Uveal melanomas

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• Uveal melanomas using MLPA for acquired
  disorders
• Trudie Cottrell
• Liverpool DNA Lab

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Overview

• Uveal melanomas- diagnosis and treatment.
• Chromosomal copy number changes and clinical
  significance.
• Sample types, DNA preparation and testing.
• MLPA- technical issues and data analysis.
• Future of Uveal melanoma diagnosis.

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• Uveal melanomas- cancer of the eye arising from
  pigmented melanocytes.

Ciliary melanoma
Iris melanoma

• Tumours progress from low grade into a high grade
  state.
• Liver metastases are common.
• Mortality rate 50 after 10-15yrs.

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Chromosomal changes in Uveal melanomas

• Chromosomal changes are non-random and relatively
  simple.
• Analysing tumour karyotype makes it possible to
  make predictions about the clinical outcome for
  the patient.
• Changes in copy number of chromosomes 3 and 8 are
  clinically important.
• Monosomy 3 is strongly correlated with metastatic
  spread (liver usually involved).
• Gains in copy number of chromosome 8 correlate
  with monosomy 3.

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Diagnosis and treatment

• Very rare condition- General Ophthalmologist may
  see 1-2 cases per year.
• 3 specialist ocular oncology centres in England-
  Liverpool, Sheffield and London.
• The melanomas are treated by either enucleation
  or local resection.
• Enucleation Removal of entire eye.
• Incisional biopsy Small piece of tumour is cut
  out.
• Excisional biopsy Entire tumour is removed.

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• Trans-vitreal biopsy a fine gauge vitreous
  cutter (vacuum) is passed through the eye to the
  middle of the tumour to remove small samples for
  analysis.

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• Prior to 2007- cytogenetic analysis was preformed
  by FISH on touch preps and on cell cultures. FISH
  probes for chromosomes 3 and 8 were used to look
  for aberrant copy number.
• In spring 2007 the Liverpool DNA lab introduced
  MLPA diagnostic testing of Uveal Melanomas. Today
  we test using MLPA in conjunction with FISH
  analysis by the Cytogenetics department in
  certain circumstances.
• We use the Salsa MLPA kit P027 Uveal Melanoma
  from MRC Holland.
• The excel spreadsheet for analysis was provided
  by Andrew Wallace at St. Marys hospital,
  Manchester.

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Salsa MLPA kit P027 Uveal Melanoma

• The probe mix contains probes for several regions
  that
• often show aberrant copy number in uveal
  melanomas.
• Chromosomal regions covered
• Chromosome 1p
• Loss of 1p may indicate decreased survival rates
  in conjunction with monosomy 3.
• Chromosome 3
• Monosomy 3 predicts metastatic spread.
• Chromosome 6
• Clinical significance of increases in copy number
  of chromosome 6 is not currently known. The
  frequency of 6p gains may be higher in
  non-metastasizing tumours.
• Chromosome 8
• This region is over expressed in high grade
  tumours, increased copy number of the long arm is
  often seen alongside monosomy 3.

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Normal result MLPA histogram showing dosage
ratios of equal heights. Two copies of each
chromosome gives each probe a dosage ratio of 1.
Normal chromosome copy number for 1p, 3, 6 8.
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Positive result Aberrant copy number of
chromosomes 3, 6q and 8.

• 2 copies chromosome 1p
• 1 copy chromosome 3
• 2 copies chromosome 6p
• 1 copy chromosome 6q
• 1 copy chromosome 8p
• 7 copies chromosome 8q

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Classic positive result- monosomy 3 with trisomy
8.
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Copy number can increase through structural
rearrangements such as isochromosomes. 1 copy
of the short arm of chromosome 8 has been lost
and the formation of isochromosomes has lead to 7
copies of 8q. Suggests 1 normal chromosome 3
isochromosomes.
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Advantages over FISH

• Many samples can be set up for MLPA testing
  simultaneously, saving time.
• FISH is very expensive.
• The FISH probes only look at chromosomes 3 and 8
  (and occasionally 6) whereas MLPA also looks at
  chromosome 1.
• FISH can fail to detect partial deletions. FISH
  probes detect the centromere.
• MLPA probes can detect a gain or loss of a
  chromosome arm or part of an arm.
• partial monosomy 3 could be missed in FISH
  analysis.

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Technical problems with MLPA

• Sample size and quality
• The lab receives solid tissue or cells as a fine
  needle aspirate.
• For large samples Cytogenetics retain some tissue
  to make touch preps.
• In the DNA lab, tissue is digested then extracted
  on the EZ1 robot.
• Samples digest at different rates.
• Yield and quality vary immensely. Fine needle
  aspirates can produce very low DNA yield.
• Evaporation during MLPA hybridization step
  (denature in plates then transfer to tubes).

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Monosomy 3 sample showing a high proportion of
normal cells.
The peak height for chromosome 3 would be
expected to drop by half. Due to a mixed cell
population of normal and tumour cells, chromosome
3 probes have only dropped to approx 0.7
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Example of a sample which failed internal quality
due to irregular control probe. Repeat testing of
the sample produced the same pattern. Poor
internal quality may be a genuine result and
should not be rejected outright.
Internal quality failure
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Post-MLPA

• All monosomy 3 results are confirmed by FISH
• - ? Polyploidy
• Any equivocal MLPA results are also set up for
  FISH analysis in cytogenetics.
• The future
• Structural rearrangements cannot be characterized
  by MLPA.
• Clinical significance of chromosomes 1 6?
• Collating MLPA data will help to create a better
  picture of the clinical significance of copy
  number changes and small deletions/ duplications.