Microdissection genotyping of gliomas:
therapeutic and prognostic considerations
Deepak Mohan1,*, Sydney D Finkelstein1, Patricia A Swalsky1, Eizaburo Sasatomi1,
Clayton Wiley2, Ronald L Hamilton2, Frank Lieberman3and Marta E Couce2
1Department of Pathology, Division of Anatomic Pathology;2Division of Neuropathology, University of
Pittsburgh Medical Center, Pittsburgh, PA, USA and3Division of Hematology Oncology, University of
Pittsburgh Cancer Center, Brain Tumor Center, University of Pittsburgh, Pittsburgh, PA, USA
Molecular anatomic pathology represents the blend of traditional morphological methods and the multigene
approach to determine cancer-related gene alterations for diagnostic and prognostic purposes. Microdissec-
tion genotyping was utilized to characterize 197 gliomas with targeted microdissection of 2–7 areas spanning
the spectrum of histologic types and grades. The methodology described herein is complementary to the
existing realities of pathology practice. The technique utilizes paraffin-embedded fixative-treated tissue of small
sample size after the primary morphological examination by the pathologist. Molecular information derived
from microdissection genotyping in combination with the traditional histological information, results in an
enhanced understanding of glioma formation and biological progression leading to improvements in diagnosis
and prediction of prognosis. In all, 100% or 32 of 32 cases with at least partial treatment response was observed
in neoplasms possessing the 1p or 1p/19q loss. The 19q loss alone without coexisting 1p showed no
improvement in treatment response. Gliomas lacking 1p loss with only allelic loss involving 3p, 5q, 9p, 10q and
17p showed unfavorable outcome of only 35%, or six of 17 cases with treatment response. In addition, the
determination of fractional allelic loss (favorable/unfavorable), was a very good independent predictor of
biological behavior. These findings emphasize the importance of determining the cumulative pattern of
mutational damage on 16 distinct sites or more, especially in the presence of 1p loss which in isolation or in
combination with 19q is a favorable prognostic factor for therapeutic response.
Modern Pathology (2004) 17, 1346–1358, advance online publication, 4 June 2004; doi:10.1038/modpathol.3800194
Keywords: microdissection genotyping; molecular anatomic pathology; glioma; loss of heterozygosity; fractional
allelic loss; 1p/19q
Molecular anatomic pathology represents the blend
of traditional histopathological methods and the
novel multigene approach, to determine cancer-
related gene alterations for diagnostic and prognos-
system of microscopic tissue sampling coupled
with mutational analysis for allelic imbalance. This
technique measures the extent of molecular damage
at multiple morphologically distinct sites while
operating within the fundamental constraints of
any modern surgical pathology laboratory. The
method optimally utilizes residual formalin fixa-
tive-treated tissue of small sample size after the
primary examination by the pathologist. The muta-
tional profiling of the glioma provides the patho-
logist and clinician with a genotypic correlate of
extent of glioma-associated mutational change pro-
vides valuable information on the stage of tumor
progression as well as treatment responsiveness.
Central nervous system gliomas encompass a
diverse collection of neoplastic entities spanning a
spectrum of biological behavior from indolent forms
to highly aggressive tumors.1–3These defined sub-
types of glioma are recognized by most observers
when present in pure form and tend to be treated in
a uniform manner with predictable outcome. The
revise the classification of gliomas based upon
well-known microscopic characteristics and some
known genotypical features.1,3The challenges in
Received 22 January 2004; revised and accepted 7 April 2004;
published online 4 June 2004
Correspondence: Dr D Mohan MD, Department of Pathology and
Laboratory Medicine, Cedars-Sinai Medical Center, 8700 Beverly
Boulevard, Los Angeles, CA 90048, USA.
Presented in part as a proffered paper at the annual United States
and Canadian Association of Pathologists meeting in Vancouver,
BC, Canada, March 2004 (Mod Pathol 2004;1:1337A).
*From July 2004 is with the Department of Pathology, Cedars-
Sinai Medical Center, Los Angeles, CA, USA.
