Molecular profiling

Intro
1. potential to revolutionise cancer diagnosis and treatment
  1. completions of the human genome sequence
  2. in conjunction with newer, cheaper, and more reliable methods of gene expression analysis
  3. by providing a molecular portrait of an individual cancer
    1. this technology will allow clinicians to determine
      1. origin of cancer
      2. potential for metastasis
      3. specific drug responsivenss
      4. probability of recurrence
2. currently when cancer is diagnosed it is classified according to the gross morphological appearance of the cells and surrounding tissue
  1. limitations
    1. relies on a subjective review of the tissue that is dependent on the knowledge and experience of a pathologist
      1. therefore may not be reproducible
    2. classification is discrete rather than continuous
      1. meaning that patients are classified into broad treatment groups
        e.g. low, medium, or high probability of recurrence
        limited ability to determine the individual recurrence risk
    3. current pathology reports either completely lack, or offer very little information regarding the potential drug treatment regime to which a cancer will respond
    4. tumours with identical pathology may have different origins and respond differently to treatment
    5. overcome by classification by molecular profiling
general
1. molecular profile determines the level of gene expression within cancer
  1. does this by hybridising the cellular RNA with known genes
    1. currently microarray technology used to provide information on thousands of genes simultaneously
  2. once the gene expression pattern is determined, information is compared to the expression profiles of cancers with known outcomes using a predetermined algorithm
    1. algorithm places the cancer into an outcome class based on similar gene expression patterns, or will return a survival probability
2. gene expression profiling:
  1. can provide a global perspective of cancer
  2. enhance the level of understanding of tumour development and progression
  3. facilitate the discovery of new diagnostic entities
  4. facilitate the development of novel anti-cancer therapeutics based on a molecular understanding of the cancer phenotype
3. the potential of molecular profiling is illustrated in the following examples (below)
  1. diffuse large B-cell lymphoma
    1. <50% 5-year survival rat
  2. breast cancer
    1. 80% average 5-year survival rate, but affects 1 in 8 females (much higher incidence)
  3. using such disparate cancers highlights the limitations of classical cancer classifications and the potential of molecular profiling
Diffuse B-Cell Lymphoma
1. current classification scheme distinguishes this type on non-Hodgkins lymphoma using characteristics of cellular morphology from biopsy specimens
  1. tumour is ranked according to stage and grade depending on the extent of cellular spread throughout the tissue and the degree of cellular differentiation respectively
    1. this information, along with the age of the patient and lactate dehydrogenase concentration is used in the International Prognostic Index to determine if the cancer has low, intermediate or high risk of recurrence
  2. even with good prognostic indicators, 36% do not respond to treatment
2. molecular profiling using Lymphochip, an expression array designed for lymphomas
  1. one algorithm successfully distinguished patients with two subtypes of lymphoma originating from different progenitor cells
    1. one with a 76% response to chemotherapy
    2. the other with a 16% chemotherapy response
    3. SAWYERS et al, Nature, 2000
    4. strong predictor of survival even in patients classified in the low risk group according to standard tumour typing methods
      1. by identifying patients who are unlikely to respond to standard treatment, more aggressive alternatives can be sought earlier in the course of therapy
3. another development in 2002 was made by Van't Veer et al, who examined expression of 25,000 transcripts; 78 node-negative patients
  1. this paper found 70 genes that could predict outcome in 65/78 patients
  2. noted that the most useful genes for prediction were involved in cell cycle, signal transduction, invasion and metastasis
Breast Cancer
1. molecular profiling can be of benefit even in cancers that, traditionally, are highly curable.
2. standard treatment regimes for breast cancer rely on the grade and stage of the tumour, as well as oestrogen receptor and HER2/neu expression status
  1. However, all oestrogen receptor positive breast cancers are not the same
    1. molecular profiling of a variety of breast cancers separated tumours into 5 different classes
      1. oestrogen receptor positive tumours fell into two distinct classes with different survival profiles
3. another problem with breast cancer treatment is over-treatment with chemotherapy
  1. using current pathology based methods of determining chemotherapy for breast cancer patients only 3% of those afflicted show a survival benefit related to chemotherapy
    1. ~83% of these individuals would have remained cancer-free without treatment, representing a large population of unnecessarily treated patients.
