FRCPath+picture-based+questions (1)

Matthew Evans (matthew.evans7@nhs.
net)
31/05/2021
FRCPath questions: picture-based

questions
Picture-based question 1
A 55-year-old man is diagnosed with colorectal cancer. MMR immunohistochemistry is
performed.
Images adapted from VENTANA MMR IHC Panel Interpretation Guide for Staining of Colorectal Tissue
Which is the correct interpretation?

A The results are equivocal and testing must be repeated
B This case needs BRAF mutation testing
C This is a normal result
D This patient requires genetics referral
E This result excludes Lynch syndrome
Page 1 of 39
Matthew Evans (matthew.evans7@nhs.net)
31/05/2021
A 52-year-old woman is diagnosed with endometrioid endometrial carcinoma. MMR
immunohistochemistry is performed.

A This case needs BRAF mutation testing
B This case needs MLH1 promoter methylation testing
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You are validating MMR staining in your laboratory using a variety of archival colorectal
cancers. The results of staining of four different colorectal cancers (1-4) are shown below.

A Specimen (1) shows loss of expression of MLH1
B Specimen (2) shows loss of expression of MLH1
C Specimen (3) shows preserved expression of MLH1
D Specimen (4) shows preserved expression of MLH1
E Specimen (4) shows uninterpretable staining
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Four colorectal cancer samples undergo MSI testing (tumours 1-4), alongside sample
normal tissue. The results of MSI testing for the markers BAT-25 and BAT-26 are
provided.
Images adapted from https://www.ous-research.no/home/lothe/methods/2766

A Testing for tumour 3 has failed
B Tumour 1 shows microsatellite instability with respect to both markers
C Tumour 2 shows microsatellite instability with respect to both markers
D Tumour 3 shows microsatellite instability with respect to BAT-25
E Tumour 4 shows microsatellite instability with respect to both markers
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A colorectal cancer sample is sent to you for KRAS, NRAS and BRAF mutation testing. An
H&E-stained tumour cellularity assessment slide is produced.
Images adapted from https://www.ous-research.no/home/lothe/methods/2766

A A technique with a limit of detection of 20% mutant alleles is likely to detect
mutations present
B If a technique with a minimum tumour cell content of 50% detects a mutation, the
result may not be valid
C If a technique with a minimum tumour cell content of 70% detects a mutation, the
result will be valid
D This sample will not be amendable to macrodissection
E Tumour cell content here is around 50%
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A tumour sample undergoes Sanger/direct sequencing. An excerpt of the result is
provided. You may assume that base 1 in the results is at the start of the gene being
sequenced.
Images adapted from Ding, Juan & Zhao, Dandan & Du, Renqian & Zhang, Yuehua & Yang, Haipo & Liu, Jieyu & Yan,
Chuanzhu & Zhang, Feng & Xiong, Hui. (2015). Clinical and molecular genetic analysis of a family with late-onset LAMA2-
related muscular dystrophy. Brain & development. 38. 10.1016/j.braindev.2015.08.005.
Images adapted from https://biology.stackexchange.com/questions/45545/deducing-amino-acid-sequence-from-a-dna-

sequence

A c.10C>G
B c.14A>T
C c.3G>T
D p.G10C
E p.L4V
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A tumour sample undergoes next-generation sequencing. An excerpt of the result is
provided.

A Base position 93,830,157 has been sequenced with a likely lower degree of
confidence than 93,830,150
B Most base positions here have been sequenced with a read depth of 10×
C There is a C>T substitution at base position 93,830,137
D There is a T>C substitution at base position 93,830,137
E There is evidence of a germline substitution mutation at base position 93,830,137
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A tumour sample undergoes next-generation sequencing. An excerpt of the result is
provided.
Non-synonymous tumour mutation burden: 4.6 mutations/MB
Images adapted from https://www.genomicsengland.co.uk/wp-content/uploads/2016/11/image-1.jpg

A This is a highly mutated tumour
B This patient is female
C This patient is likely to respond to immune checkpoint inhibitors
D This tumour likely has a BRCA mutation
E This tumour likely has an MMR defect
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A tumour sample undergoes pyrosequencing. An excerpt of the result is provided, along
with the reference sequence.
Reference sequence ATCGAATGTT
Images adapted https://www.sciencedirect.com/science/article/pii/B9780124104716000037

A There is evidence of a C deletion
B There is evidence of a GG insertion
C There is evidence of a substitution
D There is evidence of an A deletion
E There is no evidence of a mutation
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A 46-year-old woman is diagnosed with EGFR-mutated lung adenocarcinoma. She is
treated with erlotinib, but a year later progresses. She undergoes repeat biopsy. Real-time
PCR is used to test for an EGFR T790M mutation. You may assume that any signal which
passes threshold on this graph is a valid signal.
Cycle number
Images https://www.spandidos-publications.com/10.3892/ol.2016.4263

A The internal control has failed and so the T790M signal cannot be regarded as
genuine
B The T790M signal does not meet threshold and so cannot be regarded as
genuine
C The T790M signal is detected earlier than the internal control signal
D The T790M signal is not as strong as the internal control and so cannot be
regarded as genuine
E This tumour harbours a T790M mutation
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A 65-year-old man is diagnosed with colorectal cancer. BRAF V600E
immunohistochemistry is undertaken.

A This patient does not have Lynch syndrome
B This result needs to be confirmed by PCR
C This tumour is unlikely to have a KRAS mutation
D This tumour may have a BRAF mutation outside codon 600
E This tumour shows loss of nuclear expression of BRAF
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A 48-year-old man is diagnosed with non-small cell lung cancer. ALK
immunohistochemistry is undertaken.
Images adapted from VENTANA anti-ALK (D5F3) Rabbit Monoclonal Primary Antibody Interpretation Guide for Non-Small
Cell Lung Carcinoma (NSCLC)

A The tumour is very likely to be an adenocarcinoma
B The tumour likely has an underlying ROS1 translocation
C There is no staining in internal controls and so the result is not valid
D This confirms the presence of an ALK1-EML4 translocation
E This result requires confirmation by FISH or NGS
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A 45-year-old woman is diagnosed with ductal carcinoma NST of the breast. HER2
immunohistochemistry is undertaken. You may assume that this is representative of the
entire tumour.
Images adapted from http://www.pathologyoutlines.com/imgau/breastmalignanther2Reisenbichler03.jpg

A HER2 expression is heterogeneous and so it is good practice to undertake FISH
B There is no nuclear staining and so this is not genuine HER2 expression
C This confirms that the tumour is invasive rather than DCIS
D This patient is likely to respond to trastuzumab
E This result requires confirmation by FISH
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A 62-year-old man develops a rash. Biopsies from two separate areas shows a heavy
lymphoid infiltrate, with a differential diagnosis between a cutaneous T-cell lymphoma and
a reactive lymphoid infiltrate. TCR clonality testing is undertaken on both biopsies.
Images adapted from https://link.springer.com/article/10.1007/s12308-008-0013-9/figures/6

A A reactive proliferation would not give rise to this result
B The fact that the same results are seen in both biopsies suggests that the result is
artefactual
C The signal occurs too late in the reaction to be considered genuine
D This finding supports a diagnosis of a reactive lymphoid proliferation
E This finding supports a diagnosis of T-cell lymphoma
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A 54-year-old woman is diagnosed with lung adenocarcinoma. ROS1
immunohistochemistry shows patchy positivity. ROS1 FISH is undertaken using dual-
colour break-apart probes. The green probe hybridises to the ROS1 gene and the orange
probe to a control region. You can assume that this is representative of the entire sample.
Images adapted from https://www.molecular.abbott/int/en/products/oncology/vysis-alk-break-apart-fish-probe-kit

A All signals are fused, so there is no evidence of ROS1 translocation
B All signals are split, consistent with ROS1 translocation
C Two signals are split and one is fused, consistent with ROS1 translocation
D Only one signal is present, so this nucleus cannot be assessed
E Only two signals are split, so this is insufficient for diagnosis of ROS1
translocation
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A 20-year-old man is diagnosed with a small round blue cell tumour of the tibia. EWSR1
FISH using dual-colour break-apart probes confirms the presence of an EWSR1
translocation. ESWR1-FLI1 translocation testing is undertaken using dual-colour dual-
fusion probes (green EWSR1, orange FLI1). You can assume that this is representative of
the entire sample.
Images adapted from http://store.biogenex.com/us/applications/fish/efish-probes/oncology-probes/cmyc-igh-dual-color-dual-

fusion-probe-3943.html

A FISH testing for alternative fusion partners is advisable
B This raises the possibility of synovial sarcoma
C This casts doubt over the original EWSR1 FISH result
D This confirms a diagnosis of Ewing sarcoma
E This confirms the presence of a translocation involving FLI1
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A 65-year-old woman is diagnosed with breast cancer. HER2 FISH is performed. The
green probe hybridises to CEP17 and the orange probe to the HER2 gene. You can
assume that this is representative of the entire sample.
Images adapted from http://www.ikonisys.com/media/cms_page_media/47/oncoFISH%20her2%20b.jpg

A HER2 copy number is 10
B HER2 copy number is 4
C HER2 copy number is 8
D HER2:CEP17 ratio is 8
E There is evidence of chromosome 17 aneusomy
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A 58-year-old man is diagnosed with a high-grade mature B-cell lymphoma. FISH using
dual-colour break-apart probes for MYC, BCL2 and BCL6 rearrangement is performed (A,
B and C, respectively).
Images adapted from

http://www.demosmedical.com/media/samplechapters/9781620700945/mobile/9781620700945_Chapter1.html

A B-cell acute lymphoblastic leukaemia
B Burkitt lymphoma
C Diffuse large B-cell lymphoma
D High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements
(‘double-hit lymphoma’)
E High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements
(‘triple-hit lymphoma’)
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A 24-year-old woman is diagnosed with cervical squamous cell carcinoma. An oncologist
requires definitive assessment of HPV status. You perform high-risk HPV RNA ISH. A
medium- and high-power image is provided.
Images adapted from Interpretation Guide for Ventana INFORM® HPV Probes In Situ Hybridization (ISH) Staining of
Cervical Tissue

A p16 immunohistochemistry could have been used as an alternative
B The tumour is positive for high-risk HPV
C There is no strong, diffuse nuclear staining and so this cannot be regarded as
genuine positivity
D This staining could indicate low-risk HPV infection
E This staining could indicate non-productive HPV infection
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There is preserved nuclear staining in the tumour cells (as well as in the internal control
stromal and inflammatory cells) for MLH1 and PMS2. For MSH2 and MSH6, there is
preserved staining in the stromal and inflammatory cells demonstrating that staining has
worked. However, there is loss of staining for both markers in the tumour cells. This is
most likely caused by a germline mutation in either the MSH2 or MSH6 genes (although
other mechanisms are possible), and therefore there is a high likelihood that the patient
has Lynch syndrome. This patient therefore requires genetics referral. The geneticists will
sequence the MMR genes from non-tumour tissue to identify a germline mutation.
A The results are equivocal and testing must be repeated

There is good, strong staining in internal controls for all four proteins so staining
has definitely worked. There is absolutely no staining in the tumour cell nuclei for
MSH2 and MSH6, and so there is no doubt that this is genuine loss.
B This case needs BRAF mutation testing
Had there been loss of expression of MLH1 and PMS2, this would be correct.
BRAF mutation testing is needed in the first instance, followed by MLH1 promoter
methylation testing if BRAF is wildtype. This helps to distinguish between
sporadic and germline MMR defects. For cases showing MSH2 and MSH6 loss,
the cause is highly likely to be germline and so genetics referral is required
instead.
A normal result would be nuclear staining for MLH1, PMS2, MSH2 and MSH6 in
tumour cells which is at least as strong as the staining seen in internal controls.
Correct
No MMR result excludes Lynch syndrome. Even if expression of all four proteins
is preserved, there is a slim possibility that there is an underlying germline
mutation which has not resulted in loss of staining. It is therefore important that
clinicians are not falsely reassured if there is a strong family history.
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There is loss of nuclear staining in the tumour cells for MLH1 and PMS2, with strong
staining in the internal control stromal and inflammatory cells (indicating that staining has
worked); MSH2 and MSH6 staining is preserved. There are many possible causes for this,
but only three are likely: sporadic hypermethylation of the MLH1 promoter region, a
germline mutation in MLH1, or a germline mutation in PMS2. The first step is to perform
BRAF mutation testing. If there is a BRAF mutation, it is highly likely that the MMR defect
is due to sporadic hypermethylation of the MLH1 promoter and no further testing is
required. If BRAF is wildtype, direct testing of the level of methylation of the MLH1
promoter is required. If this shows hypermethylation (generally > 20% methylation, or
significantly higher methylation than seen in non-tumour tissue), the defect can be
considered sporadic; otherwise there is a high likelihood of Lynch syndrome and the
patient requires genetics referral.
A This case needs BRAF mutation testing

Correct
B This case needs MLH1 promoter methylation testing
This is not an unreasonable answer. It would be reasonable to skip out BRAF
mutation testing and simply perform MLH1 promoter methylation testing.
However, MLH1 promoter methylation testing is more costly and complex than
BRAF mutation testing, and needs to be performed only in a minority of cases if
BRAF mutation testing is performed.
A normal result would be nuclear staining for MLH1, PMS2, MSH2 and MSH6 in
tumour cells which is at least as strong as the staining seen in internal controls.
The majority of cases which show MLH1 and PMS2 loss have sporadic MLH1
promoter hypermethylation. Referring all these cases for genetics review would
therefore generate a large amount of unnecessary work.
No MMR result excludes Lynch syndrome. Even if expression of all four proteins
is preserved, there is a slim possibility that there is an underlying germline
mutation which has not resulted in loss of staining. It is therefore important that
clinicians are not falsely reassured if there is a strong family history.
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A Specimen (1) shows loss of expression of MLH1
In specimen (1) there is no staining for MLH1 in the tumour cell nuclei. However,
there is also absolutely no staining in the internal control stromal and
inflammatory cells. In order to consider a case as showing loss of expression,
there must be significantly weaker (or totally lost) staining in the tumour cells than
is seen in the internal controls. As such, staining is this case is uninterpretable. It
should be repeated (in a biopsy specimen, if this is a resection, in the hope of
there being better fixation). If this does not work, MSI testing may be a helpful
alternative.
B Specimen (2) shows loss of expression of MLH1
Correct. Here, the internal control stromal and inflammatory cells are
stained, and so staining has worked. There appears to be some staining in
the tumour cell nuclei, but this is far less extensive and far weaker than is in
the internal controls. This is therefore regarded as loss of expression. It is
important to remember that – although loss of MMR protein expression is
normally total – relative loss of expression can occur and has the same
implication as total loss; they key is to compare the staining in the tumour
cells with the staining in internal controls.
C Specimen (3) shows preserved expression of MLH1
Specimen (3) shows only weak cytoplasmic staining in the context of well-stained
internal controls. Cytoplasmic staining is not sufficient to consider a tumour MMR-
proficient. There is therefore loss of expression.
D Specimen (4) shows preserved expression of MLH1
Specimen (4) shows only weak cytoplasmic staining in the context of well-stained
internal controls. Cytoplasmic staining is not sufficient to consider a tumour MMR-
proficient. There is therefore loss of expression.
E Specimen (4) shows uninterpretable staining
There are well-stained internal control stromal and inflammatory cells in this case,
and so the staining has technically worked. In rare cases, cytoplasmic staining
may make it impossible to determine whether there is genuine nuclear staining; in
these cases, repeat staining on a different sample or MSI testing may be useful.
In this case, the cytoplasmic staining is weak, and it is easy to see that there is no
nuclear staining.
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MSI testing involves assessing the length of a set of microsatellites within a tissue sample.
In a microsatellite stable tissue, all the alleles of each microsatellite with be of a simple
length; if plotted, they will form an approximate normal distribution. If there is an MMR
defect, errors will be induced in the process of DNA replication which will allow for
microsatellites to contract and expand. Over time, therefore, all the alleles of each
microsatellite in a sample will be of widely varying lengths and the normal distribution will
be lost. In most cases, MSI testing involves assessing the lengths of five microsatellites,
and requires a non-tumour tissue sample from the same patient for comparison. Instability
in at least two microsatellites is regarded as MSI-H; instability in one microsatellite is
regarded as MSI-L (which, though controversial, is generally managed as MSI-H); stability
in all microsatellites is regarded as MSS (microsatellite stability).
A Testing for tumour 3 has failed

Tumour 3 shows normal distributions for both microsatellites, which closely
resemble those seen in the normal tissue. There is no indication that there is a
problem with the test. Both microsatellites are stable.
B Tumour 1 shows microsatellite instability with respect to both markers
For tumour 1, BAT-26 testing shows a normal distribution which resembles that
seen in the normal tissue; BAT-26 is stable. However, for BAT-25 the plot shows
two broad peaks which is in contrast to the normal distribution in the normal
tissue. This sample therefore shows instability with respect to BAT-25 only.
Assuming that all other markers are stable, this tumour would be regarded as
MSI-L; the patient would generally be worked up as if the tumour were MSI-H,
although this is controversial.
C Tumour 2 shows microsatellite instability with respect to both markers
For tumour 2, both markers show normal distributions which closely resemble
those seen in the normal tissue. Both microsatellites are therefore stable.
D Tumour 3 shows microsatellite instability with respect to BAT-25
For tumour 3, both markers show normal distributions which closely resemble
those seen in the normal tissue. Both microsatellites are therefore stable.
E Tumour 4 shows microsatellite instability with respect to both markers
Correct. For tumour 4, both markers show two separate peaks, whereas the
normal tissue shows a single normal distribution for both markers. This
indicates that both markers show microsatellite instability. Irrespective of
the other markers, this tumour is MSI-H.
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This sample shows a lymph node which is involved by metastatic adenocarcinoma. All
nucleic acid-based techniques have a limit of detection. This is the minimum proportion of
mutant alleles which can reliably be detected in a sample (e.g. if a test has a limit of
detection of 10%, but the sample contains only 5% mutant alleles the test may well miss
the mutation). Normally, all somatic cells harbour two copies (alleles) of a given gene and
in the context of a somatic mutation, only one allele will be mutated; therefore, the
minimum tumour cell content will be twice the limit of detection (e.g. limit of detection of
10% translates into a minimum tumour cell content of 20%). In the example tissue
present, there are a few malignant glands in a background of a lymph node which
contains huge numbers of lymphocytes. For every one malignant cell in this section, there
are at least ten non-tumour cells. Tumour cell content is therefore far less than 10%.
A A technique with a limit of detection of 20% mutant alleles is likely to detect

mutations present
A technique with a limit of detection of 20% will be expected to require a minimum
tumour cell content of 10%. In the image provided, tumour cell content is far less
than 10%. If testing reveals a mutation, the result can be believed. However, if the
technique fails to detect a mutation, it could be that this represents a false
negative result. The report of testing must include a caveat to this effect.
B If a technique with a minimum tumour cell content of 50% detects a mutation, the
result may not be valid
A minimum tumour cell content of 50% translates into a minimum tumour cell
content of 25%. This sample has a tumour cell content of less than 10%.
Therefore, if testing returns a wildtype result this cannot be entirely trusted.
However, if a mutation is detected, this result can be believed and no caveat
needs to be provided in the report.
C If a technique with a minimum tumour cell content of 70% detects a
mutation, the result will be valid
Correct. A minimum tumour cell content of 70% translates into a minimum
tumour cell content of 35%. If this technique returned a wildtype result, this
could represent a false negative result. However, if a technique returns a
mutant result – even if the sample cellularity is lower than the technique’s
required tumour cell content – this result can be believed.
D This sample will not be amendable to macrodissection
Macrodissection can be used to improve the tumour cell content of a sample. An
H&E assessment slide is examined and any non-tumour tissue marked. Sections
are then cut for testing, and the H&E slide is used as a guide to scrape the non-
tumour tissue off the section using a blade or pipette tip. In this way, the tumour
cell proportion of the tissue can be increased. Macrodissection is not feasible for
samples where small areas of tumour are intermingled with areas of non-tumour
(e.g. a cytology cell block). Here, the upper half of the lymph node could be
removed from the section, removing most of the lymphoid tissue and thereby
significantly increasing the tumour cell content.
E Tumour cell content here is around 50%
Tumour cell content here is less than 10%. Even with macrodissection, tumour
cell content could only be increased to around 10%. Lymph node metastases are
often very poor samples for nucleic acid-based testing because the large
numbers of small lymphocytes results in very low tumour cell content.
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Sanger/direct sequencing involves determining the actual sequence of a nucleic acid.
Here, there is one peak for a nucleotide corresponding to each base position in the
sequence. However, there appear to be two peaks at base position 10. This indicates that
two different nucleotides are present at this base position and, by extension, that there is
evidence of a mutation here. You are told that a G should be present at this base position
according to the reference sequence, but there is also a C signal. This is indicative of a G
to C substitution at base position 10. Using standard nomenclature, this is described as
c.10C>G. Each set of three nucleotides (a codon) encodes an amino acid. The first codon
(GTG) encodes valine; the second (TGA) encodes a stop codon (this is only an example);
the third (GCT) encodes alanine. The fourth codon (GTG) should normally encode valine;
by G to C substitution changes the codon to CTG which encodes leucine. There is
therefore a valine to leucine change in codon four, which is described as p.V4L.
A c.10C>G
Correct
B c.14A>T
C c.3G>T
D p.G10C
E p.L4V
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In most cases, NGS involves first amplifying the nucleic acid sample and randomly
cleaving it into many short fragments. Each of these fragments is then sequenced; each of
these sequences is called a ‘read’. Because the nucleic acid was initially amplified, each
point in the sequence should be covered by multiple reads. Bioinformatics software aligns
all these reads with each other and with a reference human genome sequence. Errors can
occur during sequencing and some positions may not be sequenced at all. If a particular
base position has been sequenced in only one read, this base cannot be ‘called’ with any
confidence because it may simply be an error. The more times a given base position is
read, the greater the confidence with which that base can be called; this is referred to as
the ‘depth’ or ‘coverage’. If a given base position has been called in twenty reads, it is said
to have a depth of 20×; the higher the depth, the greater the confidence.
A Base position 93,830,157 has been sequenced with a likely lower degree of
confidence than 93,830,150
Correct. In the image provided, position 93,830,157 has not been covered in
four reads; 93,830,150 has been covered in all. Position 93,830,157 has
therefore lower coverage/depth than position 93,30,150 and any results
from sequencing it will have a lower degree of confidence. The difference
between these two positions is small, though, and so there is little
difference in confidence.
B Most base positions here have been sequenced with a read depth of 10×
29 read tracks are shown here, and since most base positions here are included
in all the reads, most base positions have been sequenced with a read depth of
29×.
C There is a C>T substitution at base position 93,830,137
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The reference sequence shows that at this position, there should ‘normally’ be a
T. Therefore, if anything, this could only be regarded as a T>C substitution, but
even this is unlikely (see D, below).
D There is a T>C substitution at base position 93,830,137
At position 93,830,137 almost all the reads have returned the T which is expected
at this position from the reference sequence. A single read has returned a C at
this position. While it is of course possible that this could represent a genuine
substitution mutation present in a very small proportion of alleles present, it is
more likely that this simply represents an error in sequencing. Bioinformatics
software would generally disregard this as background noise and would not flag it
as a potential variant.
E There is evidence of a germline substitution mutation at base position 93,830,137
For a heterozygote with a germline mutation, one would expect to see that
mutation in half of all reads because half of the alleles (either maternal or
paternal) would be mutant. For a homozygote with a germline mutation, one
would expect to see that mutation in all reads. Therefore, in order for a mutation
to be considered germline, it should be seen in at least half of all reads. At
position 93,830,137 there is a C rather than the expected T in only one of 29
reads, and so this is unlikely to be a genuine mutation, let alone a germline
mutation.
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This is a Circos plot. It is a visual representation of data from whole genomic sequencing.
Each of the chromosomes is laid out around the plot. The lines running through the centre
of the plot represent large-scale rearrangements of DNA. Other than this, there is no
consistent way of designing Circos plots. Around the periphery, most Circos plots also
visualise single nucleotide pleomorphisms (SNPs) and copy number variation. It therefore
gives an approximate representation of the amount of damage in a genome. Below the
plot is a measurement of non-synonymous tumour mutation burden (TMB). This is a
measurement of the number of mutations in the genome which would be expected to
produce different amino acids; synonymous mutations are mutations which do not result in
a different amino acid and so would not be expected to have any functional effect. TMB is
measured in the average number of mutations per megabase of DNA. The higher the
TMB, the more abnormal proteins a tumour cell is likely to express and the greater the
potential immune response against the tumour.
A This is a highly mutated tumour

10 mutations/MB is generally taken to be the cut-off for a highly mutated tumour,
although this is only a rough figure. The Circos plot is also not particularly ‘busy’.
B This patient is female
Correct. There is essentially nothing in the part of the Circos plot
corresponding to the Y chromosome. This sample does not contain a Y
chromosome and the patient is therefore female.
C This patient is likely to respond to immune checkpoint inhibitors
Highly mutated tumours typically show good responses to immune checkpoint
inhibitors; this is because the large numbers of mutations result in large numbers
of abnormal proteins which are detected by the immune system. Immune
checkpoint inhibitors release the brakes on immune responses, and so allow
immune cells to attack the tumour. This tumour has a fairly low TMB and so would
generally not be expected to respond well to checkpoint inhibitors. However,
many poorly understood factors determine response to checkpoint inhibitors, and
so this is not definite.
D This tumour likely has a BRCA mutation
BRCA1/BRCA2 are involved in repairing double stranded DNA breaks. BRCA
mutations therefore result in severe DNA damage. This is not reflected in either
the Circos plot or the TMB. A BRCA mutation is therefore unlikely.
E This tumour likely has an MMR defect
The MMR system is involved in repairing mismatch errors introduced during DNA
replication. MMR defects therefore result in extensive DNA damage. These
tumours tend to have TMB higher than 10 mutations/MB (and often much higher
than this). An MMR defect is therefore unlikely.
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In pyrosequencing, nucleotides are added in turn. When a nucleotide binds, light is
produced; the intensity of the light is proportional to the number of nucleotides which bind.
In this example, A is added first and there is a peak indicating that binding has occurred. T
is then added and binds. C is added and binds. Then G is added; there is a peak here
which is around three times larger than the peaks for A, T or C, and so it can be inferred
that three G have bound. C is then added but there is no light detected, indicating that
there is no C at this position. Overall, then, the detected sequence is ATCGGGAATGTT.
The reference sequence is ATCGAATGTT. There is therefore evidence of a GG insertion.
A There is evidence of a C deletion

B There is evidence of a GG insertion
Correct
C There is evidence of a substitution
A substitution would manifest as two less-than-full-height peaks next to each
other, together adding up to approximately a full height peak.
D There is evidence of an A deletion
E There is no evidence of a mutation
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EGFR-mutated lung cancers treated with TKIs inevitably develop secondary resistance,
usually after around twelve months. In around half of cases, this is because the tumour
cells acquire a secondary T790M mutation in the EGFR gene. This is important because
changing the patient to a third generation TKI can re-establish tumour control if a T790M
mutation is present. Patients who progress on anti-EGFR TKI therapy therefore often
undergo repeat biopsy with EGFR mutation testing to assess for a secondary T790M
mutation.
Real-time PCR uses the fluorescence from probes to detect specific mutations of interest;
it is therefore different from sequencing which can be used to detect any mutations
present in the material sequenced. It is possible to use multiple probes for different
mutations in a single reaction to test for multiple different mutations in parallel. For each
probe, there will be a fluorescence curve. If the curve crosses a threshold within a certain
number of PCR cycles of the control (ΔCQ), it is considered a valid signal and the
mutation is considered present. If the curve does not cross the threshold or if it only
crosses the threshold a long time after the control, the mutation cannot be regarded as
having been detected.
A The internal control has failed and so the T790M signal cannot be regarded as
genuine
The internal control has formed an appropriate curve which crosses the threshold,
and so has worked.
B The T790M signal does not meet threshold and so cannot be regarded as
genuine
The T790M signal crosses the threshold (the horizontal green line). Had this
crossed the threshold with too high a ΔCQ, it would be considered invalid;
however, you are told that anything which crosses the threshold on the provided
graph can be considered genuine.
C The T790M signal is detected earlier than the internal control signal
The x-axis indicates the PCR cycle number. The internal control signal crosses
the threshold at around cycle 21, whereas the T790M signal crosses around cycle
28. The T790M signal is therefore detected later than the internal control signal.
D The T790M signal is not as strong as the internal control and so cannot be
regarded as genuine
The overall height of the curve does not matter, as long as the signal cross the
threshold with a sufficiently low ΔCQ.
E This tumour harbours a T790M mutation
Correct. Because there is a valid internal control signal and the T790M
signal crosses the threshold with a sufficiently low ΔCQ, a valid T790M
signal has been detected and there is evidence of a T790M mutation.
Page 30 of 39
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In some cases, immunohistochemistry can be used to detect the protein changes
downstream of a molecular alteration, and thereby to infer the presence of the molecular
alteration. BRAF V600E immunohistochemistry can be used to detect the protein product
of the V600E mutated BRAF gene. Here, there is convincing cytoplasmic staining in the
tumour cells which is consistent with the presence of a BRAF V600E mutation. There is no
staining with the protein product of either the wildtype or non-V600E-mutated forms of the
gene.
A This patient does not have Lynch syndrome

BRAF mutation status in itself provides no information about whether or not the
patient has Lynch syndrome. Had you been told that the tumour showed loss of
expression of MLH1 and PMS2, positive staining with BRAF V600E
immunohistochemistry would confirm that the MMR defect is likely sporadic rather
than germline, but this information is not provided here.
B This result needs to be confirmed by PCR
BRAF V600E immunohistochemistry is highly concordant with PCR, and so
positive staining definitively confirms the presence of a BRAF V600E mutation.
However, negative staining only indicates that there is no BRAF V600E mutation;
it does not exclude the possibility of there being a non-V600E BRAF mutation
(although these mutations are uncommon). It is therefore advisable that negative
results be confirmed with PCR.
C This tumour is unlikely to have a KRAS mutation
Correct. This result indicates that the tumour harbours a BRAF V600E
mutation. In colorectal cancer, mutations in BRAF, KRAS and NRAS are
essentially always mutually exclusive. It is extremely unlikely that the
tumour also has a KRAS mutation.
D This tumour may have a BRAF mutation outside codon 600
BRAF V600E immunohistochemistry is specific for the BRAF V600E mutation.
Therefore, positive staining indicates that a V600E mutation is present. If staining
is negative, however, all that can be inferred is that there is no V600E mutation; it
is entirely possible that there is a non-V600E BRAF mutation present and, if this
could be clinically important, PCR needs to be performed for confirmation.
E This tumour shows loss of nuclear expression of BRAF
BRAF V600E manifests as cytoplasmic staining. This case therefore shows
positive cytoplasmic staining indicating the presence of a BRAF V600E mutation.
Page 31 of 39
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In some cases, immunohistochemistry can be used to detect the protein changes
downstream of a molecular alteration, and thereby to infer the presence of the molecular
alteration. In the case of ALK translocations, the translocation results in overexpression of
the ALK protein. As such, tumours which have ALK translocations will demonstrate strong
cytoplasmic staining which is usually homogeneous. Importantly, if ALK
immunohistochemistry is being performed with a view to prescribing targeted therapy in
non-small cell lung cancer, it is essential that the VENTANA D5F3 assay is used; other
assays are not validated for this purpose. This tumour shows strong cytoplasmic staining
which indicates that there is an underlying ALK translocation.
A The tumour is very likely to be an adenocarcinoma

Correct. ALK translocations are found in around 2% of non-small cell
carcinomas, essentially all of which are adenocarcinomas. ALK
translocations are essentially never found in squamous cell carcinomas.
Identification of an ALK translocation in a biopsy- or cytology-diagnosed
squamous cell carcinoma suggests that the tumour may actually be an
adenosquamous carcinoma in which the adenocarcinomatous component
has not been sampled.
B The tumour likely has an underlying ROS1 translocation
The presence of strong cytoplasmic staining indicates that there is an underlying
ALK translocation. EGFR mutations, ALK translocations and ROS1 translocations
are almost always mutually exclusive. It is extremely unlikely that the tumour also
has a ROS1 translocation.
C There is no staining in internal controls and so the result is not valid
ALK is not generally expressed by normal epithelium, stroma or inflammatory
cells. Unlikely MMR immunohistochemistry, therefore, whether staining has
worked cannot be inferred by staining in the background tissue. Usually separate
on-slide control tissue is used to confirm that staining has worked appropriately;
this is often appendix, since the ganglion cells in its wall should be positive for
ALK.
D This confirms the presence of an ALK1-EML4 translocation
This staining confirms the presence of a ALK overexpression, and from that it can
be inferred that there is a translocation involving the ALK gene. This does not,
however, give any indication of what the fusion partner is. EML4 is the
commonest ALK fusion partner, but the fact that this is an ALK-EML4 fusion
cannot be determined using immunohistochemistry. This would require
sequencing or FISH.
E This result requires confirmation by FISH or NGS
FISH was originally the gold standard technique for testing for ALK translocations.
It is now known that ALK immunohistochemistry (strictly using the VENTANA
D5F3 assay) is superior in predicting response to anti-ALK drugs. This is likely
because not all ALK translocations detected by FISH will result in ALK
overexpression; targeting these uncommon FISH-positive IHC-negative ALK
translocations will therefore not result in clinical benefit. ALK
immunohistochemistry is therefore now the gold standard technique and does not
require confirmation. RNA NGS has so far been shown to be roughly equivalent
to immunohistochemistry but immunohistochemistry is still considered the gold
standard technique.
Page 32 of 39
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HER2 gene amplification in breast cancer predicts response to trastuzumab (Herceptin).
ISH techniques are considered the gold standard for assessing for HER2 amplification.
However, at the extremes, immunohistochemistry for HER2 is an accurate surrogate
marker for HER2 gene amplification. Assessment is based on the proportion of cells
expressing membranous HER2, the completeness of staining, and the intensity of
staining. Cases which show HER2 expression scored 0 or 1+ can confidently be said not
to harbour HER2 gene amplification; those with a score of 3+ can confidently be said to
have underlying HER2 gene amplification. For those with a score of 2+, confirmation with
ISH is required. This case shows strong, membranous expression of essentially all tumour
cells, and so is scored 3+; ISH is not required.
A HER2 expression is heterogeneous and so it is good practice to undertake FISH

It is true that cases which show heterogeneous staining with
immunohistochemistry may benefit from FISH. In general, if a biopsy sample
shows heterogeneous staining, staining is best repeated on the resection
specimen. If the staining still is heterogeneous and difficult to score, FISH may
help to clarify the situation. This case does not show heterogeneity and so FISH
is not required.
B There is no nuclear staining and so this is not genuine HER2 expression
Only membranous staining is assessed for HER2.
C This confirms that the tumour is invasive rather than DCIS
Both invasive carcinoma and DCIS can express HER2, and HER2
immunohistochemistry cannot help in distinguishing between them. It is important
that only invasive carcinoma is scored. It may therefore be useful to assess HER2
staining alongside myoepithelial markers, to ensure that in situ disease is not
inadvertently being scored.
D This patient is likely to respond to trastuzumab
Correct. This case is scored 3+, and so the patient is likely to respond to
HER2-targeted therapy.
E This result requires confirmation by FISH
Cases scored 3+ on immunohistochemistry can confidently be regarded as
having underlying HER2 gene amplification. There is no need to perform FISH.
Page 33 of 39
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It may be difficult to distinguish between reactive and neoplastic B-cell and T-cell
proliferations. This is particularly true for cases of cutaneous T-cell lymphomas where it is
frequently difficult to exclude the possibility of a reactive T-cell infiltrate. Here TCR
clonality may be useful. The TCR genes undergo rearrangement in T-cell development. In
a reactive proliferation, each cell will undergo different TCR rearrangement resulting in
TCR genes of different lengths; if these lengths are plotted, they will form a normal
distribution – a polyclonal pattern. However, in a neoplastic proliferation, most cells will
come from the same clone and so will have undergone the same TCR rearrangement – all
the neoplastic cells will have the same TCR gene length, resulting a monoclonal pattern.
This can help, alongside clinical information, morphology and immunohistochemistry, to
determine whether a proliferation is reactive or neoplastic. In this case, TCR clonality
testing reveals very sharp peaks with look identical in testing of both biopsies; this is
suggestive of a neoplastic T-cell proliferation. In a reactive proliferation, one would expect
to see broad normal distributions which may look slightly different in both biopsies.
A A reactive proliferation would not give rise to this result

A reactive proliferation would be unlikely to give rise to this result, but it would not
be impossible. This can occur particularly in cases which have limited numbers of
lymphoid cells (‘pseudoclonality’). It is therefore important that the results of
clonality testing be interpreted alongside all other available information.
B The fact that the same results are seen in both biopsies suggests that the result is
artefactual
Although it is possible to see ‘monoclonal’ clonality results in a reactive
proliferation, it would be very unusual to see a monoclonal spike occurring at the
same amplicon length in two biopsies. It is therefore useful for clinicians to take
more than one biopsy in cases of suspected cutaneous lymphoma and for both of
these to be used for clonality testing. Identification of a monoclonal spike at
different amplicon lengths would be suggestive of artefact; the same amplicon
length would support this being a neoplastic proliferation.
C The signal occurs too late in the reaction to be considered genuine
The x-axis here is amplicon length. It has nothing to do with the time which has
elapsed during the reaction (unlike real-time PCR).
D This finding supports a diagnosis of a reactive lymphoid proliferation
A reactive lymphoid proliferation would typically show a broad spread, resembling
a normal distribution.
Images adapted from https://link.springer.com/article/10.1007/s12308-008-0013-9/figures/6

