Microarray gene expression utilized to

identify putative serum biomarkers of NF1

associated tumors

Connective Tissue Oncology SocietyNovember 14, 2008

Trent Hummel, Yonatan Mahler, Shyra Miller, Timothy Cripe, Walter Jessen, Bruce Aronow, Nancy Ratner

Cincinnati, OH, USA

Background

• 30% of NF1 patients develop plexiform neurofibromas (PNF) – these typically arise in childhood– Benign tumors involving larger nerves– Can invade critical structures causing death and significant morbidity– Irregular borders make growth calculations difficult with CT and MRI scans– Surgery is the only reliable treatment– Plexiform neurofibromas can undergo transformation to malignant peripheral

nerve sheath tumors (MPNST)

Huson SM, et al. Brain 1988Rasmussen SA, et al Am J Epidemiol 2000

Background• Malignant peripheral nerve sheath tumors (MPNST)

• Highly aggressive sarcomas – 5 year survival approximately 30%

• Lifetime risk in NF1 patients is 8-13%

• No reliable method to detect transformation of plexiform neurofibromas to MPNSTs

Huson SM, et al. Brain 1988Rasmussen SA, et al Am J Epidemiol 2000

Hypotheses

• There are unique serum proteins that mark malignant peripheral nerve sheath tumors

• There are unique proteins in the peripheral blood of NF1 patients with PNF that correlate with tumor burden/growth.

NF1 Microarray Consortium 86 microarrays in 9 batches (77 samples plus 9 references)

45 Cell culture samples

NHSC

dNFSC +/-

dNFSC -/-

pNFSC

MPNST cell

10 Normal human Schwann cell

2 Dermal neurofibroma NF1 +/- Schwann cell

9 Dermal neurofibroma NF1 -/- Schwann cell

11 Plexiform neurofibroma Schwann cell

13 MPNST cell line

32 Tumor samples

dNF

pNF

MPNST

13 Dermal neurofibroma

13 Plexiform neurofibroma

6 MPNST

• Gene expression from 10 independent cultures of primary human Schwann cells were compared to 67 NF1 tumor samples • Included cultured neurofibroma Schwann cells and primary tumors on an Affymetrix platform

NF1 Microarray Consortium - Nancy Ratner, Shyra Miller, Bruce Aronow, Walter Jessen,Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page

6.0

0.0

dNF

pNF

MPNST

Normal

dNFSC +/-

dNFSC -/-

pNFSC

MPNST cell

• The degree of differential expression is expressed by color

• Each row represents a single gene expression profile • Genes in blue exhibit down regulation

• Genes in red signify upregulated expression as compared to normal human Schwann cells.

• For this project, genes that demonstrated differential upregulation between NHSC and neurofibromas and/or MPNSTs were chosen for further analysis.

Methods

Test candidates in human model

Filter these up-regulated genes through a database of secreted proteins

Validate candidates withquantitative RT-PCR

Confirm secretion of proteins in cell media

Methods

• Using microarray data from NF1 Microarray Consortium (Miller S, et al. submitted)

• Compared differentially expressed probesets with the Secreted Protein Database– List of probesets that encode secreted

proteins that may be released into the extracellular milieu

Chen Y, et al. Nucleic Acids Res. 2005

9473 genes differentially expressedbetween cell culture classesNormal Human Schwann celldermal NFSC, plexiform NFSC,MPNST cell line (FDR=0.05)

3721 genes from the Secreted Protein Databasewith a confidence rank of 0-3

1664 of 3721 genes upregulated from Normal Human Schwann cell to Neurofibromaor Normal Human Schwann cell to MPNST

Methods

• Analysis yielded 666 genes that encode putative secreted proteins

•Differentially upregulated between NHSC and neurofibromas and/or MPNST

• Subsequent cluster analysis across both tumors and cell lines reduced the number of candidates to 132

•Alternatively spliced forms of genes were removed

•92 genes that encode putative secreted proteins and that are upregulated in MPNST and/or neurofibromas as compared to normal human Schwann cells

Top Candidates

• Initial Candidates Selected base on:– Robust expression in microarray data in all

cell lines when compared to NHSC– Commercially available reagents

– Adrenomedullin (ADM)