    2. 14% would die despite receiving chemotherapy, representing a population that would benefit from early identification for aggresseve or experimental treatments
  2. One molecular profiling algorithm developed for breast cancer
    1. decreases the number of patients placed in the high-risk category by 33-38%
      1. thus significantly reducing the number of patients undergoing needless chemotherapy
    2. In addition, a large proportion of the patients classified as low risk with current pathology classifications were found to have poor prognosis via molecular profiling
      1. these candidates might benefit from early, aggressive treatments or experimental treatments
4. despite the promise demonstrated with molecular profiling, several barriers must be overcome prior to routine diagnostic implementation for patient intervention
  1. One barrier is the cost of microarray technology for determine the molecular profile of the tumour
    1. this technology is expensive
    2. requires special handling procedures
    3. lacks standardisation within the research community
    4. In the US a single microarray analysis can cost more that $900 in materials alone
  2. To obtain the RNA necessary for molecular profiling of a tumour, a tissue sample must be snap frozen in liquid nitrogen at the time of excision
    1. adding a step to the standard excision and formalin fixing procedure
  3. In addition, standard microarrays, necessary for clinical application and interpretation of results, have not been created
    1. Of the 409 genes used in one study to form a clinical profile for breast cancer, only 67% and 37%, respectively, were used in two other breast cancer tumour studies, limiting the usefulness of the profile because of differences in the microarray used
  4. In addition to these technical hurdles, a large data set of existing microarray analyses of cancer with known outcomes is necessary to build a robust algorithm for determining outcomes in the malignancy to be profiled.
    1. these algorithms often consist of analyses of hundreds of genes simultaneously, with only some of the genes contributing to the prognostic value of the test
future/summary
1. efforts are underway to reduce the problems associated with molecular profiling in order to bring this technology from bench to bedside.
2. there are issues with RNA preparation that need to be taken into consideration
  1. time interval to being frozen/extracted
  2. use of formalin-fixed, paraffin-embedded tissues
  3. requirement for enriched cell populations
  4. requirement for amplification of target material (low yield)
  5. possible distortion of signal
3. laser capture microdissection provides mechanism for obtaining enriched populations of cells, it is flexible and user-friendly, the fixation process can affect downstream analysis, and however, it is suitable for DNA, RNA, and protein analysis
4. in an attempt to overcome the cost limitation of microarray technology, a small number of genes taken from a larger expression data set can be tested for clinical relevance
  1. recently, clinically relevant outcomes have been predicted for diffuse large B-cell lymphoma using quantitative reverse transcription-polymerase chain reaction (RT-PCR: a technology routinely used for laboratory testing) of only 6 genes
    1. RT-PCR can be performed on formalin-fixed, paraffin-embedded tissue and can be designed to examine multiple genes per reaction, making it much cheaper that a microarray analysis
    2. It is also highly sensitive and reproducible in other lab environments
5. another interesting application being investigated is attempting to determine a handful of genes that are differentially expressed in aggressive tumours of various cellular origins
  1. a single gene expression pattern, representing an activated wound healing phenotype, was a significant indicator of metastasis and death in breast, lung and gastric cancer
    1. this gene expression pattern, known as the core serum response, consisted of 512 genes, including genes that were involved in the cell cycle, with cell motility, extracellular remodelling, cell-cell signalling, and myofibroblast phenotype development
6. In the post-genomic era, a cancer patient would not only know the grade and stage of the tumour or malignancy, but also the relevant gene expression pattern
  1. this information will become a tool for selecting the most promising drug regime, predicting the metastatic potential of the cancer and allowing the patients and physicians to weigh the relative merits of aggressive treatment earlier in the course of disease
  2. this potential, while not yet commercially available, will quickly become a reality.
    1. in the case of large B-cell lymphoma, this information can be obtained with a 6-gene RT-PCR assay that can be easily standardised in a clinical laboratory
    2. the potential of molecular profiling is not limited to lymphoma and breast cancer, progress is being made with molecular profiling of lung and prostate caner, as well as acute leukaemia
    3. the potential of molecular profiling is clear, and the hurdles for the implementation of this powerful new tool are rapidly being overcome

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Molecular profiling

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