ONCOLOGY CYTOGENETICS USER GUIDE
Document Title Oncology Cytogenetics User Guide
Document Ref. No. [MI-CG-Christie-User Guide]
Edition No. Version 2.0
Date of Issue January 2007
Review Interval Bi-annual
Author N. Telford & Oncology Cytogenetics staff
Authorised By N. Telford
Reviewed By N. Telford & Oncology Cytogenetics staff
Review History Version 1.0 Issued May 2005
Reviewed Sept 2006 – Jan 2007
Address; Oncology Cytogenetics
Christie Hospital NHS Trust
Manchester M20 4BX
General Enquiries; 0161 446 3165
FAX; 0161 446 3051
Main Departmental Contacts
Head of Department/ Nick Telford Tel; 0161 446 3163
Consultant Clinical Cytogeneticist email@example.com
Secretary Liz Parker Tel; 0161 446 3165
Laboratory Manager/ Yvonne Cook Tel; 0161 446 3165
Principal Cytogeneticist Markella Mikkelsen Tel; 0161 446 8608
Clinical Cytogeneticists Mike Green
Cytogenetic Technologists Pragnya Patel
Hours of Operation
Monday to Friday 8.30am to 5pm
Weekends There is no routine service at weekends. Samples
requiring special attention should be arranged in advance.
Bank Holidays The department is not routinely staffed on Bank Holidays.
Contact details for an on-call Cytogeneticist are left at
Christie Hospital switchboard (0161 446 3000) for the
receipt of urgent samples.
A letter is sent in advance detailing arrangements at
Christmas and Easter.
2 [MI-CG-Christie-User Guide]
Bone Marrow is the tissue of choice to investigate patients suspected of having leukaemia or
Peripheral Blood can be sent if disease cells are present in sufficient numbers to allow cell
Other tissues can be analysed and lymph nodes, spleen, ascitic fluid, CSF and solid tumours
are occasionally received.
Paraffin Embedded Tissue for FISH on lymphoma, breast or brain tumour patients is preferred
as 3µ sections but can be sent as paraffin blocks if preferred.
The laboratory will provide containers to regular referrers for bone marrow and blood collection.
These contain heparinised tissue culture medium with antibiotics to facilitate the transport of the
small amount of bone marrow and avoid desiccation.
An allocation of specimen bottles will be issued at the beginning of each month based on the
number of samples usually received. More bottles can be sent upon request, at any time, by
hospital transport or by post.
In emergency, a blood tube containing lithium heparin could be used.
Use only heparinised containers.
Please DO NOT use other anticoagulants such as EDTA, which is toxic to cells.
Solid tissues should be placed in a sterile liquid such as culture medium, Hank’s balanced salt
solution or saline.
The department is happy to advise on the use of alternative specimen containers.
Despatch of Samples
All sample bottles should be fully labelled and placed in a plastic specimen bag with request
card in separate pocket. Samples should be packed in sufficient absorbent packing to soak up
the entire contents in the event of leakage and placed in cardboard sample box or
Samples should be sent to the laboratory as soon as possible, preferably on the day of
collection. Samples not being sent immediately should be refrigerated overnight at 4oC and sent
at the earliest opportunity the following day.
First class post is usually acceptable. At times, it may be necessary to send specimens to the
laboratory by taxi, to avoid delays, especially approaching weekends and bank holidays
It is advisable that all Friday samples arrive on the day of collection to ensure that the samples
are cultured before the weekend.
Please send samples at the earliest opportunity.
It is advisable to telephone about any samples that could arrive at the laboratory late in
the day or out of hours.
The Duty Scientist may advise sending the sample the following day.
SAMPLES DETERIORATE RAPIDLY IN HOT WEATHER. IF SAMPLES MUST TRAVEL A
LONG DISTANCE IN HOT TEMPERATURES, PLEASE CONSIDER SENDING THE SAMPLE
IN A REFRIGERATED BOX.
3 [MI-CG-Christie-User Guide]
Please fill in all the patient demographics on the request card.
The reason for referral is important to determine what culture types need to be set up, what
tests to perform, numbers of cells to analyse and sample prioritisation. All relevant clinical and
haematological information and likely diagnosis can be included. It is important to give trial
information as certain trials can have specific requirements, such as levels of analysis by
cytogenetics and/or FISH.
