, n10 pelvis, n5 orbit ear pharynx testis
buttock
ARMS, n9 extremity, n6 axilla unknown, n2
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Gene Expression Chips

• Affymetrix HuFl
• gt 20 25mers of 6,800 known expressed gene
  seqeuence tiled across chip, with paired
  mismatches
• Fluorerscent cmRNA hybridized to chip
• Fluorescence intensity quantitated and analyzed
  by defined algorithms (Gene Chip Suite)

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Specimen Processing
B) Extract RNA C) Synthesis of double-stranded
cDNA D) In-vitro transcription w/
biotinylated nucleotides E) Size confirmation of
cRNA transcripts
A) Cut pilot section of OCT embedded
frozen tissue
500 bp
dissection of tumor tissue when possible
F) Fragmentation of cRNA
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Gene Expression Alv vs. Emb RMS
ARMS vs. ARMS
ERMS vs. ERMS
22
Gene Expressioin Pattern of Embryonal vs
Alveolar RMS
23
Gene Expression Pattern Embryonal vs Alveolar
RMS
H19 RagA Alpha globin Beta globin EIF-2-assoc p67
homolog 16p13.1-3 seq
Endothelin 3 MHC class II ag Carnitine
palmitoyltransferase 1 CD14
24
Gene Expression Pattern of Metastatic vs
Primary RMS
25
Gene Expression Pattern Metastatic vs
Primary RMS
AML1-Evi-1 oncogene 12p15 sequence mullerian
inhib factor precur
HOX3D EGF kinesin-like BP homeobox
gene hepatocyte GF
26
Tumor Heterogeneity - Alveolar RMS with Focal
Embryonal RMS
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Tumor Heterogeneity - Alveolar RMS with Focal
Embryonal RMS
Vs. All ARMS
Vs. All ERMS
Vs. All ERMS
Vs. All ARMS
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TUMOR HETEROGENEITY - ALVEOLAR RMS WITH FOCAL
EMBRYONAL RMS
retinol binding protein keratin type
II fibulin-2 IGFBP-45 heat-shock protein
c-fos ets--like protein c-kit OB-cadherin TGF-beta
binding protein
29
TUMOR PROGRESSION
POPLITEAL METASTASIS
PRIMARY
LUNG METASTASIS
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TUMOR PROGRESSION
POPLITEAL METASTASIS
cadherin-8 protein kinase inhibitor titin nebulin
rb-1 RBP2 MUM-1 4p16.3 frag
PRIMARY
rb-1 MUM-1 (mel ub mut pr) lumican MEF2C
LUNG METASTASIS
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TUMOR CLASSIFICATION
ATYPICAL PARATESTICULAR RHABDOMYOSARCOMA
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Gene Expression Pattern of Atypical Paratesticu
lar Rhabdomyosarcoma
c/w All ERMS
c/w All ARMS
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Pax3,7/FKHR Translocation Analysis
H20
19
20
23
30
81
82
83
87
27
500 bp
t(213) (n4)
ARMS (n8)
t(113) (n1)
translocation negative (n3)
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Gene Expression Patterns c/w Translocation Status
axonemal dynein (P7) ficolin (P7) myosin, light
chain (P3) rad2 (P3) topoisomerase II (P3)
RAR prot (P3) RAR (P3) TGF-beta (neg) APC
(neg) EGF (neg)
axonemal dynein (P7) RAR responsive prot
(P7) heregulin alpha (P7) myosin, heavy light
(neg) troponin (neg)
35
Studies In Progress
Confirmatory analyses Immunohistochemistry
(protein) In situ hybridization
(localization) Quantitative RT-PCR Data
mining Expression profiles Pathway
Prognosis


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Preliminary Cluster Analysis
TRANSLOC STATUS PAX3/FKHR NEGATIVE NEGATIVE PAX7/F
KHR NEGATIVE PAX3/FKHR PAX3/FKHR
PROGNOSIS CLUSTER POOR POOR INTERMED FAVORABLE POO
R POOR POOR
OUTCOME DEAD DEAD DEAD ALIVE / 2.5yr DEAD DEAD DEA
D
TUMOR ARMS1 ARMS2 ARMS3 ARMS4 ARMS5 ARMS6 ARMS7
37
ARMS Survival by Translocation Status
IRS IV Patients, all metastatic ARMS with tissue
available
1.0
0.9
Log Rank Test p0.0026
0.8
PAX7
0.7
0.6
Proportion
0.5
Negative
0.4
0.3
PAX3
0.2
0.1
0.0
0
1
2
3
4
5
6
7
8
9
10
Overall survival (years)
Data Jim Lynch, IRSG
38
Conclusions

• Gene Expression Profiles can diagnose
  histologic types of rhabdomyosarcoma
• These profiles also identify atypical variants
  may allow re-classification
• They also detect genetic variants (eg, PAX3 vs.
  PAX7 and suggest re-classification of fusion
  negative cases
• Preliminary data even suggest an association with
  biologic behavior!