The
growing momentum of molecular analysis has enormous
promise for diagnostics and numerous research applications.
The vast arsenal of newer modalities in the field
of molecular pathology available today is bewildering
and ever expanding. They provide opportunities to
study etiopathogenesis, to aid diagnosis, to prognosticate
tumours and to devise targeted therapy.
Molecular diagnostics
Immunohistochemistry helps classify soft tissue and
bone tumors, but a proportion of pediatric tumors
defy such attempts at classification. Specific translocation
based molecular “signatures” of these
tumors have the potential to resolve such diagnostic
dilemmas.
Ewing sarcoma family of tumors (ESFTs) are being characterized
by non-random gene rearrangements between the EWS
gene on chromosome 22q12 and various members of the
ETS gene family such as FLI1 (chromosome 11), ERG
(chromosome 21), ETV1 (chromosome 7), E1AF (chromosome
17) and FEV (chromosome 2). Accurate diagnosis of
ESFT is crucial for the most appropriate clinical
management of patients. Desmoplastic small round cell
tumor is characterized by t(11;22) (p13;q12) resulting
in EWS-WT1 fusion. Alveolar rhabdomyosarcoma demonstrates
PAX3-FKHR and PAX7-FKHR fusion. t(X;18) (p11;q11)
is associated with > 90% of both monophasic and
biphasic synovial sarcomas; SYT-SSX1 and SYT-SSX2
fusion transcripts correlate with biphasic and monophasic
morphology respectively.
Sensitive technique like RT-PCR has made it possible
to evaluate tiny biopsies, archived paraffin blocks
and needle aspiration cytology material for the presence
of specific translocations and arrive at a definitive
diagnosis. The use of Fluorescence in Situ Hybridization,
FISH, on interphase nuclei, allows the detection of
nucleic acid targets while preserving the morphology
of the tissue. Many chromosomal abnormalities such
as duplication, deletion or translocation, are diagnosable
using FISH. This technique requires less tissue, can
be performed on archival tissue, and there is visual
control of the tumour. Investigating aneuploidy at
chromosomes 3, 7, 17, and 9p21 (e.g. loss at 9p21)
using a multitarget FISH performed on urine specimens
is a promising approach and useful adjunct to urine
cytology and cystoscopy for the non-invasive detection
of recurrent urothelial tumours.
Molecular
diagnostics, however, are generally viewed as expensive
and labor intensive, which means that advances to
improve automation, throughput, scalability, and reliability
are critical.
Prognostic uses
– The fusion transcript type in the evaluation
of ESFT may be prognostically useful with the presence
of EWS-FLI1 type1 transcript being associated with
improved outcome compared with that in patients with
other fusion transcript types.
– Alveolar rhabdomyosarcoma with PAX3-FKHR fusion
is associated with poor prognosis as compared to those
with PAX7-FKHR translocation.
– SYT/SSX1 variant of synovial sarcoma might
be less favorable, associated with higher tumor proliferating
rate and reduced overall survival as compared to SYT/SSX2.
– N-myc amplification is a major prognostic
factor connoting poor prognosis in neuroblastoma.
– HER-2/neu, ERBB2, a proto-oncogene located
on chromosome 17 has become an important prognostic
indicator in breast cancer. FISH based assay uses
probes for chromosome 17 centromere and HER-2 gene
simultaneously, and HER-2 gene amplification is defined
as a ratio of HER-2 gene copies per chromosome 17
copy equal to or greater than 2.0. Amplification and
/or overexpression of HER-2 occurs in 15% to 25% of
human breast cancers and is associated with a poor
clinical outcome. Over expression of the HER-2 protein
on FISH is closely correlated with amplification of
the HER-2 gene in tumours scored immunohistochemically
as 3+, but not in tumours scored as 2+. It has been
documented in the literature that HER-2 gene amplification
determined by FISH is a better predictor of response
to trastuzumab-based therapy than HER-2 overexpression
determined by immunohistochemical scores of 2+ to
3+. The accurate assessment of HER-2 gene amplification
is critical in the management of these tumours.