The department operates a Specimen Acceptance Policy [LP-PathGen-Christie SpecAccept
(Policy ID:1016). The following details are essential requirements for request cards and
Request Card Specimen bottle
1. Patients full name and date of 1. Patients full name, with hospital
birth. number (or NHS number) and/or
2. Hospital number and/or NHS date of birth.
number. 2. Specimen type and site of
3. NHS number for external specimen to distinguish multiple
4. Reason for referral/clinical 3. High-risk label (if appropriate)
5. Specimen type.
6. Consultant name or initials and
7. Requestor’s name and signature.
8. Date specimen was taken.
9. High risk status (if appropriate).
10. Private patient (if appropriate).
Please use the current version of the request card.
Please see following picture of version 2.0
4 [MI-CG-Christie-User Guide]
Request Form v2.0
ONCOLOGY CYTOGENETICS REQUEST ONCOLOGY CYTOGENETICS
Lab. No. PATHOLOGY DEPARTMENT
STICK PATIENTS IDENTITY LABEL HERE CHRISTIE HOSPITAL
MANCHESTER M20 4BX
OR GIVE THE FOLLOWING DETAILS:
HOSPITAL CONSULTANT SURNAME
DATE TAKEN TIME TAKEN SPECIMEN FIRST NAME
CLINICAL DIAGNOSIS & RELEVANT DETAILS ADDRESS
Current treatment: POSTCODE
DIAGNOSIS/ REMISSION/FOLLOW UP NHS No.
PROGRESSION TRIAL: DATE OF BIRTH HOSPITAL NUMBER
HIGH INFECTION RISK? YES / NO (PLEASE CIRCLE)
DOCTORS SIGNATURE PRINT DOCTORS NAME OTHER REFERENCE No.(e.g.Path No.) SEX NHS
MANDATORY INFORMATION LF-CG-Christie-Request Form v2.0
In submitting this sample, the clinician confirms that consent has been obtained for testing and storage of the patient material
5 [MI-CG-Christie-User Guide]
Policy for High Risk Samples
All samples from patients at High Risk of infection referred for cytogenetic analysis should be
identified to the laboratory. The sample and request card must be clearly labelled as High Risk.
Samples at High Risk of infection (with ACDP category ≥3 pathogen) cannot be
processed by the laboratory.
HIV, Hepatitis B or Hepatitis C samples can be processed by prior arrangement,
if there is clear clinical need.
Generally, samples at risk of infection with an ACDP category 3 pathogen (or higher) will not
routinely be processed by the laboratory.
Any sample of uncertain risk status with ACDP category 3 pathogen or if satisfactory
arrangements cannot be made, will be disposed of by incineration.
Samples at risk of infection with HIV, Hepatitis B or Hepatitis C, however, may be processed by
prior agreement between the Oncology Cytogenetics laboratory and the referring consultant.
Full cytogenetic analysis will only be considered in circumstances where a result will directly
affect patient management. Processing of such samples may incur additional cost to the
HIV, Hepatitis B and Hepatitis C samples received where no arrangements are made or without
strong indication for cytogenetic analysis, will be fixed uncultured for possible FISH only.
Consequently, a conventional cytogenetics result will not be possible.
In all instances, a record of the actions taken will be made and a report issued to the referring
6 [MI-CG-Christie-User Guide]
Policy of Consent for Testing and Retention of Samples
In submitting a sample to Oncology Cytogenetics, the clinician confirms that consent has been
obtained for testing and storage of the patient material.
This material is tested and retained for possible Oncology Cytogenetics use and only in
connection with the original reason for referral. It will not be passed on to other parties or used
for research or purposes other than the reason that they were originally referred.
The Oncology Cytogenetics department currently retains fixed cell suspensions from samples
for 10 years. This allows for further testing of samples and is particularly useful for additional
FISH tests or testing a diagnostic sample to establish the FISH signal pattern, to enable testing
of a current post-treatment sample.
Used microscope slides from routine cytogenetic analysis are retained for 10 years.
A sample not analysed at the time of referral can be reactivated at any time, if required.
A full year of samples and slides are disposed of in the January following the completion of a full
ten years of age.
7 [MI-CG-Christie-User Guide]
All new acute leukaemias, chronic myeloid leukaemias and those cases possibly relapsing
or transforming will be treated as urgent and will be analysed within one week.
All other samples will be treated as non-urgent and analysed as soon as possible, usually
within three weeks.
All samples that are not urgent with an uncertain diagnosis will be held in abeyance,
pending further information. Further details are requested on a form, which also permits you
to suggest a priority level for the referral.
This is necessary because at the time of biopsy the diagnosis may not be known and
chromosome analysis may not be required after bone marrow morphology is examined.
Consultants are requested to cooperate as fully as possible with this policy. This is to
avoid unnecessary and labour intensive analytical work and helps the laboratory to
process its large workload.
