Page 164 - Concise Pathology for Exam Preparation ( PDFDrive )

6 Neoplasia 149

2. In situ hybridization: Molecular technique by which nucleic acid sequences (cellular/
viral DNA and RNA) are localized by specifically labelled nucleic acid probes directly in
the cell rather than after DNA extraction, eg, localization of oncogenes.
3. A variety of DNA-/RNA-based techniques in which DNA/RNA are extracted and anal-
ysed, eg, DNA analysis by Southern blot and RNA analysis by Northern blot are
also available.
4. A molecular cytogenetic technique called spectral karyotyping is a highly sensitive
method that allows the examination of all chromosomes in a single experiment. This
technique, which is based on 24-colour chromosomal painting with a mixture of fluo-
rochromes, can detect all types of chromosomal rearrangements in tumours including
very small translocations and insertions.
5. Another available technique, comparative genomic hybridization, is more frequently
used in research laboratories as it requires a lot of time and effort. This technique
allows the analysis of genome amplification and chromosomal gains and losses in
tumour cells. It has been used to differentiate primary from metastatic carcinomas, and
to identify primary tumours of uncertain origin.
6. DNA microarray analysis and proteomics: These methods are used to obtain gene
expression signatures (molecular profiles) of cancer cells. DNA microarray techniques
reveal the RNA expression from as many as 30,000 different genes using gene–chip
technology. Conventional DNA probes are substituted by silicon chips that contain the
entire range of genes, and high-resolution scanners are used for measurement. Pro-
teomics determines the protein profiles of tumours. With the methods currently in use,
protein profiles from about 3,000 genes can be obtained.
7. Validation of new markers for cancer diagnosis can be done on multiple tissue
samples, using tissue arrays. In this technique, core samples are obtained from
tissues embedded in a paraffin block and used to prepare a new block that may
contain hundreds of tissue fragments. These multiple samples are then used to test
the expression of potential tumour markers by immunohistochemical or in situ
hybridization techniques.
The above molecular methods can be used for
• Analysis of molecular cytogenetic abnormalities and mutational analysis: Cer-
tain genetic alterations are associated with poor prognosis, and hence their detection
allows stratification of patients for therapy. As an example, amplification of the
N-MYC gene and deletions of 1p bode poorly for patients with neuroblastoma. These
can be detected by routine cytogenetics, and also by fluorescent in-situ hybridization
(FISH) or polymerase chain reaction (PCR) assays.
• Study of oncogenic viruses at molecular level: Oncogenic viruses can contribute
to different steps of the carcinogenic process. In addition to elucidate the aetiology
of several human cancers, the study of oncogenic viruses has been invaluable to the
discovery and analysis of key cellular pathways that are commonly rendered
dysfunctional during carcinogenesis, in general.
• Detection of minimal residual disease: After treatment of patients with leukae-
mia or lymphoma, the presence of minimal disease or the onset of relapse can be
monitored by PCR-based amplification of unique nucleic acid sequences gener-
ated by the translocation. For example, detection of BCR-ABL transcripts by PCR
gives a measure of the residual leukaemia cells in treated patients with chronic
myeloid leukaemia.
• Diagnosis of hereditary predisposition to cancer: As was discussed earlier,
germline mutations in several tumour suppressor genes, including BRCA1, BRCA2
and the RET proto-oncogene are associated with a high risk of developing specific
cancers. Thus, detection of carriers of these mutations in family members of af-
fected patients or in those at high risk of carrying the mutation can be achieved
by molecular methods.

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