Modern Pathology (2004) 17, 1346–1358
& 2004 USCAP , Inc All rights reserved 0893-3952/04 $30.00
glioma classification and prognostication are in
many ways no different from that encountered in
organizing diverse subtypes of other forms of human
cancer. Histopathologic evaluation has been thought
by some as being somewhat subjective and does not
take into account some of the unique molecular
attributes that determine biological behavior and
patients.4,5This is not to say that microscopic
cellular examination is without merit. The integra-
tion of morphologic features and molecular attri-
butes offersthe potential
improvement in glioma diagnosis, prognostication,
classification and treatment.
To address these issues and to improve glioma
classification, recent efforts have been directed to
supplementing histopathologic diagnosis with mo-
lecular characterization of mutational damage.5–12In
general these approaches fall into two broad cate-
gories; (1) those focused on defining one or a small
number of specific glioma-associated gene altera-
tions5,6and (2) those having genome wide capabil-
ities providing information on the status of vast
numbers of human genes at one time.7–9The latter,
which included RNA expression microarrays,11,12
comparative genomic hybridization13,14and proteo-
mic technology,15,16have attracted much interest
given the comprehensive scope of molecular char-
acterization which can serve both as a tool for gene
discovery as well as a potential means for tissue
Specific individual gene alterations have been
demonstrated to be critical in the development and
progression of gliomas. No single gene mutation,
however, can fully account for glial tumorigenesis or
be used by itself for comprehensive glioma diag-
nosis or prognostication in combination with micro-
specific gene targets appear to be involved in glioma
formation and progression, techniques that focus on
one or a small number of gene targets inevitably
prove insufficient to meet the needs for comprehen-
sive tumor characterization.17–20
Genome- or proteome-wide techniques for glioma
characterization, while powerful in scope, requires
relatively large amounts of fresh tissue for effective
performance.13,15This often proves incompatible
with existing pathology practice that requires all
available tissue in a given case to be initially subject
to optimal chemical fixation so that careful and
thorough microscopic examination can be per-
formed. Since histopathologic evaluation requires
only several 4mm thick microscopic sections, resi-
dual tissue is often present; however, it is chemi-
cally fixed in a manner that precludes many
molecular biologic techniques. Moreover, the quan-
tity of diagnostic tissue in neuropathology is often
very small given the highly functional nature of
brain tissue. Finally, even in that minority of
situations when abundant tumor tissue has been
removed, the intrinsic heterogeneity in neoplastic
progression may be such that sampling from multi-
ple regions is necessary for relevant correlative
molecular analysis. To be most effective, molecular
analysis must be broad in scope yet effective on
minute fixed tissue specimens so that it may
augment traditional histopathology in a manner that
enables close correlation between molecular find-
ings and cellular characteristics.21,22
With these operational considerations, we have
pursued microdissection-based genotyping as a
diagnostic approach for use in human gliomas23–25
as well as many other forms of cancer.21,25–30The
approach involves three sequential steps; tissue
microdissection, PCR amplification of a broad array
of genomic targets in search of allelic imbalance,
and DNA quantization by capillary electrophoresis.
Automated equipment can be readily configured to
accurately sample critical sites within a given
neoplasm, generate adequate amounts of gene-
specific DNA through robust PCR and detect muta-
tional damage of various types with a high degree of
precision. Given the recent progress made in under-
standing the molecular pathogenesis of glioma
development, progression and treatment responsive-
ness, this specific form of human cancer is very
suitable for a comprehensive integrated pathology/
molecular diagnostic approach designed specifically
for clinical application at this time.
Tissue microdissection methods have progressed
greatly over the past 5 years assisted by the
availability of equipment-assisted and manual ap-
proaches easily adapted for use with histologic and
cytologic material.31–34High throughput systems are
now also available for nucleic acid amplification,
amplicon size fractionation and DNA quantization
that are precise, reliable and relatively inexpensive
Over the past decade, the relationship between
oligodendroglial growth pattern, 1p/19q genomic
deletion and heightened treatment responsiveness
to certain forms of combination chemotherapy has
been intensively studied, with many important
diagnostic and therapeutic implications forthcom-
ing.38–41Empirically, gliomas composed of neoplas-
tic oligodendrocytes often demonstrate dramatic
response to radiation therapy and/or chemotherapy.