Adrenomedullin

• Adrenomedullin (ADM) is a secreted peptide (52 AA)• Stimulates angiogenesis

– ADM knockout mice die at E14 because of severe vascular defects

– ADM expression correlates with vascularity in RCC and breast carcinomas

• Hypoxia up-regulates ADM in human glioma cell lines

• GBM Xenograft tumor model - anti-ADM antibody greatly reduced both tumor growth and vascularity

Zhao et al 1998 Jougasaki and Burnett, 2000Kitamuro et al, 2000 Garayoa et al, 2000Shindo et al., 2001 Oehler et al., 2002Ouafik et al., 2002

Validation of Array Data with RT-PCR

ADM fold change by RT-PCR inMPNST cell lines compared to NHSC

0

50

100

150

200

216

48

156

3119

193

85

1

* sporadic MPNST cell lines

Fo

ld c

han

ge

ADM fold change by RT-PCRin NFSC compared to NHSC

NHSC

NFSC +/-

NFSC +/-

NFSC -/-

NFSC -/-

0

20

40

60

8070

55

37

13

1F

old

Ch

ang

e

•Quantitative RT-PCR confirms microarray data in MPNST cell lines and NFSC (NF1 +/- and -/-) cell lines as compared to Normal Human Schwann cells

•Greater than 3 fold change considered significant

ADM in Conditioned Media

Results using a competitive ELISA adrenomedullin detection kit (Phoenix Pharmaceuticals) following the manufacturer’s protocol

ADM level in serum free MPNSTconditioned media

NHSC CM

8814

CM

26T C

M

S462

CM

S462-

TY CM

T265

CM

88-3

CM

0.0

0.5

1.0

1.5

*

*

*

*

*

* = p<0.0013

*

** = p<0.04

**

ng

/ml

Methods

Test candidates in human model

Filter these up-regulated genes through a database of secreted proteins

Validate candidates withquantitative RT-PCR

Confirm secretion of proteins in cell media

CharacteristicsNF1 w/o

PNFNF1 w/

PNFNF1

MPNST*Controls

Total 10 17 5 25

SexFemale 7 6 Unknown 15

Male 3 11 10

AgeRange 13-42 5-42 Unknown 4-19

Mean 21 19 13

Median 17 16 15*4/5 MPNST serum samples courtesy of Victor Mautner, 1/5 was a 16 y/o

who had serum collected for presumed PNF and found to have MPNST.

4/5 MPNST samples courtesy Victor Mautner

Serum Concentration of Adrenomedullinin NF1 patients with and without tumor burden

Controls

NF1 with

PNF

NF1 w/o

PNF

NF1 M

PNST

0.0

0.1

0.2

0.3

0.4

ng/ml

p<0.0001

p=0.03p<0.0001

p<0.0001

p=0.0006•ADM elevated in patients with NF1 regardless of tumor status

•No difference in ADM concentration when comparing plexiform neurofibroma tumor burden, tumor location, age, or gender

•Adrenomedullin is a potential serum biomarker of NF1

•Increased serum levels of ADM may correlate with presence of MPNST, though small sample size limits the power of this analysis.

Conclusions

• Feasible to utilize microarray gene expression data to generate a list of putative secreted biomarkers

– Confirming expression with secondary technique such as RT-PCR

– Testing protein expression/secretion in cell lines and subsequently in human serum

• Adrenomedullin

– Potential biomarker for NF1

– Also potential serum biomarker for NF1 related MPNSTs

Future Directions

• Continue investigating list of 92 genes

• Obtain more human MPNST serum samples

• Further investigate the biological effects of adrenomedullin on MPNST cell lines

• Envision a panel of proteins used to delineate between growth of plexiform neurofibromas and transformation to malignant peripheral nerve sheath tumors– Add to clinical decision making – Add to treatment decisions regarding therapy

Thanks to…

• Nancy Ratner• Shyra Miller• Members of NF1 Microarray consortium - Bruce Aronow,

Walter Jessen, Marco Giovannini, Anat Stemmer-Rachamimov, Peggy Wallace, Conxi Lazaro, and Grier Page

• Jon Williams• John Perentesis• Elizabeth Schorry• Tim Cripe• Yoni Mahler

SPD• Entries

ranked according to prediction confidence (rank 0 -3)

Chen Y, et al. Nucleic Acids Res. 2005