Diagnosis of Recurrent Disease
Urinary cytology has been the reference test for the
evaluation of symptomatic patients, for the detection
of lesions in high-risk patients, and for the follow-up
of patients with a prior history of urothelial carcinoma.
Although routine cytology has a relatively high level
of specificity for the detection of high-grade in
situ and invasive lesions, the sensitivity for patients
with low-grade TCC is low. Urine cytology is further
limited by poor specificity for clinically significant
lesions in cases that are classified as atypical on
the basis of cells with equivocal cytologic features.
Therefore, the clinical outcomes for individual cases
cannot be accurately determined by cytologic examination
alone. As a result, many patients undergo unnecessary
procedures to rule out malignancy based on limited
specificity for clinically significant lesions. The
U.S. Food and Drug Administration (FDA) recently approved
the UroVysionTM Bladder Recurrence Cancer Test (Vysis;
Downers Grove, IL) for the detection of urothelial
carcinoma in urine. The UroVysionTM test has been
designed for interphase cell quantification of chromosomes
3, 7, 17, and 9p21 region (p16/CDKN2A gene), which
are recognized as frequently involved in numerical
anomalies in bladder cancer. Interestingly, the UroVysionTM
test also was proven to be effective in detecting
tumor recurrence before evidence of urothelial carcinoma
was found on biopsy, likely due to the ability of
voided urine to represent the entire urothelium versus
the regional evaluation of the biopsy. Patients with
atypical/suspicious findings on urine cytology and
negative FISH may be subjected to longer intervals
of cystoscopic surveillance. Thus negative FISH is
also considered a valuable negative finding. The assay
also holds hope for a group of patients with hematuria
and dysuria with negative cystoscopic findings and
urine cytologic findings. Patients with positive FISH
but negative biopsy should be carefully followed,
as they are at risk of harboring occult disease. Anticipatory
positive cases (FISH pos/CYSTO neg/HISTO neg) recur
at twice the rate and in half the time as true negative
cases.
Molecular Therapeutics
The emergence of molecularly targeted therapeutics
in oncology has placed increased importance on molecular
diagnostic tests in guiding therapeutic intervention.
EGFR Activity in Cancer
The EGFR is a member of a family of four receptors
[EGFR (HER1 or ErbB1), ErbB2 (HER2/neu), ErbB3 (HER3),
and ErbB4 (HER4)].
These receptors are large proteins which reside in
the cell membrane. When a ligand, for example EGF
or TGF-a, binds to the EGFR, it causes receptor dimerisation
and autophosphorylation of the internal receptor domain.
This initiates a cascade of cellular reactions that
result in increased cell division and influences other
aspects of malignant progression of the tumor - angiogenesis,
metastasis and an inhibition of apoptosis.
Inhibition of the EGFR thus provides a rational target
for anticancer therapy. Currently, two approaches
are in clinical development: monoclonal antibodies
and small molecule inhibitors of the EGFR tyrosine
kinase enzyme.
A monoclonal antibody to HER2/neu (trastuzumab) has
proved successful in limiting the growth of tumors
and has been licensed for the treatment of advanced
breast cancers that overexpress this receptor. The
measurement of p185HER-2/neu expression has gained
new importance based on the approval of trastuzumab
for the treatment of patients with metastatic breast
cancer. Treatment with Trastuzumab alone or in combination
with chemotherapy is indicated only for patients whose
tumours over express p185HER-2/neu or demonstrate
HER-2/neu gene amplification based on FISH.
The combination of trastuzumab with cytotoxic chemotherapy
is also considered to improve other clinical endpoints,
including response duration, time to tumour progression,
and overall survival, compared with the chemotherapy-only
treatment option.