It is important to bear in mind that even this kind of polyclonal pattern can be seen
in a neoplastic situation. This is particularly true in T-cell lymphomas where most
of the lymphoid population can be reactive, obscuring the monoclonal result from
the neoplastic population.
E This finding supports a diagnosis of T-cell lymphoma
Correct. This shows a monoclonal peak which would support a diagnosis of
T-cell lymphoma assuming that the clinical picture, morphology and
immunohistochemistry all suggest T-cell lymphoma.
Page 34 of 39
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The current gold standard for ROS1 translocation testing in non-small cell lung cancer is
by FISH. Immunohistochemistry can be used as an initial screening tool. It has high
sensitivity but low specificity, which means that negative staining can generally be
considered genuine negativity, but positivity requires confirmation with FISH. Here, dual-
colour break-apart probes have been used. The normal situation would be to have two
fused signals. The presence of split signals indicates the presence of a translocation
(because the ROS1 gene has been ‘broken apart’ from the sequence which is normally
found next to ROS1).
A All signals are fused, so there is no evidence of ROS1 translocation

This image shows one fused signal (yellow) and two split signals (green and
orange). The normal situation for a dual-colour break-apart probe is to have two
fused signals, but that is not what is seen here.
B All signals are split, consistent with ROS1 translocation
This is not what is shown here. In any case, four split signals (two green and two
orange) would be very unusual. A ROS1-translocated tumour will generally only
have a translocation involving one of the ROS1 alleles. ROS1 translocations are
uncommon as it is (around 1% of non-small cell lung cancers); the chances of
having biallelic ROS1 translocation are therefore extremely slim.
C Two signals are split and one is fused, consistent with ROS1 translocation
Correct. There are indeed two split signals (green and orange) in addition to
one fused signal (yellow). This indicates that a ROS1 translocation is
present.
D Only one signal is present, so this nucleus cannot be assessed
This image shows three signals (two split, one fused). This is a fully assessable
situation.
E Only two signals are split, so this is insufficient for diagnosis of ROS1
translocation
As for (B), identification of four split signals (indicating ROS1 biallelic
translocation) would be extremely unusual. The detection of one fused and two
split signals is the typical finding in a ROS1-translocated cell.
Page 35 of 39
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The differential diagnosis of a small round blue cell tumour is broad and the first step in
making a diagnosis is immunohistochemistry. This is chiefly to exclude possibilities like
carcinoma, lymphoma and melanoma, and partly to narrow down the possibilities within
sarcoma. Many small round blue cell sarcomas can be distinguished on the basis of their
translocations. The vast majority of Ewing sarcomas harbour translocations involving
EWSR1. It is common practice to perform FISH using dual-colour break-apart probes, and
then to use dual-colour dual-fusion probes and/or RT-PCR to confirm the fusion partner if
this information is needed. FISH with dual-colour dual-fusion probes involves using a
probe for each of the genes of interest. Normally, these genes should be separate (split); if
they are translocated to form a fusion gene they will be close to each other (fused). In this
case, there are four split signals (the green and orange signals are separated by more
than one signal thickness) indicating that there is no evidence of an EWSR1-FLI1 fusion;
however, you are told that the presence of an ESWR1 translocation has been confirmed
by dual-colour break-apart probe FISH. It is likely that there is an EWSR1 translocation
involving a gene other than FLI1 (e.g. ERG, ETV1).
A FISH testing for alternative fusion partners is advisable

Correct. You are told that an EWSR1 fusion has been detected, but this
shows that it is not an EWSR1-FLI1 fusion. If the fusion partner needs to be
known, more dual-colour dual-fusion probes need to be used (e.g. for ERG,
ETV1). RT-PCR or NGS could alternatively be used to identify the fusion
partner if needed.
B This raises the possibility of synovial sarcoma
You have been told that this is a small round blue cell tumour with an EWSR1
translocation. Synovial sarcoma is generally spindled and harbours SS18
translocations. The fact that an EWSR1-FLI1 fusion specifically has not been
identified does not suggest that this is a synovial sarcoma.
C This casts doubt over the original EWSR1 FISH result
An EWSR1 translocation was identified using dual-colour break-apart probes. The
fact that this FISH testing has failed to demonstrate a fusion simply suggests that
there is not an EWSR1-FLI1 fusion. There is likely an alternative EWSR1 fusion
partner (e.g. ERG, ETV1). In this context, detection of an EWSR1 translocation
(irrespective of its fusion partner) supports a diagnosis of Ewing sarcoma.
D This confirms a diagnosis of Ewing sarcoma
The information provided here adds essentially nothing to the diagnosis in this
case. In this setting, prior detection of an EWSR1 translocation using dual-colour
break-apart probes large confirms the diagnosis. Failure to detect an EWSR1-
FLI1 fusion neither supports nor militates against this diagnosis.
E This confirms the presence of a translocation involving FLI1
These dual-colour dual-fusion probes show four split signals, indicating that the
EWSR1 and FLI1 genes are not next to each other. Therefore, whatever the
EWSR1 fusion is, it is not an EWSR1-FLI1 fusion.
Page 36 of 39
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HER2 FISH involves using a probe against the HER2 gene (on chromosome 17) and a
probe against CEP17 (in the centromeric region of chromosome 17). HER2 amplification
can be determined by either an increased HER2 copy number or an increased
HER2:CEP17 ratio. Calculation of the HER2:CEP17 ratio allows for ‘pure’ HER2
amplification to be distinguished from chromosome 17 aneusomy (increased numbers of
copies of chromosome 17). HER2 copy number is simply the number of HER2 signals in
the nucleus. This nucleus contains two CEP17 and eight HER2 signals.
A HER2 copy number is 10

