Molecular pathology
The application of molecular pathology techniques has revolutionized pathological
diagnosis, especially for lung and colorectal cancer (70% activity), but also for sarcomas,
melanomas and lymphomas.
Cytogenetics
Cytogenetics is the study of chromosomes and related disease states caused by an
abnormal chromosome number and/or structure. The main drawback of this
technique is the need for fresh tissue, that must be stimulated to undergo cell division
in vitro in order to visualize chromosomes in mitosis with light microscopy; other
drawbacks are the inability to detect base substitutions and indels and inability to
identify the mutations responsible for malignant transformation. Cytogenetics is still
used for chorionic villi, amniotic fluid, peripheral blood, skin fibroblasts and bone marrow
and it is important in the diagnosis and management of hematological malignancies.
Immunohistochemistry (and immunocytochemistry) – IHC and ICC
This technique exploits the
specificity of antigen-antibody
reaction; nowadays we use indirect
analyses with secondary antibodies
that contain a chromogen, binding
the primary antibody and
precipitating making the reaction
visible even with the naked eye. IHC is still used nowadays thanks to its prognostic and
predictive utility in:
- breast cancer to assess estrogen and progesterone receptors, HER2 and Ki67.
- lung cancer, to assess ALK, ROS1, and MET
- NETs, for SSTR2A and Ki67
- gastric cancer for HER2 and
EBV
- pancreatic and biliary cancer
for ROS1 mutations
- urothelial cancer: ARID1A
- prostate cancer: PTEN, Ki67
- cancer in general: PDL-1, MSI
→ impact on therapy!!! MSI
can also be assessed with PCR or NGS but this is more expensive.
Precision medicine in oncology aims to match individual patients with the right treatment
at the right time, based on the patient’s biologic and molecular characteristics – this
can be done nowadays thanks to the development of new checkpoint, kinase or PARP
inhibitors e.g. Trastuzumab for HER2+ breast cancer or Imatinib mesylate for BCR-ABL
translocation in CML and c-KIT mutations in GISTs that have greatly improved patient
survival, and anti-EGFR TKIs (target intracellular domain) in NSCLC or anti-EGFR mAbs
, (targeting extracellular domain) in CRC based on the finding of gene mutations in KRAS
and EGFR upon hot-spot testing.
Next generation sequencing
The first approach used for DNA
sequencing was the Sanger
technique, whereby chain-
terminating dideoxynucleotides are
incorporated by DNA polymerase during in-vitro DNA replication. This includes 3 steps:
denaturation at 95°C.,
annealing of primers at 44
°C, and at 72°C several times.
A single PCR product is mixed
with a DNA polymerase, a
specific primer and four dead-
end nucleotides labeled with
fluorescent probes. The
ensuing reaction produces a ladder of DNA molecules of all possible lengths, each
labeled with a tag corresponding to the base at which the reaction stopped due to
incorporation of a terminator nucleotide. After size separation by electrophoresis, the
sequence is “read” and compared with the normal sequence to detect mutations.
Sanger sequence is the gold standard for complete sequencing due to its high sensitivity
and precision, but major drawbacks are that it
is time consuming and cannot detect
deletions, translocations or copy number
changes. This is why methods used currently
for detection and profiling of nucleic acid
sequence variation include PCR, NGS and
hybrid capture; these three approaches have
many overlapping aspects and are not
mutually exclusive but have allowed the
analysis of an entire gene, the whole exome,
or the entire genome, rather than the single
gene.
NGS: Hybrid capture technique
The hybrid capture technique involves the hybridization of selected fragments of DNA
or RNA that represent the target region with a shotgun genomic DNA library (for DNA-
based assay) or libraries of expressed transcripts (for RNA-based assays). It has been
used for comprehensive genomic profiling of large gene panels (>400 genes) in large
patient populations to screen for genetic alterations that include base substitutions,
MSI and TMB (tumor mutational burden). Hybrid capture method has the most uniform
coverage and highest sensitivity for variant calling.