Cytogenetics is the microscopic study of chromosomes, which can show abnormalities that
represent genetic defects in the DNA that they contain. These abnormalities can be numerical
(loss or gains of chromosomes) or structural (e.g. translocations, inversions, deletions).
Chromosome abnormalities can confirm a clonal disease and can often suggest a more specific
diagnosis or a prognosis. The abnormalities can be used to monitor remission and diagnose
relapse, transformation or secondary disease. Increasingly, cytogenetic abnormalities indicate
specific and targeted treatment regimes.
Conventional cytogenetic analysis relies on the culture of cells to produce metaphase
chromosomes, where individual chromosomes can be visualised. Tissue therefore needs to be
as fresh as possible with viable disease cells. Cells are processed and stained using ‘banding’
techniques to produce a karyotype.
Fluorescence in situ hybridisation (FISH) uses fluorescently labelled gene probes to detect
specific gene sequences on the microscope slide. This can be used to confirm specific genetic
abnormalities at the molecular level. FISH can be used with metaphase chromosomes but is
also applicable to interphase cells and so cell culture is not always necessary. Different
strategies are possible with multiple fluorochromes, which can be used in conjunction with other
fluorescent labelling techniques.
8 [MI-CG-Christie-User Guide]
Telephone enquiries are welcome. Cytogenetics’ staff will be pleased to process samples by
request if required urgently to determine treatment or by specific appointments.
Hard-copy reports will be sent to the referring consultant.
Reports will be addressed elsewhere if requested to do so in writing.
Policy for Faxing Reports
Oncology Cytogenetics is obliged to follow the Fax policy of Christie Hospital NHS Trust.
For security reasons and because this is time-consuming, this procedure can only be used
occasionally and therefore only when urgent reports are required immediately.
All other reports will be sent by post/hospital transport and will usually be received within a day
Faxing will entail;
Ring the recipient before we send the Fax.
Check with the recipient that the Fax number and the Fax is available.
Ask the recipient to stand by the Fax machine.
Fax the header sheet only first.
The recipient must phone when the header sheet is received.
When we receive confirmation by phone, the rest of the Fax with the report will be sent.
9 [MI-CG-Christie-User Guide]
Summary of Services Offered for Routine Cytogenetics and FISH
Disease group Cytogenetics FISH
Chronic Myeloid leukaemia √ √ (BCR/ABL)
Chronic myeloid leukaemia √ √
(follow-up) Screen for Ph plus additional
abnormalities including for
Ph –ve clones
Acute myeloid leukaemia √ To confirm specific abnormality
(at diagnosis) found by cytogenetics or as
indicated by morphology.
Del(5) and del(7) on failed
APL √ Rapid (same-day) FISH for
(at diagnosis) PML/RARA
MDS √ Del(5) and del(7) on failed
?MDS Χ (on request) Χ
ET Χ √ (BCR/ABL)
PRV √ Χ
Myelofibrosis √ Χ
CMML √ Χ
Hypereosinophilia √ PDGFRα & PDGFRβ.
ITP Χ Χ
Aplastic anaemia √ Χ
BMT patients (sex-mismatched) Χ √ (X/Y)
(only if % recipient cells is
significant by FISH)
BMT patients (sex-matched) √ (if abnormal at diagnosis) √ (if abnormal at diagnosis)
All follow-ups (except CML) √ (if abnormal at diagnosis) √ (if abnormal at diagnosis)
Acute lymphoblastic leukaemia √ ALL panel: BCR/ABL, MLL,
(B-cell) (Paediatric <20 years old) TEL/AML1
Acute lymphoblastic leukaemia √ BCR/ABL
Acute lymphoblastic leukaemia √ Χ
CLL/SLL Χ (on request only) CLL panel: P53, ATM, D13S319,
Lymphoma (on staging bone Χ (on request, if lymphocytosis) Χ (on request if lymphocytosis or
marrow aspirate) if FISH abnormal on other tissue)
Lymphoma (on lymph node √ √ (if indicated)
Lymphoma (on paraffin N/A √ (if indicated)
Multiple Myeloma/MGUS √ IGH/FGFR3, del(13q) and others
Breast (on paraffin embedded N/A HER2 amplification status
Oligodendroglioma (on paraffin N/A 1p- & 19q- deletion status
10 [MI-CG-Christie-User Guide]
Service Specification and Indications
Chronic Myeloid Leukaemia
Bone marrow cytogenetics is essential at diagnosis, on a pre-treatment specimen, to
demonstrate the translocation t(9;22), which gives rise to the Philadelphia (Ph) chromosome.
This distinguishes typical CML from Ph negative MPD and reactive marrows. Additional clonal
chromosome abnormalities may be present that could change prognosis. Confirmation of Ph
positivity is an absolute requirement for treatment with imatinib. Diagnostic samples will be
treated urgently and a result will be available within 7 days.
FISH is used to detect the BCR-ABL gene rearrangement in the 5% of cases that show normal
cytogenetics but have a cryptic BCR-ABL abnormality. A further 5% of cases show variant
translocations and involve other chromosomes, with or without visible involvement of
chromosome 9. These may also require FISH, as will the small number of cases that fail to grow
in culture. In around 11% of cases, FISH demonstrates deletion of ABL and/or BCR sequences
from around the chromosome breakpoint on the abnormal chromosome 9 [der(9)]. This is a
candidate adverse prognostic factor at diagnosis.1,2
Early cytogenetic response is the most important prognostic factor in CML. Chromosome
analysis is used to monitor the initial response to treatment. However, more sensitive methods
would be required (such as RT-PCR) to monitor patients after cytogenetic remission is
achieved. Post-treatment patients will be monitored for Ph positivity and the common
abnormalities seen at transformation (see below). Additional clonal chromosome abnormalities
in the Ph negative cell line are a recognised phenomenon in a significant proportion of CML
patients treated with imatinib. The majority of these patients are asymptomatic but a small
number develop MDS, with monosomy 7 and trisomy 8 appearing to confer a higher risk5.
Emerging consensus strategies for monitoring CML recommend that cytogenetic studies should
be part of the periodic review, such as performing cytogenetics at least every 6 months until
complete cytogenetic remission is achieved and then every 12 months to detect Ph negative
clones and MDS.3,4 80% of cases at transformation show clonal evolution with recognised
additional chromosomal changes. Bone marrow samples should be sent for cytogenetic studies
if disease acceleration is suspected or if there is rising level of BCR-ABL status by RT-PCR.
• Full karyotype at diagnosis to detect t(9;22) and additional abnormalities
• FISH at diagnosis to detect cryptic BCR-ABL
• Post-treatment bone marrows screened for Ph’ by cytogenetics and/or FISH.
Screen for clonal abnormalities in Ph’ negative cells.
• Full karyotype at possible relapse or transformation for t(9;22) and additional
1. Huntly BJ, Bench A, Green AR. Double jeopardy from a single translocation: deletions of the derivative chromosome 9 in
chronic myeloid leukemia. Blood. 2003 Aug 15;102(4):1160-8. Epub 2003 May 1.
2. Fourouclas N, Campbell PJ, Bench AJ, Swanton S, Baxter EJ, Huntly BJ, Green AR. Size matters: the prognostic
implications of large and small deletions of the derivative 9 chromosome in chronic myeloid leukemia. Haematologica.
3. Hughes T, Deininger M, Hochhaus A, Branford S, Radich J, Kaeda J, Baccarani M, Cortes J, Cross NC, Druker BJ,
Gabert J, Grimwade D, Hehlmann R, Kamel-Reid S, Lipton JH, Longtine J, Martinelli G, Saglio G, Soverini S, Stock W,
Goldman JM. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and
11 [MI-CG-Christie-User Guide]
recommendations for harmonizing current methodology for detecting BCR-ABL transcripts and kinase domain mutations
and for expressing results. Blood. 2006 Jul 1;108(1):28-37.
4. Baccarani M, Saglio G, Goldman J, Hochhaus A, Simonsson B, Appelbaum F, Apperley J, Cervantes F, Cortes J,
Deininger M, Gratwohl A, Guilhot F, Horowitz M, Hughes T, Kantarjian H, Larson R, Niederwieser D, Silver R, Hehlmann
R. Evolving concepts in the management of chronic myeloid leukemia. Recommendations from an expert panel on behalf
of the European Leukemianet. Blood. 2006 Sep 15;108(6):1809-20.
5. Kovitz C, Kantarjian H, Garcia-Manero G, Abruzzo LV, Cortes J. Myelodysplastic syndromes and acute leukemia
developing after imatinib mesylate therapy for chronic myeloid leukemia. Blood. 2006 Oct 15;108(8):2811-3.
An abnormal karyotype is found in approximately 60% of AML cases at presentation. A large
number of chromosome abnormalities have been identified that can aid the diagnosis of AML
and identify subtypes. The WHO classification defines certain groups by their cytogenetic
abnormalities. Importantly, many have prognostic implications and are therefore used in
stratified treatment regimes.