There are eight HER2 signals, so HER2 copy number is 8.
B HER2 copy number is 4
There are eight HER2 signals, so HER2 copy number is 8.
C HER2 copy number is 8
Correct. There are eight HER2 signals, so HER2 copy number is 8.
D HER2:CEP17 ratio is 8
There are eight HER2 signals and two CEP17 signals. HER2:CEP17 ratio is
8÷2=4.
E There is evidence of chromosome 17 aneusomy
There are two CEP17 signals, indicating that there are two copies of chromosome
17. There is no evidence of chromosome 17 aneusomy.
Page 37 of 39
31/05/2021
All three FISH tests have been performed using dual-colour break-apart probes. The
normal situation is to have two fused signals; the presence of split signals indicates the
presence of a translocation. MYC FISH shows one fused and two split signals, indicating
that there is a MYC translocation. BCL2 FISH is a little unusual in that it shows two fused
and two split signals, indicating a BCL2 translocation and also suggesting possible
increased numbers of copies of the BCL2 gene. BCL6 FISH shows two fused signals,
indicating that there is no BCL6 translocation.
A B-cell acute lymphoblastic leukaemia

You are told that the histology is of a mature lymphoma, which rules out B-cell
acute lymphoblastic leukaemia.
B Burkitt lymphoma
Burkitt lymphomas harbour MYC translocations, but not translocations involving
BCL2 or BCL6.
C Diffuse large B-cell lymphoma
DLBCL should classically lack translocations involving any of MYC, BCL2 or
BCL6.
D High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6
rearrangements (‘double-hit lymphoma’)
Correct. A high-grade mature B-cell lymphoma with translocations involving
MYC and one of BCL2 or BCL6 is a ‘double-hit’ lymphoma.
E High-grade B-cell lymphoma, with MYC and BCL2 and/or BCL6 rearrangements
(‘triple-hit lymphoma’)
A ‘triple-hit’ lymphoma harbours translocations involving all three of MYC, BCL2
and BCL6.
Page 38 of 39
31/05/2021
RNA ISH is used in a similar way to immunohistochemistry, but rather than detecting
proteins it detects RNA sequences. By detecting RNA rather than proteins, it can
specifically detect cells expressing a gene rather than detecting protein which may have
been secreted into the extracellular matrix or onto other (non-expressing) cells. HPV ISH
is used to detect cells which are actively expressing high-risk HPV RNA sequences, rather
than cells which harbour non-productive HPV nucleic acid sequences. Positivity manifests
as staining in cell nuclei. It may be strong and cover the entire nucleus (episomal pattern)
or may only manifest as small spots in the nucleus (integrated pattern).
A p16 immunohistochemistry could have been used as an alternative

p16 overexpression is non-specific. Besides being a surrogate marker of high-risk
HPV infection, it can be expressed by any rapidly-proliferating cell. Therefore
although in the correct setting p16 overexpression can be interpreted as
representing high-risk HPV infection, HPV RNA ISH is more specific and so the
two are not truly interchangeable.
B The tumour is positive for high-risk HPV
Correct. There is nuclear staining (albeit with the less obvious integrated
pattern) indicating productive high-risk HPV infection.
C There is no strong, diffuse nuclear staining and so this cannot be regarded as
genuine positivity
This pattern would be considered the episomal pattern which does indeed
indicate positivity:
Images adapted from Interpretation Guide for Ventana INFORM® HPV Probes In Situ Hybridization (ISH)
Staining of Cervical Tissue
However, the less obvious integrated pattern seen here is still regarded as
positivity.
D This staining could indicate low-risk HPV infection
The probes used for HPV RNA ISH are specific for high-risk HPV subtypes. This
therefore cannot indicate low-risk HPV infection.
E This staining could indicate non-productive HPV infection
The fact that there is positivity indicates that HPV RNA is present within the cells.
This means that viral genes are actively being transcribed and therefore that there
is productive HPV infection.
Page 39 of 39

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FRCPath questions: picture-based

Which is the correct interpretation?

Which is the correct interpretation?

Which is the correct interpretation?

Images adapted from https://www.ous-research.no/home/lothe/methods/2766

Which is the correct interpretation?

Images adapted from https://www.ous-research.no/home/lothe/methods/2766

Which is the correct interpretation?

Images adapted from https://biology.stackexchange.com/questions/45545/deducing-amino-acid-sequence-from-a-dna-

Which is the correct interpretation?

Which is the correct interpretation?

Non-synonymous tumour mutation burden: 4.6 mutations/MB

Images adapted from https://www.genomicsengland.co.uk/wp-content/uploads/2016/11/image-1.jpg

Which is the correct interpretation?

Reference sequence ATCGAATGTT

Images adapted https://www.sciencedirect.com/science/article/pii/B9780124104716000037

Which is the correct interpretation?

Which is the correct interpretation?

Which is the correct interpretation?

Which is the correct interpretation?

Images adapted from http://www.pathologyoutlines.com/imgau/breastmalignanther2Reisenbichler03.jpg

Which is the correct interpretation?

Images adapted from https://link.springer.com/article/10.1007/s12308-008-0013-9/figures/6

Which is the correct interpretation?

Images adapted from https://www.molecular.abbott/int/en/products/oncology/vysis-alk-break-apart-fish-probe-kit

Which is the correct interpretation?

Images adapted from http://store.biogenex.com/us/applications/fish/efish-probes/oncology-probes/cmyc-igh-dual-color-dual-

Which is the correct interpretation?

Images adapted from http://www.ikonisys.com/media/cms_page_media/47/oncoFISH%20her2%20b.jpg

Which is the correct interpretation?

Images adapted from

Which is the correct interpretation?

Which is the correct interpretation?

A The results are equivocal and testing must be repeated

A This case needs BRAF mutation testing

A Testing for tumour 3 has failed

A A technique with a limit of detection of 20% mutant alleles is likely to detect

A This is a highly mutated tumour

A There is evidence of a C deletion

A This patient does not have Lynch syndrome

A The tumour is very likely to be an adenocarcinoma

A HER2 expression is heterogeneous and so it is good practice to undertake FISH

A A reactive proliferation would not give rise to this result

Images adapted from https://link.springer.com/article/10.1007/s12308-008-0013-9/figures/6

A All signals are fused, so there is no evidence of ROS1 translocation

A FISH testing for alternative fusion partners is advisable

A HER2 copy number is 10

A B-cell acute lymphoblastic leukaemia

A p16 immunohistochemistry could have been used as an alternative

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