Moreover, glioma subsets manifesting 1p/19q allelic
loss appear to account for the majority of anaplastic
glioma patients with long disease free and overall
A similar tendency for treatment
responsiveness has been appreciated in mixed
gliomas composed of cells that in part display
oligodendroglial growth patterns.39,40This led to a
greater awareness among neuropathologists for
histologic recognition of oligodendroglial differen-
tiation as part of the routine microscopic evaluation
of glial neoplasms.
Cytogenetic studies have demonstrated the fre-
quent occurrence of 1p and/or 19q in pure oligoden-
drogliomas or mixed oligodendroglial/astrocytic
neoplasms.41–43Working from a cytogenetic perspec-
Glioma mutational profiling
D Mohan et al
Modern Pathology (2004) 17, 1346–1358
relatively higher degree of precision. Further opti-
mization of the panel of markers with addition of
novel techniques to enable detection of gene
amplification and DNA methylation alterations in
concert with allelic imbalance will improve micro-
dissection genotyping for more effective tumor
While the challenge of parallel genotyping of 16 or
greater specific cancer-associated genetic alterations
applied to multiple different sites within a given
tumor may seem daunting, the availability of high
throughput molecular biologic technologies such as
robotic PCR, automated capillary electrophoresis
and quantitative PCR make this feasible and cost
effective. Automated slide-based techniques such as
immunohistochemistry and in-situ hybridization are
now commonplace within the pathology laboratory.
Similarly, integrated system for tissue microdissec-
tion, large-scale PCR and high-volume genotyping
will find an equivalent position within the patho-
logy laboratory. With this system in place, patholo-
gists will then be free to make the greatest use of
these methods in a fashion fully consistent and
congruent with established histopathology practice
thereby making the best use of precious tissue
specimens both small in size and subject to current
modalities of fixation.
predict treatment responsivenesswitha
This paper is dedicated to the memory of Dr A Julio
Martinez, whose everlasting efforts will forever
influence our study of neuropathology.
1 Kleihues P, Cavenee WK, (eds). WHO Classification of
Tumors: Pathology and Genetics of Tumors of the
Nervous System. IARC Press: Lyon, 2000.
2 Schiffer D. Classification and biology of astrocytic
gliomas. Forum (Genova). 1998;8:244–255.
3 Kleihues P, Louis DN, Scheithauer BW, et al. The WHO
J Neuropathol Exp Neurol 2002;61, 215–225; 226–229.
4 Pollack IF, Biegel J, Yates A, et al. Risk assignment in
childhood brain tumors: the emerging role of molecu-
lar and biologic classification.
5 Caskey LS, Fuller GN, Bruner JM, et al. Toward a
molecular classification of the gliomas: histopathology,
molecular genetics, and gene expression profiling.
Histol Histopathol 2000;15:971–981.
6 Collins VP. Progression as exemplified by human
astrocytic tumors. Semin Cancer Biol 1999;9:267–276.
7 Zagzag D, Friedlander DR, Margolis B, et al. Molecular
events implicated in brain tumor angiogenesis and
invasion. Pediatr Neurosurg 2000;33:49–55.
8 Henson JW. Early genetic events in the formation of
astrocytomas. Curr Opin Neurol 2000;13:613–617.
9 Holland EC. Gliomagenesis: genetic alterations and
mouse models. Nat Rev Genet 2001;2:120–129.
10 Maher EA, Furnari FB, Bachoo RM, et al. Malignant
glioma: genetics and biology of a grave matter. Genes
11 Sallinen SL, Sallinen PK, Haapasalo HK, et al.
Identification of differentially expressed genes in
human gliomas by DNA microarray and tissue chip
techniques. Cancer Res 2000;60:6617–6622.
12 Rickman DS, Bobek MP, Misek DE, et al. Distinctive
molecular profiles of high-grade and low-grade glio-
mas based on oligonucleotide microarray analysis.
Cancer Res 2001;61:6885–6891.
13 Nishizaki T, Ozaki S, Harada K, et al. Investigation of
genetic alterations associated with the grade of astro-
cytic tumor by comparative genomic hybridization.
Genes Chromosomes Cancer 1998;21:340–346.
14 Paunu N, Sallinen SL, Karhu R, et al. Chromosome
imbalances in familial gliomas detected by compara-
15 Rohlff C. Proteomics in molecular medicine: applica-
tions in central nervous systems disorders. Electro-
16 Rutka JT, Taylor M, Mainprize T, et al. Molecular
biology and neurosurgery in the third millennium.
17 Rutka JT, Akiyama Y, Lee SP, et al. Alterations of the
p53 and pRB pathways in human astrocytoma. Brain
Tumor Pathol 2000;17:65–70.
18 Nagane M, Lin H, Cavenee WK, et al Aberrant receptor
signaling in human malignant gliomas: mechanisms
19 Ivanchuk SM, Mondal S, Dirks PB, et al. The INK4A/
ARF locus: role in cell cycle control and apoptosis and
implications for glioma growth. J Neurooncol 2001;51:
20 Finkelstein SD, Przygodzki R, Swalsky PA. Microdis-
section-based p53 genotyping: concepts for molecular
testing. Mol Diagn 1998;3:179–191.
21 Pollack IF, Finkelstein SD, Woods J, , et al, Children’s
Cancer Group. Expression of p53 and prognosis in
children with malignant gliomas. N Engl J Med 2002;
22 Chozick BS, Weicker ME, Pezzullo JC, et al. Pattern of
mutant p53 expression in human astrocytomas sug-
gests the existence of alternate pathways of tumori-
genesis. Cancer 1994;73:406–415.
23 Pollack IF, Hamilton RL, Finkelstein SD, et al. The
relationship between TP53 mutations and overexpres-
sion of p53 and prognosis in malignant gliomas of
childhood. Cancer Res 1997;57:304–309.
24 Pollack IF, Finkelstein SD, Burnham J, , et al,
Children’s Cancer Group. Age and TP53 mutation
frequency in childhood malignant gliomas: results in
a multi-institutional cohort. Cancer Res 2001;61:
25 Finkelstein SD, Hasegawa T, Colby T, et al. 11q13
allelic imbalance discriminates pulmonary carcinoids
from tumorlets. A microdissection-based genotyping
approach useful in clinical practice. Am J Pathol 1999;
26 Safatle-Ribeiro AV, Ribeiro Jr U, Sakai P, et al.
Integrated p53 histopathologic/genetic analysis of
premalignant lesions of the esophagus. Cancer Detect
Glioma mutational profiling
D Mohan et al
Modern Pathology (2004) 17, 1346–1358
27 Cong WM, Bakker A, Swalsky PA, et al. Multiple
genetic alterations involved in the tumorigenesis of
human cholangiocarcinoma: a molecular genetic and
clinicopathological study. J Cancer Res Clin Oncol
28 Raja S, Finkelstein SD, Baksh FK, et al. Correlation
between dysplasia and mutations of six tumor sup-
pressor genes in Barrett’s esophagus. Ann Thorac Surg
29 Fan CY, Liu KL, Huang HY, et al. Frequent allelic
imbalance and loss of protein expression of the DNA
repair gene hOGG1 in head and neck squamous cell
carcinoma. Lab Invest 2001;81:1429–1438.
30 Rolston R, Sasatomi E, Hunt J, et al. Distinguishing de
novo second cancer formation from tumor recurrence:
mutational fingerprinting by microdissection geno-
typing. J Mol Diagn 2001;3:129–132.
31 Rubin MA. Use of laser capture microdissection, cDNA
microarrays, and tissue microarrays in advancing our
understanding of prostate cancer. J Pathol 2001;195:
32 Mariani L, McDonough WS, Hoelzinger DB, et al.
Identification and validation of P311 as a glioblastoma
invasion gene using laser capture microdissection.
Cancer Res 2001;61:4190–4196.
33 Mariani L, Beaudry C, McDonough WS, et al. Death-
associated protein 3 (Dap-3) is overexpressed in
invasive glioblastoma cells in vivo and in glioma cell
lines with induced motility phenotype in vitro. Clin
Cancer Res 2001;7:2480–2489.
34 Smit ML, Giesendorf BA, Heil SG, et al. Automated
extraction and amplification of DNA from whole blood
using a robotic workstation and an integrated thermo-
cycler. Biotechnol Appl Biochem 2000;32:121–125.
35 Weiler J, Gausepohl H, Hauser N, et al. Hybridisation
(PNA) oligomer arrays. Nucleic Acids Res 1997;25:
36 Belgrader P, Devaney JM, Del Rio SA, et al. Automated
polymerase chain reaction product sample preparation
for capillary electrophoresis analysis. J Chromatogr B
Biomed Appl 1996;683:109–114.
37 Perry JR, Louis DN, Cairncross JG. Current treatment of
oligodendrogliomas. Arch Neurol 1999;56:434–436.
38 Burton E, Prados M. New chemotherapy options for
the treatment of malignant gliomas. Curr Opin Oncol
39 Perry JR. Oligodendrogliomas: clinical and genetic
correlations. Curr Opin Neurol 2001;14:705–710.
40 Croteau D, Mikkelsen T, Rempel SA, et al. New
innovations and developments for glioma treatment.
Clin Neurosurg 2001;48:60–81.
41 Bigner SH, Rasheed BK, Wiltshire R, et al. Morpho-
logic and molecular genetic aspects of oligodendroglial
neoplasms. Neuro-oncol. 1999;1:52–60.
42 Biegel JA. Cytogenetic and molecular genetics of
childhood brain tumors. Neuro-oncol 1999;1:139–151.
43 Cairncross JG, Ueki K, Zlatescu MC, et al. Specific
genetic predictors of chemotherapeutic response and
survival in patients with anaplastic oligodendroglio-
mas. J Natl Cancer Inst 1998;90:1473–1479.
44 Paleologos NA, Cairncross JG. Treatment of oligoden-
droglioma: an update. Neuro-oncol 1999;1:61–68.
45 Sehgal A. Molecular changes during the genesis of
human gliomas. Semin Surg Oncol 1998;14:3–12.
46 Rasheed BK, Wiltshire RN, Bigner SH, et al. Molecular
pathogenesis of malignant gliomas. Curr Opin Oncol
47 Goussia AC, Agnantis NJ, Rao JS, et al. Cytogenetic and
molecular abnormalities in astrocytic gliomas (Re-
view). Oncol Rep 2000;7:401–412.
48 Weiss WA. Genetics of brain tumors. Curr Opin Pediatr
49 Takeshima H, Sawamura Y, Gilbert MR, et al. Applica-
tion of advances in molecular biology to the treatment
of brain tumors. Curr Oncol Rep 2000;2:425–433.
50 Hill JR, Kuriyama N, Kuriyama H, et al. Molecular
genetics of brain tumors. Arch Neurol 1999;56:
51 Fueyo J, Gomez-Manzano C, Yung WK, et al. The
functional role of tumor suppressor genes in gliomas:
clues for future therapeutic strategies. Neurology
52 Nozaki M, Tada M, Kobayashi H, et al. Roles of the
functional loss of p53 and other genes in astrocytoma
tumorigenesis and progression. Neuro-oncol. 1999;1:
53 Bredel M, Pollack IF, Hamilton RL, et al. Epidermal
growth factor receptor expression and gene amplifica-
tion in high-grade non-brainstem gliomas of child-
hood. Clin Cancer Res 1999;5:1786–1792.
54 Sung T, Miller DC, Hayes RL, et al. Preferential
inactivation of the p53 tumor suppressor pathway
and lack of EGFR amplification distinguish de novo
high grade pediatric astrocytomas from de novo adult
astrocytomas. Brain Pathol 2000;10:249–259.
55 Darling JL, Warr TJ. Biology and genetics of malignant
brain tumours. Curr Opin Neurol 1998;11:619–625.
Glioma mutational profiling
D Mohan et al
Modern Pathology (2004) 17, 1346–1358