Recent studies have shown promising results with tyrosine
kinase inhibitors of EGFR (ZD1839 and OSI-774) in
patients with esophageal and lung carcinomas. In non-small-cell
lung carcinomas, ZD1839 (Iressa®; AstraZeneca;
London, UK) produced objective responses in about
15%-20% of patients with advanced disease who had
failed one or two chemotherapy regimens (at least
one platinum based) and in 10% of patients who had
failed two or more prior chemotherapy regimens containing
platinum and docetaxel, with a favorable adverse event
profile. These findings raised considerable excitement
regarding the potential role of such therapies for
lung cancer patients with less advanced disease and
their potential to improve survival.
Genomics
A major leap in functional genomic investigations
would be the ability to perform global gene expression
analysis with in vivo-derived genetic material originating
from morphologically distinct cellular populations
that constitute the various stages of cancer progression.
The recent advent of high-density DNA microarray technology,
with its capacity for simultaneous monitoring of thousands
of genes, provides a unique opportunity for high-throughput
genetic analysis of tumours.
Proteomics
Molecular diagnostics, however, are generally viewed
as expensive and labor intensive, which means that
advances to improve automation, throughput, scalability,
and reliability are critical.
Prognostic uses
– The fusion transcript type in the evaluation
of ESFT may be prognostically useful with the presence
of EWS-FLI1 type1 transcript being associated with
improved outcome compared with that in patients with
other fusion transcript types.
– Alveolar rhabdomyosarcoma with PAX3-FKHR fusion
is associated with poor prognosis as compared to those
with PAX7-FKHR translocation.
– SYT/SSX1 variant of synovial sarcoma might
be less favorable, associated with higher tumor proliferating
rate and reduced overall survival as compared to SYT/SSX2.
– N-myc amplification is a major prognostic
factor connoting poor prognosis in neuroblastoma.
– HER-2/neu, ERBB2, a proto-oncogene located
on chromosome 17 has become an important prognostic
indicator in breast cancer. FISH based assay uses
probes for chromosome 17 centromere and HER-2 gene
simultaneously, and HER-2 gene amplification is defined
as a ratio of HER-2 gene copies per chromosome 17
copy equal to or greater than 2.0. Amplification and
/or overexpression of HER-2 occurs in 15% to 25% of
human breast cancers and is associated with a poor
clinical outcome. Over expression of the HER-2 protein
on FISH is closely correlated with amplification of
the HER-2 gene in tumours scored immunohistochemically
as 3+, but not in tumours scored as 2+. It has been
documented in the literature that HER-2 gene amplification
determined by FISH is a better predictor of response
to trastuzumab-based therapy than HER-2 overexpression
determined by immunohistochemical scores of 2+ to
3+. The accurate assessment of HER-2 gene amplification
is critical in the management of these tumours.
Suggested references
1. Hicks DG, Dawson A, Mattingly S, Crowe JP. The
unmet clinical need for new molecular genetic markers
in the prognosis and therapeutic management of breast
cancer. Arch Pathol Lab Med 2005; 129: 1372-1374.
2. Kipp BR, Karnes RJ, Brankley SM, Harwood AR, Pankratz
VS, Sebo TJ, Blute MM, Lieber MM, Zincke H, Halling
KC. Monitoring intravesical therapy for superficial
bladder cancer using fluorescence in situ hybridization.
J Urol 2005; 173: 401-404.
3. Ladanyi M. Translocation-Based Molecular Diagnosis
of Sarcomas. Am J Surg Pathol 2003; 27: 414-415.
4. Ladanyi M. Translating research into cancer molecular
diagnostics and patents. J Clin Pathol 2005; 58: 793-4.
5. Lal P, Salazar PA, Hudis CA, Ladanyi M, Chen B.
HER-2 testing in breast cancer using immunohistochemical
analysis and fluorescence in situ hybridization. A
single-institution experience of 2,279 cases and comparison
of dual-color and single-color scoring. Am J Clin
Pathol 2004; 121: 631-636.
6. Roblick UJ, Auer G. Proteomics and clinical surgery.
Br J Surg 2005; 2:1464-5.