Certain chromosome abnormalities are used to assign a risk group in AML trials;
• t(15;17), t(8;21) and inv16/t(16;16) are associated with specific morphology and
relatively good prognosis and are detectable by routine cytogenetics or FISH.
• Complex karyotypes, rearrangements of 3q, deletions of 5q and loss of chromosomes 5
or 7 are associated with a poor prognosis.
• Many other chromosome abnormalities, of varying specificity, are recognised that can
aid diagnosis and may suggest prognosis.
All patients should have conventional cytogenetics performed at diagnosis to identify favourable
and unfavourable prognostic abnormalities1. Cytogenetics can be used to monitor response to
treatment and can confirm relapse or indicate disease progression.
• Full karyotype at diagnosis
• Rapid FISH for t(15;17) at diagnosis (usually <24 hours)
• FISH at diagnosis to detect t(8;21), inv(16) as suggested by morphology or as
• Post-treatment bone marrows screened for previous abnormality by routine
cytogenetics or FISH, as appropriate.
• Full karyotype at possible relapse for recurrence of previous abnormality, clonal
evolution or new disease.
1. British Committee for Standards in Haematology: Milligan DW, Grimwade D, Cullis J O, Bond L, Swirsky D, Craddock C, Kell J,
Homewood J, Campbell K, McGinley S and Wheatley K, G Jackson. Guidelines on the management of acute myeloid leukaemia in
adults. British Journal of Haematology 2006. 33, 451–458.
Approximately 40% of confirmed, de novo MDS cases have karyotype abnormalities at
diagnosis, which helps to confirm the presence of a clonal disorder and aids the distinction
between MDS and reactive causes of dysplasia. Numerous abnormalities have been described,
the commonest of which are trisomy 8, monosomy 7/deletion of 7q, monosomy 5/ deletion of 5q,
and deletions of 9q and 20q. However, none are specific to MDS and are found in AML and
other myeloid diseases. There are therefore no specific markers for MDS subtypes or to confirm
transformation to acute leukaemia.
12 [MI-CG-Christie-User Guide]
However, some chromosome abnormalities indicate more aggressive disease and therefore
have major prognostic value. Karyotype is therefore included as a criterion in prognostic
schemes, e.g. International Prognostic Scoring System (see Table below)1. Cytogenetics is
recommended in all cases where bone marrow examination is indicated.2
Good prognosis Normal karyotype or –Y, del (5q), del (20q) as sole abnormalities.
Intermediate prognosis + 8 and others.
Poor prognosis Complex karyotype (≥ 3 abnormalities), chromosome 7
Recently developed new therapeutic agents (such as 5-azacytidine, lenalidomide and
decitabine) have been developed for the treatment of MDS and have been reported to be
effective in specific cytogenetic subgroups.
Macrocytic anaemias and other cytopaenias are referred for investigation of MDS. It would be
helpful if cytogenetics requests were called off (to avoid unnecessary analyses) if the diagnosis
of MDS is excluded or cytogenetics is no longer required after the examination of bone marrow
Samples that fail to grow in culture will have FISH for 5q and 7q deletions.
• Full karyotype.
• FISH for 5q and 7q deletions on cases failing to grow in culture.
1. Greenberg P, Cox C, LeBeau MM, Fenaux P, Morel P, Sanz G, Sanz M, Vallespi T, Hamblin T, Oscier D,
Ohyashiki K, Toyama K, Aul C, Mufti G, Bennett J. International scoring system for evaluating prognosis in
myelodysplastic syndromes. Blood. 1997 Mar 15;89(6):2079-88. Erratum in: Blood 1998 Feb 1;91(3):1100.
2. Bowen D, Culligan D, Jowitt S, Kelsey S, Mufti G, Oscier D, Parker J; UK MDS Guidelines Group. Guidelines
for the diagnosis and therapy of adult myelodysplastic syndromes. Br J Haematol. 2003 Jan;120(2):187-200.
The most common cytogenetics abnormalities in AA are trisomy 8 and aberrations of
chromosome 7. The finding of a cytogenetic abnormality in AA can be a strong indication of
clonal evolution to MDS. Cytogenetics is performed on AA upon request.
Cytogenetic abnormalities are found in 10 20% of patients with PRV. The abnormalities are
general myeloid markers including trisomy of chromosomes 8 and 9, del (20q), del (13q) and del
(1p). An abnormal karyotype, a marker of clonality, is a major diagnostic criterion1. Patients who
progress to myelodysplastic syndrome or acute leukaemia almost always have a karyotypic
abnormality and there is an increase in frequency of abnormalities and increasing karyotype
complexity in disease progression. However, there is no specific abnormality that will confirm
13 [MI-CG-Christie-User Guide]
1. McMullin et al ‘Guidelines for the diagnosis, investigation and management of polycythaemia/erythrocytosis’ British
Journal of Haematology Volume 130 Issue 2 Page 174 - July 2005
• Full karyotype on confirmed primary polycythaemia
An abnormal karyotype is unusual in ET (~5% of cases) and the abnormalities, when present,
are general myeloid markers (+8, +9, del(13q)). A full cytogenetic analysis is rarely helpful but
can be performed on difficult cases.
BCR-ABL should be performed to exclude CML with thrombocytosis only, with FISH being the
method of choice. This satisfies the requirements of the PT1 trial.
• FISH for BCR-ABL performed on all cases of ET and high platelets
• Full karyotype of confirmed cases on special request
Chromosome abnormalities are found in 60% of cases but again are not specific. However, an
abnormal karyotype at diagnosis is an adverse prognostic factor. There is no specific marker for
transformation but a complex karyotype and abnormalities of chromosomes 5 and 7 is strongly
• Full karyotype
Cytogenetic abnormalities are found in up to 40% of cases but are rarely specific; the
commonest being trisomy 8, monosomy 7 or deletion of 7q and rearrangements of 12p. The
rare translocations t(5;12) and t(8;13) appear to define a subset of patients with a specific
disease entity including eosinophilia (see below). Distinction between CMML and atypical
chronic myeloid leukaemia may be difficult and BCR-ABL FISH will be performed on request.
• Full karyotype
• FISH for BCR-ABL performed on request
Chromosomal translocations have been described in eosinophilic MPD that are interesting
because the diseases are reported to respond to imatinib. The rare FIP1L1-PDGFRα gene
fusion results from an interstitial deletion of chromosome 4 and cannot be detected by routine
cytogenetics. Other chromosome abnormalities, including the related t(5;12) translocation or
variants involving the PDGFRβ gene, may be detected by routine cytogenetics.
• Full karyotype in cases where reactive eosinophilia has been excluded
• FISH for FIP1L1-PDGFRα
14 [MI-CG-Christie-User Guide]
Numerous recurrent cytogenetic abnormalities of prognostic importance are recognised in ALL
and cytogenetics should be carried out at diagnosis in all cases. ALL disease cells are notorious
for their poor survival in vitro and rapid transport to the laboratory is critical for successful
chromosome analysis and FISH. Cytogenetic abnormalities can be used to monitor response to
therapy and confirm relapse.
The Ph’ translocation, t(9;22) is more common in adults than children and is associated with a
poor prognosis. Imatinib can be incorporated into intensive treatment regimes. Translocations
involving the MLL gene, such as t(4;11), are common, especially in infants and are associated
with an unfavourable prognosis. The t(12;21) is the most common translocation in paediatric
cases and is only detectable by FISH for the ETV6-RUNX1 (TEL-AML1) gene fusion. This is
associated with a favorable outcome but overall improvement in EFS needs to be confirmed.
The t(1;19) is also a common abnormality which was initially thought to have a poorer
prognosis, which can now be largely overcome by more intensive therapy.
Gain of specific chromosomes, resulting in hyperdiploidy, is generally associated with a
favourable prognosis but uncharacteristic trisomies may modify the significance.
Loss of specific chromosomes, hypodiploidy or near haploidy, are indicators of poor prognosis.
Other chromosome abnormalities, of varying specificity, are recognised that can aid diagnosis.
Recently amplification of RUNX1 (AML1) gene, detectable by FISH, has been incorporated into
trial protocols as a high risk abnormality.
• Full karyotype at diagnosis
• BCR-ABL FISH at diagnosis for cryptic t(9;22) in adults
• BCR-ABL, MLL, TEL-AML1 FISH at diagnosis for cryptic abnormalities in children
• Post-treatment bone marrows screened for previous abnormality by routine
cytogenetics or FISH, as appropriate
• Full karyotype at possible relapse
Chromosome translocations help to define disease subtypes, the most common are the t(11;14)
associated with mantle cell lymphoma, t(14;18) with follicular lymphoma and t(8;14) with Burkitt
lymphoma. However, these rearrangements are not specific to one disease and the profile of
chromosome translocations needs to be interpreted alongside morphology and immunophenotype
for accurate diagnosis. Other translocations such as t(11;18) in MALT lymphoma and those
involving the ALK gene at 2p23 in Anaplastic Large Cell Lymphoma can be useful diagnostically.
Furthermore, t(14;18) (IGH/BCL2), t(8;14) (IGH/C-MYC), 3q27 (BCL6), 17p (p53) deletion, are
informative prognostic markers in certain situations and predict progression to high grade disease.
FISH is available for all of the above abnormalities and is the test of choice as lymphoid cells can
be difficult to grow in culture. FISH on paraffin-embedded tissue sections is usually the most
practical as fresh tissue is not usually available.
Cytogenetics can be performed on lymph nodes providing fresh material is sent in tissue culture
medium to the Cytogenetics Laboratory, as soon as possible. Alternatively peripheral blood or
bone marrow can be used as test material if these are involved. However, the limited number of
cells, which can be fully analysed by routine cytogenetic analysis, makes this test of little value in
15 [MI-CG-Christie-User Guide]
staging. Bone marrow infiltration should be confirmed by other methods and significant
involvement should be confirmed before cytogenetic analysis is performed.
• FISH on paraffin embedded tissue sections
• FISH or full karyotype on fresh primary tissue by special request
• FISH or full karyotype on bone marrow or blood, only if involved (lymphocytosis
present) or if FISH abnormal on other tissue.
Cytogenetic analysis is not essential for the diagnosis of CLL but can be helpful. In particular,
exclusion of translocation t(11;14) is indicated on CD5+ve cases with an uncertain diagnosis.
Cell culture is unreliable in CLL and FISH is the test of choice. FISH is available for all of the
following markers. Peripheral blood is suitable test material, in most cases.
FISH can identify prognostic abnormalities del(13q), trisomy 12, del(11q), del(17p). In particular,
both chromosome 11q and 17p deletions have been shown to be associated with short survival
In a univariate analysis, using FISH, patients with an isolated deletion of 13q, trisomy 12,
deletion of 11q, or of loss of one copy of the p53 gene on 17p had a median survival of 133,
114, 79 and 32 months respectively2. Alemtuzumab may be an effective therapy for patients
with CLL with p53 deletions.3
1. Oscier D, Fegan C, Hillmen P, Illidge T, Johnson S, Maguire P, Matutes E, Milligan D; Guidelines Working Group of the
UK CLL Forum. British Committee for Standards in Haematology. Guidelines on the diagnosis and management of
chronic lymphocytic leukaemia. Br J Haematol. 2004 May;125(3):294-317.
2. Dohner H, Stilgenbauer S, Benner A, Leupolt E, Krober A, Bullinger L, Dohner K, Bentz M, Lichter P. Genomic
aberrations and survival in chronic lymphocytic leukemia. N Engl J Med. 2000 Dec 28;343(26):1910-6.
3. Lozanski G, Heerema NA, Flinn IW, Smith L, Harbison J, Webb J, Moran M, Lucas M, Lin T, Hackbarth ML, Proffitt JH,
Lucas D, Grever MR, Byrd JC. Alemtuzumab is an effective therapy for chronic lymphocytic leukemia with p53 mutations
and deletions. Blood. 2004 May 1;103(9):3278-81.
• FISH panel for 11q, 13q and 17p deletions and trisomy 12 on peripheral blood or
bone marrow on all cases referred.
• FISH for t(11; 14) [IGH/CCND1] as indicated or upon request.
• Full karyotype not performed routinely, but available by special request.
Myeloma shows a range of recognised cytogenetic abnormalities which can aid
subclassification of the disease and are strong prognostic factors.1 Deletions/monosomy of
chromosome 13, non-hyperdiploidy and certain balanced translocations, t(4;14), t(14;16), have
a strong negative impact on prognosis. This can be exaggerated when identified by routine
cytogenetics as opposed to FISH2 and del(13q)/monosomy 13, when identified by routine
cytogenetic screen, is the commonest and most significant cytogenetic prognostic marker in
myeloma.3 Data from current ongoing clinical trials will clarify the prognostic significance of
cytogenetics and the relevance for choice of therapy. However, cytogenetic and/or FISH
analysis may be helpful for individual patients.4
Routine analysis comprises a screen of at least 100 cells for monosomy 13/del(13q) and gross
changes rather than a detailed analysis of a smaller number of cells. This is because of the
16 [MI-CG-Christie-User Guide]
small proportion of disease cells and their low proliferative rate in culture. t(4;14) and some
other IGH translocations are cryptic and can only be identified by FISH.
• Screen for 13q deletions and other gross karyotypic changes
• FISH for t(4;14) if plasma cell infiltration is significant
• FISH for other IGH translocations and other abnormalities on request
• FISH combined with immunuofluorescence, to identify small plasma cell
population, is not available but is being developed
1. Fonseca, R., Barlogie, B., Bataille, R., Bastard, C., Bergsagel, P.L., Chesi, M., Davies, F.E., Drach, J., Greipp, P.R.,
Kirsch, I.R., Kuehl, W.M., Hernandez, J.M., Minvielle, S., Pilarski, L.M., Shaughnessy, J.D., Jr, Stewart, A.K. & Avet-
Loiseau, H. (2004) Genetics and cytogenetics of multiple myeloma: a workshop report. Cancer Research, 64, 1546–1558.
2. Dewald GW, Therneau T, Larson D, Lee YK, Fink S, Smoley S, Paternoster S, Adeyinka A, Ketterling R, Van Dyke DL,
Fonseca R, Kyle R. Relationship of patient survival and chromosome anomalies detected in metaphase and/or interphase
cells at diagnosis of myeloma. Blood. 2005 Nov 15;106(10):3553-8.
3. Chiecchio L, Protheroe RK, Ibrahim AH, Cheung KL, Rudduck C, Dagrada GP, Cabanas ED, Parker T, Nightingale M,
Wechalekar A, Orchard KH, Harrison CJ, Cross NC, Morgan GJ, Ross FM. Deletion of chromosome 13 detected by
conventional cytogenetics is a critical prognostic factor in myeloma. Leukemia. 2006 Sep;20(9):1610-7.
4. Alastair Smith, Finn Wisloff, Diana Samson, the UK Myeloma Forum, Nordic Myeloma Study Group and British Committee
for Standards in Haematology (2006) Guidelines on the diagnosis and management of multiple myeloma 2005 British
Journal of Haematology 132 (4), 410–451.
General References and Suggested Further Reading
1. Bain B. J. Leukaemia Diagnosis 3 Edition Blackwell Science: 2003.
2. Jaffe E.S. et al (Eds.) World Health Organization Classification of Tumours. Pathology and Genetics of Tumours of
Haematopoietic and Lymphoid Tissues. IARC Press: Lyon 2001.
3. Rooney D. E. (Ed.) Human Cytogenetics: malignancy and acquired abnormalities 3 Edition: OUP 2001.
17 [MI-CG-Christie-User Guide]
The Christie Hospital
The Christie Hospital NHS Trust is the largest single-site cancer treatment centre of its kind in
Europe, and is an international leader in cancer research and development. The hospital is
based in Manchester, and as it is a specialist centre, patients travel to the Christie from all over
the North West and beyond. The Christie covers a population of 3.5 million. Over its hundred-
year history, the Christie has pioneered numerous developments in cancer diagnosis and
therapy. It works in close partnership with many organisations such as other NHS Trusts,
Manchester Universities, Cancer Research UK and the Paterson Institute for Cancer Research.
The Oncology Cytogenetics service offers chromosome analysis to Greater Manchester and the
North West of England. It started in the Paterson Institute for Cancer Research more than 15
years ago. The relevance of the techniques to modern leukaemia diagnosis has resulted in the
laboratory being incorporated into the Pathology directorate and relocation to the main Hospital
site in 1997. New, purpose-built premises were funded by the Kay Kendal Leukaemia fund and
Christie Hospital NHS Trust. Fluorescence in situ hybridisation (FISH) was formally added to the
service repertoire in 1998.
A successful bid to the Department of Health’s Pathology Modernisation Fund for 2000/01, the
department was awarded funding for a modern laboratory patient database, computer hardware
and state of the art image analysis equipment, to facilitate development of the FISH service.
The department comprises a highly skilled team of dedicated scientists and technologists that
provide a comprehensive cytogenetics service to diagnose cancer-associated genetic
abnormalities. All relevant staff are state registered and clinical scientists are formally trained in
Clinical Cytogenetics and Molecular Cytogenetics. The department performs to professional
standards and participates in UKNEQAS schemes for cytogenetics and FISH.
Greater Manchester and Cheshire Haematology Malignancy Diagnostics
Proposals have been agreed with colleagues at the Manchester Royal Infirmary to develop an
integrated (Christie and MRI) molecular diagnostic service for leukaemia and lymphoma. This will
be a core part of a comprehensive and fully coordinated Haematology Malignancy Diagnostics
(HMD) jointly provided by Christie and MRI. Routine cytogenetics and FISH-based molecular
cytogenetic tests will be provided by the Oncology Cytogenetics service and will be further
developed as required. The HMD service development will allow compliance with national strategy
as expressed in the National Institute for Clinical Excellence (NICE) “Improving Outcomes
Guidance for Haematological Cancers,” and has been designed to meet the needs in this regard of
the Greater Manchester and Cheshire Cancer Network.
18 [MI-CG-Christie-User Guide]