The platforms which use these approaches are:
The application of molecular pathology techniques has revolutionized pathological
diagnosis, especially for lung and colorectal cancer (70% activity), but also for sarcomas,
melanomas and lymphomas.
Cytogenetics
Cytogenetics is the study of chromosomes and related disease states caused by an
abnormal chromosome number and/or structure. The main drawback of this
technique is the need for fresh tissue, that must be stimulated to undergo cell division
in vitro in order to visualize chromosomes in mitosis with light microscopy; other
drawbacks are the inability to detect base substitutions and indels and inability to
identify the mutations responsible for malignant transformation. Cytogenetics is still
used for chorionic villi, amniotic fluid, peripheral blood, skin fibroblasts and bone marrow
and it is important in the diagnosis and management of hematological malignancies.
Immunohistochemistry (and immunocytochemistry) – IHC and ICC
This technique exploits the
specificity of antigen-antibody
reaction; nowadays we use indirect
analyses with secondary antibodies
that contain a chromogen, binding
the primary antibody and
precipitating making the reaction
visible even with the naked eye. IHC is still used nowadays thanks to its prognostic and
predictive utility in:
- breast cancer to assess estrogen and progesterone receptors, HER2 and Ki67.
- lung cancer, to assess ALK, ROS1, and MET
- NETs, for SSTR2A and Ki67
- gastric cancer for HER2 and
EBV
- pancreatic and biliary cancer
for ROS1 mutations
- urothelial cancer: ARID1A
- prostate cancer: PTEN, Ki67
- cancer in general: PDL-1, MSI
→ impact on therapy!!! MSI
can also be assessed with PCR or NGS but this is more expensive.
Precision medicine in oncology aims to match individual patients with the right treatment
at the right time, based on the patient’s biologic and molecular characteristics – this
can be done nowadays thanks to the development of new checkpoint, kinase or PARP
inhibitors e.g. Trastuzumab for HER2+ breast cancer or Imatinib mesylate for BCR-ABL
translocation in CML and c-KIT mutations in GISTs that have greatly improved patient
survival, and anti-EGFR TKIs (target intracellular domain) in NSCLC or anti-EGFR mAbs
, (targeting extracellular domain) in CRC based on the finding of gene mutations in KRAS
and EGFR upon hot-spot testing.
Next generation sequencing
The first approach used for DNA
sequencing was the Sanger
technique, whereby chain-
terminating dideoxynucleotides are
incorporated by DNA polymerase during in-vitro DNA replication. This includes 3 steps:
denaturation at 95°C.,
annealing of primers at 44
°C, and at 72°C several times.
A single PCR product is mixed
with a DNA polymerase, a
specific primer and four dead-
end nucleotides labeled with
fluorescent probes. The
ensuing reaction produces a ladder of DNA molecules of all possible lengths, each
labeled with a tag corresponding to the base at which the reaction stopped due to
incorporation of a terminator nucleotide. After size separation by electrophoresis, the
sequence is “read” and compared with the normal sequence to detect mutations.
Sanger sequence is the gold standard for complete sequencing due to its high sensitivity
and precision, but major drawbacks are that it
is time consuming and cannot detect
deletions, translocations or copy number
changes. This is why methods used currently
for detection and profiling of nucleic acid
sequence variation include PCR, NGS and
hybrid capture; these three approaches have
many overlapping aspects and are not
mutually exclusive but have allowed the
analysis of an entire gene, the whole exome,
or the entire genome, rather than the single
gene.
NGS: Hybrid capture technique
The hybrid capture technique involves the hybridization of selected fragments of DNA
or RNA that represent the target region with a shotgun genomic DNA library (for DNA-
based assay) or libraries of expressed transcripts (for RNA-based assays). It has been
used for comprehensive genomic profiling of large gene panels (>400 genes) in large
patient populations to screen for genetic alterations that include base substitutions,
MSI and TMB (tumor mutational burden). Hybrid capture method has the most uniform
coverage and highest sensitivity for variant calling.
The platforms which use these approaches are:
