vol. 3 no. 2 - pathcare south africa. 3 no. 2 2 3 12 14 molecular ... lyrics of gloria gaynor's...

ForumPathologyApril 2010

Vol. 3 No. 2

2 3

12 14

MolecularPathology andPersonalisedMedicine

An Introducationinto MolecularPathology

MolecularDiagnostics ofPathogens

Pathologists'andLaboratories'Contact Details

Molecular TestingOffered by PathCare(Outside Back Cover)

Personalised/IndividualisedMedicine: The Roleof in theTreatment ofMetastaticColorectal Cancer

KRAS

MolecularTesting in SolidTumours

10MolecularTechnology inthe Managementof ChronicMyelogenousLeukaemia

“If it were not for the great

variability among individuals,

medicine might as well be a

science, not an art.”

Sir William Osler, 1892

Editorial Team

Editor

Consulting Editor

Design and Layout

e-mail

A list of PathCare laboratories and

depots is available on our website at

Elandi Bishop

Dr Dawie de Beer

Tara Willey

Lindsay Willenberg

[email protected]

www.pathcare.co.za

Drs Dietrich, Voigt, Mia & Partners, PathCare Business Centre, PathCare Park

Neels Bothma Street N1 City, Goodwood 7460 • Private Bag X107, N1 City, Goodwood 7463

Tel : (021) 596 3400 Fax : (021) 596 3726

Published April 2010 by:

,

•

From the EditorThis edition of our pathology forum is of a highly technical nature and

focuses on the rapidly developing and ever-changing science of

molecular pathology, and the impact on us as pathologists and

practising clinicians alike. We trust that you will find the information

useful and that it will assist you in the understanding of the most

modern laboratory techniques which PathCare is introducing as

rapidly as possible into our test profile.

Due to the nature of this subject and with most of our clinicians more

familiar with the English terminology, this edition is only published in

English.

Ons Afrikaanssprekende ondersteuners is egter baie welkom om

enige van die outeurs direk te kontak sou u graag enige van die

onderwerpe in meer detail in u moedertaal wou bespreek.

Thank you to all the authors for the time and effort to enable us to

publish a Pathology Forum dedicated to molecular pathology. A

special thanks to professor Izak Loftus for coordinating this

publication.

For your convenience, we have compiled a comprehensive list of

molecular tests available at PathCare (see outside back cover).

Please do not hesitate to contact our pathologists regarding the

availability of any other additional tests.

CEO

Director Special Operations

Dr John Douglass

Dr Johan van Wyk

Human Resources Director

Chief Financial Officer

Chief Systems Officer

Mr Dumisani Ndebele

Ms. Julie Buissinne

Dr Clive Prior

Management

hairman

-chairman

CEO

Prof Izak Loftus

Dr Marthinus Senekal

EXTERNAL BOARD MEMBERS

Deputy

Dr John Douglass

Board of Governors

C

Adv Graham Van Der Spuy

Prof Geoff Everingham

Mr Louis Buckle

REPRESENTATIVES

Dr Frik Botha

Dr Michael Hofmeyr

Dr Ross Millin

Dr Andre Venter

Dr Braam van Greunen

Partner,

, Eastern Region

, Western Region

, Far Northern Region

r, Namibia

Northern Region

Partner

Partner

Partner

Partne

FeaturesMolecular Pathology andPersonalised MedicineProf Izak LoftusHisto and-forensicPathologist,PathCare Somerset West

An Introduction intoMolecular DiagnosticsDr Oubaas Pretorius, PhDTechnical Consultant,PathCare PCR Laboratory,Cape Town

Personalised/IndividualisedMedicine: The Role ofin the Treatment of MetastaticColorectal Cancer

KRAS

Dr Nico de Villiers, PhD(Human Genetics)Technical Consultant,PathCare PCR Laboratory,Cape Town

Molecular Testing in SolidTumoursDr Linda SteynHistopathologist,PathCare Somerset West

Molecular Technology in theManagement of ChronicMyelogenous LeukaemiaDr Illse LouwHaematology Pathologist,Paarl

Molecular Testing offeredby PathCare(Outside Back Cover)

Molecular Diagnostics ofPathogensDr Oubaas Pretorius, PhDTechnical Consultant,PathCare PCR Laboratory,Cape Town

Pathologists' andLaboratories' Contact Details

3 52 6

10 12 14

1

Access to genomic technologies and treatment will be determined by a

number of factors, of which money will most probably be the most

important single factor. This is not only limited to the cost of testing, but

also includes the cost of treatment if applicable. Health funders may

argue that it is better to spend their funds on screening procedures or

other programmes which may benefit more individuals, rather than

investing in the health of only a few at very high costs. In the South

African context, this may create an even wider division in the health

expenditure between the public- and private sector.

Privacy and confidentiality of information is very important. Genetic

testing also has the potential to (indirectly) identify whether other

family members are at risk of developing a specific condition. The

question whether those family members must be informed, or whether

it is an invasion of their privacy then needs to be answered. On the

other hand, it may also be argued that withholding such information

may put them at risk.

The release of genetic test information may adversely affect a patient as

well as his or her extended family via possible loss of health insurance

or compromised employability. However, even if broad protections

were provided for health insurance or employability, inadvertent

disclosure of genetic information can cause psychological trauma and

stigmatisation.

oncology patients and also in microbiology. During the last few years

medicine has slowly moved from a “one shoe fits all” approach to a more

personalised / individualised approach. These changes in diagnostic

medicine have forced pathologists to become more and more involved in

molecular pathology. Although it used to be primarily of importance to

microbiologists and serologists, the scope has now expanded to include

clinical genetics as well as histopathology and haematopathology. As a result

of the proliferation of often highly technical information which is readily

available on the internet, more and more patients confront their clinicians

with bundles of documentation, even more questions and false expectations.

Oncotherapy is one discipline where personalised or individualised medicine

is indeed becoming more and more relevant in the treatment of cancers. The

use of different biomarkers is aptly illustrated in breast carcinoma.

Oestrogen-receptor status in cases of breast carcinoma was one of the first

biomarkers to be used in the treatment of cancers and formed an integral part

in the treatment of these tumours during the last few decades. This was

followed by the use of Herceptin® (transtuzumab) based on the

amplification of the gene in 25-30% of these

tumours. Immunohistochemistry stains with antibodies reacting with the

above gene product are used to identify those patients who will benefit most

from the treatment. Fluorescence in-situ hybridisation (FISH) is sometimes

needed to confirm this gene amplification.

This is a highly technical field with terminology which is often foreign to most

practising clinicians. Oubaas Pretorius gives a brief introduction to molecular

diagnostics by tracing the processing of a sputum sample for tuberculosis as

an example. He also discusses the molecular diagnostics of pathogens,

including the lessons learnt from the recent outbreak of the H1N1 virus

(swine flu). Linda Steyn discusses molecular testing in solid tumours,

including colorectal carcinoma and breast carcinoma. Regarding the future

of histopathology, it is important to remember that molecular profiling is not

intended to replace the morphological assessment of a malignant neoplasm,

but rather to provide supplementary information, i.e. the risk of recurrence

and response to therapy. Nico de Villiers also discusses the role of and

in the treatment of metastatic colorectal carcinoma. The

management of chronic myelogenous leukaemia is discussed by Illse Louw

These are just a few of the interesting aspects of molecular pathology. Further

developments on the molecular front include genotyping for warfarin dosing

and also other drugs. In the case of warfarin this involves the stratification of

warfarin dosing based on the variation in the vitamin K epoxide

reductasecomplex and cytochrome P450 2C9

gene polymorphisms. As a leading pathology practice, PathCare

will keep you informed when these techniques become therapeutically

applicable.

HER2 (c-erb-B2; HER2/neu)

KRAS

BRAF

(VKORC1) (CYP450 2C9 or

CYP2C9)

PathCare Pathology Forum

Prof Izak Loftus

Molecular Pathology and Personalised Medicine

Ante-natal genetic testing has been well

established for decades; during the last

couple of years the advent of more

sophisticated molecular techniques

expanded the armamentarium of not only the

research orientated scientist, but also the

diagnostic tools available to the practising

pathologist and clinician. This ability was

further promoted when the technology

s u p p o r t i n g t h e s e a n a l y s e s w a s

commercialised and became more readily

available for everyday practice. This edition of

the PathCare Forum focuses primarily on the

use of genetic information in the treatment of

Finally, like any scientific development, molecular pathology and

medicine is not immune to ethical issues and dilemmas. The four basic

principles in medical ethics will have to be adhered to, i.e. respect for

patient autonomy, beneficence (the promotion of what is best for the

patient), non-maleficence (avoiding harm) and justice. The following

ethical issues form an integral part of genetics, more specifically

molecular pathology, and will need to be taken into account when dealing

with genetic and molecular issues:

1)

2)

3)

These are just a few of the issues we will have to deal with. Maybe the

lyrics of Gloria Gaynor's song echo the ethics of genetics and molecular

pathology:

I am what I am

I am my own special creation

So come take a look

Give me the hook

Or the ovation

It's my world

That I want to have a little pride

My world

And it's not a place I have to hide in

Life's not worth a dam

Till I can say

I am what I am……..

3


Dr Oubaas Pretorius

An Introduction into Molecular Diagnostics

of establishing specialized laboratories, it makes sense to consolidate all

molecular diagnostics activities into one unit and in the private sector that is

generally the case.

Broadly speaking, molecular diagnostics can be divided into two main

branches, one focusing on pathogens and the other on genetics.

Traditionally the pathogen-related work dominated both in the number of

available assays and the volume of tests performed per assay. However,

there is a rapid trend towards more genetic assays becoming available, both

constitutional and somatic.

The mainstay of molecular diagnostics is the ability to multiply nucleic acids

artificially to such a level that it can be detected and analysed. For instance,

instead of waiting six weeks for TB bacilli to grow, the DNA can be extracted

Although at first glance the term molecular

diagnostics seems very wide, it is reserved for

diagnostics based on nucleic acids and to a

lesser extent, proteins. It thus excludes the

target molecules that are the focus of general

chemical pathology. With the convergence of

technology used in molecular diagnostics,

classical boundaries among disciplines are

being broken down on an ongoing basis. For

instance, the tools used for detecting and

enumerating bacteria, viruses, fungi,

parasites, germline mutations and sporadic

genetic abnormalities in tumours are all the

same. For that reason and due to the expense

from the primary specimen and amplified using the polymerase chain

reaction (PCR) for detection within a few hours. Without going into

technical detail, PCR is just an in vitro version of what is happening inside

the cells anyway, namely replication of nucleic acid. It just happens

much faster and most importantly, in a targeted manner, as we can decide

which part of an organism's nucleic acid to preferentially amplify.

The diagnostic potential for PCR was seen very soon after invention of the

process, but it took a long time to mature to such a degree where it could

function at the same level as traditional tests. In the early days of

molecular diagnostics, PCR was beginning to get a bad image because of

very variable results. After the introduction of rigorous rules for

prevention of false positives due to contamination of today's reaction with

yesterday's amplified DNA, matters began improving. In the beginning,

false negatives were also a problem, caused by poor choice of target

regions. This was rectified by studying many strains of each organism to

determine the inherent variability of each species. This is an ongoing

process where the tools of bioinformatics and epidemiology converge. To

give an idea of how it works in practice, I shall trace an imaginary sample

through the whole process in the laboratory, using a sputum sample for

TB as example. As with all pathology tests, we in the laboratory become

aware of the request once it has been logged on the computer system

where it appears as a pending test. Eventually (hopefully sooner rather

than later!) the sample will reach our laboratory and the work can start.

First up is DNA extraction, which involves enzymatic lysis of all the cells in

the sample, followed by preferential binding of nucleic acids to magnetic

beads. The process is automated and can be done either in batch mode

where up to 32 samples are processed simultaneously, or it can be done

individually in the case of an urgent sample. After capture of the magnetic

beads, washing follows to thoroughly clean the nucleic acid. The purified

nucleic acid, which may be a mixture of human, pathogen and normal

flora, is then eluted from the magnetic beads in about 0.1 ml of buffer.

The extraction takes place in a dedicated laboratory, in our case called

Area 2. This is the only place where primary samples are allowed.Area 2

After the extraction is completed, it is now time to add the PCR reagents so

that the sample can be run. The reagents are stored in a separate room

known as Area 1 or the clean room. No sample material or previously

amplified nucleic acids are allowed in this room, to prevent cross-

contamination. Just the correct amounts of reagents for the number of tests

to be done are aliquoted into tubes and taken to Area 2 where the extracted

sample is waiting. For each batch that is run, a positive control and negative

control (also known as the no template control as it contains water instead of

DNA as sample) are included.

Once the reagents have been added, the tubes are taken to yet another

dedicated room, Area 3, also known as the post-PCR laboratory. This is

where the actual amplification and analysis takes place. In this laboratory

are all the thermocyclers used for amplification as well as the machines used

for analysis. This is the most dangerous area for causing contamination of

the other areas and there are strict rules in place for preventing carry-over of

amplicon (massively multiplied DNA, the product of PCR) to the other areas.

Our TB sample is then loaded into one of the machines, in this case a real-

time thermocycler which combines the thermal cycling ability of a

conventional thermocycler with an optical system capable of detecting the

formation of amplicon while it happens, hence the real-time tag. Real-time

PCR allows quantification of the target molecule, as it will become visible

above the threshold in a concentration dependent manner; the sooner it

comes up, the higher the initial concentration. If a standard curve is

constructed using a range of standards of known concentration, the absolute

number of molecules per milliliter of initial sample can be calculated. This is

very handy for monitoring treatment of viral diseases, e.g. AIDS where we

determine the number of HIV-1 RNA copies per milliliter plasma. Since

sputum is not a homogeneous matrix and since TB bacilli are not spread

evenly through the lung, quantification is difficult in this case, and we only

report on the presence or absence of TB.

When the run is finished (1-2.5 hours, depending on the specific assay), the

operator prepares a report using the analysis software. For a valid assay, the

two controls must conform: The Positive Control must be Positive and the

Negative Control must be Negative. Included in all our pathogen assays is an

internal control, which is a “dummy reaction” running in the background of

the real assay. This contains the required primers, probes and target DNA for

a complete PCR reaction, but everything is present in limiting amounts so as

not to interfere with the primary reaction aimed at the pathogen. The

internal control probe is labelled with a different dye from the primary

reaction, so that it can be distinguished from it by the machine's optical

system. In order to differentiate a failed reaction from a true negative result,

the internal control must be positive in all negative samples. In some cases,

there may be interfering substances that co-purified with the DNA, especially

from “difficult” matrixes like urine, stool or sputum. Internal controls are

only used for pathogen assays, not genetic tests. In the case of genetic tests,

it is considered a failure if the target DNA does not amplify.

Molecular diagnostics is the fastest growing discipline in pathology and there

is nothing to indicate that it may slow down in the foreseeable future. There

are many new developments waiting in the wings, unfortunately they all

suffer from being untested and/or expensive.

It usually takes longer than anticipated before any new technology becomes

mature enough to be used on a routine basis. Even so, we may see some of

the following soon:

Microarrays used instead of or complementary to classical cytogenetics.

The major advantage here is that there is no need to cultivate cells to get

them into metaphase as DNA is isolated from whole blood and

hybridised to the microarray.

High-throughput sequencing may allow diagnosis of multiple mutations

simultaneously or even (if the cost can come down enough) allow full

genome sequencing of individual patients.

Next-generation pathogen detection and genotyping. This topic will be

highlighted in the accompanying paper on pathogens (see page 11).


Area 3

Area 1

5


Dr Nico deVillers

Personalised/Individualised Medicine:The Role of in the Treatment of Metastatic Colorectal CancerKRAS

Background

Causes

Globally colorectal cancer (CRC) is the third

and fourth leading cause of cancer related

death in women and men respectively with

1,2 million new cases and an estimate of 630

000 deaths in 2007. In the South African

population the lifetime risk (0-74 years) is

1:134 and 1:91 in women and men

respectively.

Up to 30% of cases are familial and the rest

sporadic (70%) with risk factors that include

age, lifestyle, colorectal polyps, Crohn's

disease and ulcerative colitis.

The absence of mutations in tumour tissue has recently shown to

positively affect the response of colon tumors to the EGFR targeted agent

Cetuximab. Therefore tumours with mutated genes are highly

unlikely to respond to anti-EGFR treatment. Unfortunatelly 40% of

wildtype tumours does not respond to anti-EGFR treatment and the

role of other genes downstream of KRAS in the EGFR pathway is currently

under investigation to determine their role. These can include other

sequence variations in the gene than tested for, or variations in the

and genes.

These findings open the new field of individualized medicine and in the

case of metastatic CRC is already incorporated in patient selection for

anti-EGFR treatment.

CRC is common and treatable when detected early.

Most cases are detected late when cancer is metastatic

The EGFR pathway is important for targeted treatment in metastatic

CRC.

Gene mutations ( ) in this pathway can influence tumour

response to anti-EGFR targeted treatment.

Testing to identify mutations in patient tumours is available to

determine effective treatment using anti-EGFR agents.

Studies on the influence of gene defects to treatment in other cancers

are underway and open the field of individualised medicine and

personalised patient treatment

KRAS

KRAS

KRAS

KRAS

NRAS BRAF

KRAS

KRAS

In Summary

References:

Amado RG, Wolf M, Peeters M, Van Cutsem E, Siena S, Freeman DJ, Juan T, Sikorski R, Suggs S,

Radinsky R, Patterson SD, Chang DD: Wild-type is required for panitumumab efficacy in

patients with metastatic colorectal cancer. J Clin Oncol 2008, 26:16261634 Cancer association

of South Africa: Lievre A, Bachet JB, Boige V, Cayre A, Le Corre D, Buc E, Ychou M, Bouche O,

Landi B, Louvet C, Andre T, Bibeau F, Diebold MD, Rougier P, Ducreux M, Tomasic G, Emile JF,

Penault-Llorca F, Laurent- Puig P: mutations as an independent prognostic factor in patients

with advanced colorectal cancer treated with cetuximab. J Clin Oncol 2008, 26:374379, Van

Cutsem E, Kohne CH, Hitre E, Zaluski J, Chang Chien CR, Makhson A, D'Haens G, Pinter T, Lim R,

Bodoky G, Roh JK, Folprecht G, Ruff P, Stroh C, Tejpar S, Schlichting M, Nippgen J, Rougier P:

Cetuximab and chemotherapy as initial treatment for metastatic colorectal cancer. N Engl J Med

2009, 360:14081417

KRAS

KRAS

Diagnosis

Treatment

Prognosis

The Epidermal Growth Factor Receptor (EGFR) Signaling Pathway.

and its Role in Monoclonal Antibody Treatment.

Colonoscopy, sigmoidoscopy, barium enema, faecal occult blood test, faecal

DNA test

Surgery with or without chemotherapy is the most common form of

treatment and very effective in the early localised stages.

If diagnosed early in disease progression (I-IIb) the five year survival rate is

90%, but unfortunately only 39% of patients are diagnosed during this

stage.

EGFR and its ligands are cell signaling molecules that mediates molecular

events critical to cellular growth and survival. This pathway is activated in

response to ligand binding ce receptors. The

EGFR receptor is upregulated in most CRC and modulate events such as

tumor survival, growth, proliferation, adhesion and angiogenesis and this

makes it an excellent candidate for targeted treatment using monoclonal

antibodies and tyrosine kinase inhibitors that targets this pathway showing

clinical activity against CRC.

The gene was discovered more that 25 years ago as the component of

Kirsten sarcoma virus responsible for oncogenesis and encodes one of the

proteins in the EGFR signaling pathway essential for downstream regulation.

Several mutations have been detected in the gene that affects the

function of this highly regulated protein and the EGFR pathway as a whole.

The wild-type protein is only active for a short period of time due to

EGFR mediated activation whereas the mutated protein is switched on

permanently and is not influenced by EGFR activation. This cause the EGFR

signaling pathway to be permanently activated leading to unregulated

tumour growth.

Mutated genes have since been detected in solid tumours of pancreatic

cancer (90%), papillary thyroid cancer (60%), colon cancer (50%) and non-

small lung cancer (30%).

(TGF and EGF) to the cell-surfaα

KRAS

KRAS

KRAS

KRAS

KRAS

6


Dr LindaSteyn

Molecular Testing in Solid Tumours

100 CRC

15MSI-H

Table 1: Breakdown of the molecular

pathogenesis of colorectal cancers.

2HNPCCGermline DNA

mismatch repair

gene mutation

MLH1 promotor

methylation,

CIMP+

10 CIMP+

75 CIMP-,

chromosomal

instability

13 Sporadic

85MSS/MSI-L

Such screening methods can identify patients who should have additional

genetic testing and counselling. Molecular testing using a panel of

microsatellite markers can detect MSI-H tumours. Most MSI-H colorectal

cancers are sporadic and arise from epigenetic gene silencing of one of the

mismatch repair protein genes, most commonly . A minority of

MSI-H colorectal cancers are inherited (hereditary non-polyposis

colorectal cancer) and arise from germline mutation of one of the

mismatch repair genes or .

A subset of colorectal carcinomas (25%) has widespread aberrations in

DNA methylation, including promoter silencing of genes that are

important to tumour biology. Referred to as the CpG island methylator

phenotype (CIMP) this includes most sporadic MSI-H cancers with

methylation silencing of . CIMP testing can be done to detect

abnormal DNA methylation (See Table 1).

Breakdown of the molecular pathogenesis of colorectal cancers.

MLH1

MLH1, MSH2, MSH6 PMS2

MLH1

Table 1

metastasis and perineural and angiolymphatic invasion.

In the past 2 decades the analysis of protein expression by

immunohistochemical staining has become an integral tool for assessment

of pathology specimens. More recently, molecular testing is being integrated

into very specific areas in diagnostic pathology.

This overview will provide some examples of molecular tests in different

stages of clinical applicability in a few different organ systems.

Colorectal adenocarcinomas arise through different genetic pathways and

should no longer be considered one disease. The majority (85%) of

colorectal cancers arise via the chromosomal instability pathway with

dysfunction of the APC/Beta-catenin/WNT signalling pathway. However

15% to 20% of colorectal cancers arise via the microsatellite instability

pathway (MSI), owing to either a germline mutation (Lynch syndrome) or

epigenetic gene silencing secondary to hypermethylation.

Four main proteins, MLH1, MSH2, MSH6 and PMS2 comprise the

mismatch repair complex.

Carcinomas arising via the MSI pathway have characteristic pathologic

features. Antibodies to the above proteins can be used to screen for

Microsatellite unstable tumours (MSI-H) (See figure 1).

Characteristic intact nuclear staining with MLH1 in a patient with a

microsatellite stable cancer. This pattern of staining would be present for all

four mismatch repair protein antibodies (MLH1, MSH2, MSH6, and PMS2).

Molecular Pathology of Colorectal Adenocarcinoma:

Figure 1:

Molecular testing in anatomic pathology will

almost certainly become critical for providing

optimal patient care during the next 5 to 10

years, as more assays are developed that

provide valuable diagnostic, prognostic and

therapeutic information for patient

management.

Traditionally pathologists have relied on the

haematoxylin-eosin-stained slide to make a

diagnosis. Prognostic indicators were limited

to those that could be seen at the light

microscopic level and included such variables

as the surgical margin status, lymph node

Recent studies have shown that status, EGFR amplification and

expression of were associated with outcome measures in

wild-type patients treated with a cetuximab-based regimen. Subsequent

studies will be required to confirm the clinical utility of these markers.

Sequence electrophoretogram of the PCR product of exon 15

showing two overlapping peaks at position 1799, which is diagnostic of

a T A mutation at this position.

Approximately 5% to 10% of breast cancers are caused by mutations in

high penetrance breast cancer susceptibility genes and include

and . These genes confer a high risk of breast and ovarian cancer.

Two genes associated with rare cancer syndromes, and also

confer a very high risk of breast cancer. associated breast cancers

have distinct morphology, being more often medullary-like, triple negative

and showing a “basal” phenotype. and cancers are a

heterogenous group without a specific phenotype. At present the role of

and in DNA repair is being exploited to develop novel

therapies (Poly-ADP ribose polymerase inhibitors). A number of low to

moderate penetrant genes/loci and

have also been identified but their role and contribution in breast cancer

development is still under investigation.

BRAF

PTEN KRAS

BRAF

BRCA1

BRCA2

P53 PTEN

BRCA1

BRCA2 BRCAX

BRCA1 BRCA2

9FGFR2, TNRC9, MAP3K1 LSP1

Figure 2

Molecular Pathology of Breast Tumours

→


7

The clinical detection of colorectal carcinoma with deficient mismatch repair

function is desirable for 3 reasons:

1. Identification of Lynch syndrome (HNPCC). Confirmation of the

germline mutation allows for the most accurate treatment and follow-up

recommendations for the patient and allows predictive testing to be

undertaken in interested family members.

2. Microsatellite instability is also a predictor of chemosensitivity including

5-fluorouracil and irinotecan.

3. MSI-H carcinoma is associated with a more favourable prognosis.

Based on available evidence regarding risk and outcome, mismatch repair

gene mutation carriers should be offered colonoscopy every 1 to 2 years

beginning at 20 to 25 years of age.

Subtotal colectomy is generally favoured for patients with known HNPCC-

Lynch syndrome; however the potential benefit over colonoscopic

surveillance has not been studied.

Approximately 20% of patients with colorectal cancer present with

metastatic disease and an additional 30% to 40% develop metastases

during the course of their disease. Adjuvant therapy is usually used in

patients with advanced stage disease. In particular epidermal growth factor

receptor (EGFR) inhibitor therapies have emerged as effective treatments in

a subset of patients with metastatic colorectal carcinoma. Mounting

evidence has shown that these therapies are ineffective in tumours with

mutations of codons 12 and 13 of exon 2 of the gene (see Figure 2 ).

Because of this compelling data the determination of mutation status

is recommended in all patients with metastatic colorectal carcinoma who are

candidates for anti-EGFR therapy. However only half of these patients will

benefit from treatment, suggesting the need to identify additional biomarkers

for cetuximab-based treatment efficacy.

KRAS Mutation testing in Colorectal Cancer

KRAS

KRAS

From Histopathology 50:113-130, 2007

CIMP, CpG methylator phenotype; MSI-H, high frequency microsatellite instability; MSI-L low frequency microsatellite instability; MSS microsatellite

stable

Recently, a molecular-pathologic classification of colorectal carcinoma has been proposed that is based on the presence or absence of aneuploidy and the

status of mismatch repair and methylation pathways. (See Table 2)

Molecular Pathologic Classification of Colorectal Cancer

Table 2

Group Number

1

2

3

4

5

CIMP Status

CIMP

high

CIMP

high

CIMP

low

CIMP

negative

CIMP

negative

MLH1 Status

Full Methylation

Partial

Methylation

No Methylation

No Methylation

Germline MLH1

or other mutation

Microsatellite

instability status

MSI-H

MSS/MSI-L

MSS/MSI-L

MSS

MSI-H

Chromosomal

Status

Stable

diploid

Stable

diploid

Stable

diploid

Unstable

diploid

Unstable

diploid

Precursor

Serrated polyp

Serrated polyp

Adenoma /

Serrated polyp

Adenoma

Adenoma

Proportion

12%

8%

20%

57%

3%

BRAF T1799A (V600E) mutation


58

utilizes expression array analysis of 70 genes to identify patients with

good and poor prognostic signatures. A further assay, PAM50 Assay®

based on intrinsic subtypes will also be available soon.

In brain tumours loss of 1p (short arm of chromosome 1) and 19q (long

arm of chromosome 19) is associated with oligodendroglioma

differentiation, being found in up to 80% of cases (See figure 4). This

mutational profile has also been shown to correlate with responses to

chemotherapy. Anaplastic oligodendrogliomas with combined allelic

losses on 1p and 19q are typically sensitive to PCV chemotherapy.

Longer survivals have also been reported in patients with 1p and 19q

loss.

(A) This anaplastic oligodendroglioma, grade III, is more crowded with

more pleomorphic nuclei than the grade II oligodendrogliomas. Despite

their pleomorphism, nuclei tend to be round. Mitotic figures are

numerous. (H&E.)

(B) Crowded hyperchromatic nuclei do not overexpress p53.

(C) Diffuse margin of this anaplastic oligodendroglioma in gliotic brain

highlights GFAP-negative branching microvascular proliferations.

(D) One of the deletions often found in oligodendrogliomas is on the short

arm of chromosome 1 (1p).

In this fluorescent in situ hybridization (FISH) preparation, the test probe

for 1p32 is red and the reference probe for 1q42 is green. Each nucleus is

counterstained blue. The single red dot in each of the three whole nuclei

demonstrates a deletion on 1p. The reference probe shows two green

dots, reflecting a pair of chromosomes giving this signal, as expected in

these diploid interphase nuclei. FISH preparation was contributed by Dr.

Arie Perry, Division of Neuropathology, Department of Pathology,

Washington University School of Medicine,

St. Louis, Mo.

From McKeever PE. New methods of brain tumour analysis. In: Mena H,

Sandberg G, eds. Dr. Kenneth M. Earle Memorial Neuropathology Review.

Washington, DC: Armed Forces Institute of Pathology, 2004.

Oligodendroglioma and loss of 1p and 19q

Figure 4

A

B

A

C

B

D

One of the most common applications of molecular pathology in solid

tumours is to test for amplification of gene (See Figure 3).

positive breast cancer is significantly correlated with several unfavourable

pathologic tumour characteristics. Herceptin® was developed as a biologic

targeted therapeutic against the receptor.

-positive breast cancer.

A: immunostain demonstrates intense membrane staining of all

tumour cells in a chicken-wire pattern indicating protein

overexpression (3+).

B: FISH assay using a dual probe system. Red signals denote gene

copies and green signals denote copies of chromosome 17. The average ratio

of red to green signals per nucleus is >2.2; therefore, this tumour shows

gene amplification.

Two molecular tests are available that may aid in assessment of prognosis

and the prediction of response to various therapeutic modalities in individual

patients. Oncotype DX® (Genomic Health Inc.) is based on the analysis of

expression of 21 genes and provides a recurrence score that correlates with

outcome. Mammaprint ® (Agendia) is a molecular prognostic test that

HER2 HER2

HER2

HER2

HER2

HER2

HER2

HER2

Figure 3


Conclusion:

Summary of some of the commonly used molecular tests in Solid Tumours:

The above outlines a few examples of how molecular pathology is incorporated and used in clinical and pathology practice.

In the near feature, classification and diagnosis of most tumours through morphologic analysis will be supplemented by molecular information correlating to

prognosis and targeted therapy.

References:

1. Belezzi AM, Frankel WL. Colorectal cancer due to deficiency in DNA mismatch repair function. A review. Adv. Anat Path. 2009; 16(6):405-417.

2. Coleman WB, Tsongalis GJ. Molecular Pathology. The molecular basis of human disease.

3. Dabbs DJ. Diagnostic Immunohistochemistry. Theranostic and Genomic applications.

4. Hunt, Jennifer. Molecular testing in solid tumours. An Overview. Arch Pathol Lab Med. 2008;132:164-167

5. Hunt, Jennifer. Molecular testing in anatomic pathology practice. A review of basic principles. Arch Pathol Lab Med. 2007; 132:248 -260.

6. Odze RD, Goldblum JR. Surgical Pathology of the GI tract, Liver, Biliary tract and Pancreas

7. Plesec TP, Hunt JL. Mutation Testing in Colorectal Cancer. Adv Anat Pathol. 2009; 16(5): 196-203.

8. Puig P et al. Analysis of PTEN, BRAG and EFGR status in determining benefit from cetuximab therapy in wild-type KRAS metastatic colon cancer. J

Clin Oncol. 2009 1-8.

9. Tanas MR, Goldblum JR. Fluorescence in-situ Hybridization in the Diagnosis of Soft tissue Neoplasms: A review. Adv. Anat Path. 2009;

16(6):383-391.

10. Tubbs RR, Stoler MH. Cell and Tissue Based Molecular Pathology. Odze RD, Goldblum JR. Surgical Pathology of the GI tract, Liver, Biliary tract and

Pancreas

KRAS

Molecular Haematopathology Molecular Pathology of Soft Tissue Tumours

Molecular diagnostic techniques have become an important part of

modern haematopathology as several entities in haematopoietic and

lymphoid neoplasms are defined by their underlying molecular

abnormalities. Molecular pathology is used for diagnosis (neoplastic vs.

reactive), classification, prognosis and monitoring (response and early

recurrence). In routine practice most B-cell lymphoproliferative disorders

are diagnosed on the basis of morphologic and immunophenotypic

findings without need for molecular analysis. However molecular

techniques can be of clinical utility in several settings. Clonality studies

may be helpful in some cases. Several characteristic balanced

translocations are associated with distinct B-cell lymphoproliferative

disorders. Molecular studies to detect these translocations can therefore

be valuable in cases in which the morphologic and phenotypic findings

are not clearly diagnostic. Diagnosis of T-cell lymphoproliferative

disorders can be aided by T-cell receptor rearrangement and several

recurrent cytogenetic abnormalities have been described in specific types

of T-cell lymphoproliferative disorders that may offer additional

assistance in diagnosis or assessment of prognosis.

More than any subset of solid tumours, the diagnosis of soft tissue neoplasms

(mesenchymal proliferations that occur in the extra-skeletal tissues of the body)

has become heavily reliant on molecular analysis as an adjunct to light

microscopic and immunohistochemical evaluation. This is because of the

challenging nature of the discipline and the difficulty of sorting through

diagnostic entities with overlapping histological and immunohistochemical

features. In addition a number of difficult diagnostic entities have characteristic

molecular alterations. Soft tissue tumours can be classified broadly into those

neoplasms with complex and nonspecific cytogenetic and molecular genetic

features and those harbouring relatively simple cytogenetic profiles with

consistent and recurrent genetic aberrations. The most common

morphological/immunohistochemical categories in which molecular testing is

useful includes high grade round cell sarcomas, nonmyogenic spindle cell

sarcomas, low grade myxoid neoplasms, adipocytic neoplasms and

melanocytic neoplasms.

ORGAN

COLORECTUM

COLORECTUM

BREAST

BREAST

BREAST

BREAST

BREAST

HAEMATOPATHOLOGY

HAEMATOPATHOLOGY

BRAIN - OLIGODENDROGLIOMAS

SOFT TISSUE TUMOURS

TEST

microsatellite instability

testing (MSI)Identification of Lynch syndrome, Predictor of

Chemosensitivity Prognosis

Efficacy of anti-EGFR therapy

Hereditary breast cancer treatment

Herceptin treatmentR

Recurrence score correlates with outcome and

response to chemotherapy

Prognosis

Based on intrinsic subtypes. Prognosis

Diagnosis of T-cell lymphoproliferative disorders.

Diagnosis, Classification, Prognosis, Monitoring of

response to treatment and early recurrence

Response to Chemotherapy, Prognosis

Diagnostic

KRAS and BRAF

BRCA1 and BRCA2

Her-2 amplification

Oncotype DX

Mammaprint

PAM50 Assay

T-cell receptor rearrangements and

cytogenetic abnormalities

Specific Translocations

B-cell lymphoproliferative disorders

Loss of 1p and 19q

Cytogenetic Profiles -

genetic aberrations

APPLICATION

9


In the 1990's a compound now known as imatinib mesylate was

synthesized. This agent functions through competitive inhibition at the

ATP-binding site of the enzyme, which leads to the inhibition of tyrosine

phosphorylation of proteins involved in BCR/ABL1 signal transduction

(7).

For adult patients who present with chronic myeloid leukaemia (CML) in

chronic phase it is now generally agreed that initial treatment should start

with the tyrosine kinase inhibitor (TKI), imatinib. Five years after starting

imatinib about 60% of patients will be in complete cytogenetic response

(CCyR) (see insert2) and an appreciable proportion of these will have

achieved a major molecular response (MMR), (1).

Resistance to imatinib has been reported and the molecular mechanisms

involve escape of BCR/ABL1 inhibition, either through kinase domain

mutation within and resultant impairment in the ability of

imatinib to bind, or presumed overproduction of BCR/ABL1 via genomic

amplification or the acquisition of additional Ph chromosomes in the

resistant clone (8). For patients treated with imatinib, a rising level of

is a trigger for kinase domain mutation analysis. The

characterization of inhibitor-resistant mutations is important

to direct therapeutic intervention because it is now apparent that each

resistant mutation functions as a distinct protein with unique biological

properties that may confer a gain or loss of function (9).

In the era of highly successful BCR/ABL1 kinase inhibitor therapy for

patients with CML, molecular monitoring is essential to establish

response and to guide mutation and cytogenetic analysis for the

investigation of resistance (8, 9).

BCR/ABL1

BCR/ABL1

BCR/ABL1

1

5

10

The disease known as chronic myelogenous leukaemia (see insert 3) was

described in the 1840s, first in France and subsequently in Edinburgh and

Berlin (1). In 1960, CML was the first disease where a single chromosomal

abnormality, the Philadelphia chromosome (Ph ) was demonstrated as

fundamental to the aetiology of the disease (2). In 1973 it was shown that

the Ph chromosome results from a reciprocal translocation between the long

arms of chromosome 9 and 22 (3). The molecular consequence of this

translocation is the generation of the fusion protein BCR/ABL1, a

constitutively activated tyrosine kinase. Studies have established that

BCR/ABL1 alone is sufficient to cause CML and that the tyrosine kinase

activity of the protein is required for its oncogenic activity (4, 5, 6). For these

reasons, it was thought that an inhibitor of the BCR/ABL1 tyrosine kinase

should be an effective and selective treatment for CML (7).

1

1

Dr Illse Louw

Molecular Technology in the Managementof Chronic Myelogenous Leukaemia

Major advances have been made in applying

molecular technology to the practice of

laboratory medicine. The techniques are used

as diagnostic and prognostic aids and in

monitoring of disease. It has lead to the

development and validation of novel forms of

therapy. This is very aptly illustrated by the

advances in the diagnosis and therapy of

chronic myelogenous leukaemia (CML) where

routine haematology tests are complemented

by cytogenetic analysis, fluorescent

hybridization (FISH), polymerase chain

reaction (PCR) and mutation analysis in the

management of these patients.

in-situ

11


Insert 1: Recommendations for monitoring individual patients (1, 9)

Insert 2: Definition of response (10)

Hematologic

response

Cytogenetic

response

Molecular

response

Complete: Platelets < 450 x 10 /l; WBC < 10 x 10 /l,

no immature granulocytes and < 5% basophils.

9 9

Ph1 Pos metaphase

Complete

Partial

Minor

Minimal

Complete

Major

0%

1-35%

36-65%

66-95%

Transcripts not detected

< 0.1%

Insert 3:

References

1. Goldman JM. Initial treatment for patients with CML.

Hematology 2009, 453

2. Nowell PC, Hungerford N. A minute chromosome in human

chronic granulocytic leukemia. Science. 1960; 132:1497.

3. Rowley, J.D. (1973) A new consistent chromosomal

abnormality in chronic myelogenous leukaemia identified by

quinacrine fluorescence and Giemsa staining. Nature,

243, 290293.

4. Daley GQ, Van Etten RA, Baltimore D. Induction of chronic

myelogenous leukemia in mice by the P210bcr/abl gene of the

Philadelphia chromosome. Science 1990; 247:824-30.

5. Kelliher MA, McLaughlin J, Witte ON, Rosenberg N. Induction

of a chronic myelogenous leukemia-like syndrome in mice with

v-abl and BCR/ABL. Proc Natl Acad Sci U S A 1990;87:6649-

53. [Erratum, Proc Natl Acad Sci U S A 1990; 87:9072.]

6. Heisterkamp N, Jenster G, ten Hoeve J, Zovich D, Pattengale

PK, Groffen J. Acute leukaemia in bcr/abl transgenic mice.

Nature 1990; 344: 251-3.

7. Druker BJ et al. Efficacy and safety of a specific inhibitor of the

bcr-abl tyrosine kinase in chronic myeloid leukemia. NEJM

2001 34 1031-7.

8. Shah N P. Medical management of CML. Hematology

2007, 371.

9. Branford, S. Chronic myeloid leukemia: Molecular monitoring

in clinical practice. Hematology 2007, 376.

10. Goldman JM Recommendations for the Management of

-positive Chronic Myeloid Leukaemia British Committee

for Standards in Haematology. BCSH 2008

11. Sokal JE, Cox EB, Baccarani M, et al. Prognostic

discrimination in 'good-risk' chronic granulocytic leukemia.

Blood. 1984 63: 789-799.

12. WHO classification of tumours of haematopoietic and

lymphoid tissues. 4th Ed. Ed. Swerdlow S H et al. WHO, Lyon,

2008. P32-37.

BCR-

ABL

Chronic myelogenous leukaemia is a myeloproliferative neoplasm

consistently associated with the fusion gene. The

worldwide annual incidence is 1-2 cases per 100 000 population. CML

may occur at any age, but the median age is the 5 and 6 decades of

life. There is a slight male preponderance. The patients may present in

chronic phase with weight loss, fatigue, night sweats, anaemia and

splenomegaly. Blood counts reveal a leukocytosis with left shift,

basophilia and eosinophilia. Twenty to forty percent of patients may be

asymptomatic and are identified through incidental blood count. In

patients treated with a tyrosine kinase inhibitor the current 5 year

progression free survival and overall survival is between 80-95% (12).

BCR/ABL1

th th

The following are guidelines in assessing response and recommendations on

further management.

At Diagnosis

Thereafter

At 3 months

At 6 months

Thereafter

FBC

Sokal Score (11)

Bone marrow analysis with cytogenetic analysis

FISH for BCR/ABL1 in the absence of a

Philadelphia chromosome on cytogenetic analysis

RQ-PCR for BCR/ABL1 transcripts (optional)

Blood counts at intervals of 2 or more weeks

Liver function tests

Blood count

Bone marrow cytogenetics

RQ-PCR for BCR/ABL1 transcripts

Blood count

Bone marrow cytogenetics

RQ-PCR for BCR/ABL1 transcripts

Thereafter bone marrow aspirates are only required

if CCyR has not been achieved

RQ-PCR for BCR/ABL1 transcripts at 3 monthly

intervals - indefinitely

Bone marrow cytogenetics > 12 monthly intervals if

MMR achieved, immediately upon loss of MMR

Mutation analysis on failure of treatment

suboptimal response, rise in BCR/ABL1 transcripts


12

Dr Oubaas Pretorius

Molecular Diagnostics of Pathogens

a full review of everything going on currently. One of the most common

groups of fastidious organisms that we test for are the Rickettsiae. They are

exceedingly difficult to cultivate and the standard serological tests are not

reliable, so the only sure way to detect them is by PCR. These bacteria are

cell-associated, so that buffy coat preparations need to be made prior to DNA

isolation. Even so, the amount of DNA is very low and can be missed. The

golden standard sample type is a biopsy from an eschar, since the organisms

concentrate there, but it is an invasive procedure leaving a scar and is seldom

used for diagnosis. Another example of where molecular diagnostics makes

a difference is that of , a common lung pathogen in AIDS

patients. Before PCR, it was diagnosed by immunofluorescense microscopy,

which is labour intensive and operator-dependent and required a good

quality sample to enable the antigen to stand out from the background. Now

the sputum or lavage is put through the DNA extraction process and PCR

specific for the organism is done.

Pneumocystis jiroveci

One of the major application areas of

molecular diagnostics is in clinical

microbiology in its widest sense. The ability

to look inside an organism has opened new

possibilities of refining and adding value to

diagnostics. Not only does it allow us to

detect emerging pathogens (e.g. H1N1) for

which no other assays exist, but it can also

quantify (e.g. HIV-1) and genotype (e.g. HCV)

pathogens. For some fastidious organisms

(e.g. the Rickettsia spp) it is the only reliable

way to detect them. This is a rapidly growing

field and this article is an attempt to highlight

a few examples only, as space does not allow

Molecular diagnostics is also very powerful for detecting organisms

involved in outbreaks, where no other assays have been developed yet. A

case in point is the outbreak of Influenza A H1N1 2009, popularly known

as swine flu, during 2009. Serology was of no use as the current

antibodies could only detect Influenzavirus A without distinguishing

between the seasonal variant and H1N1. Molecular diagnostics was the

only tool available and it had challenges of its own. Since the outbreak

happened so fast, there was no time to properly develop and validate an

assay that would be foolproof. Instead, the CDC in the USA gave

emergency approval for a specific set of primers and a probe that

successfully identified all isolates studied up to April 2009. It was later

found that even as early as June 2009, this assay was missing some of

the newly emerging variants of H1N1 2009. Also, laboratories stocked

up on reagents for H1N1, leading to shortages of reagents and even

swabs for taking the samples. Some manufacturers switched to other

assay designs to improve specificity, but this caused discordant results

between laboratories, with no one being sure who was correct. This

episode showed what could happen when a new disease strikes and

valuable lessons were learnt in the process, the most important of which

is to improve the vigilance of the bodies responsible for emerging disease

monitoring to enable earlier intervention.

The ability to quantify pathogens is one of the major contributions of real-

time PCR and it is in constant use for monitoring the effect of treatment on

several viral diseases. In South Africa with its high AIDS burden, the

monitoring of HIV-1 viral load is a major operation, both in the state and

private sectors. For such high-throughput assays, full automation makes

sense and there are several systems on the market capable of taking care

of the whole process, from extraction through to quantification and

reporting of the results, all with minimal operator input. As technology

improves, the lower limit of quantification and the linear range of these

assays are stretched more and more. Not long ago, 400 copies per ml

was the lower limit of HIV-1 quantification, it is currently 40-50 copies

per ml with a new generation capable of pushing it down to 20. With the

advent of real-time PCR, the distinction between quantitative and

qualitative PCR began to disappear. Real-time PCR is by nature

quantitative, if a target can be amplified, it can be quantified.


13

This will amplify any bacterial DNA present in a sample. The next step is

to identify the amplicon that has been created. This is done by using high-

accuracy mass spectroscopy. Extensive research has indicated that it is

not necessary to know the exact nucleotide sequence of an amplicon to

identify it, as the base composition is nearly just as informative. A

particular mass can almost always be arrived at by a unique base

composition. Researchers have constructed databases containing

multiple isolates of all known pathogens likely to be found in a particular

sample. For instance, several hundred bacterial and several dozen fungal

species can be identified in a blood sample. It is somewhat more difficult

to replicate this approach for viruses, but a number of virus families can

be identified already. A very promising application of this technology is in

the diagnosis of TB. The assay is designed in the form of a multiplex

containing PCR primers for identifying TB as well as looking for mutations

coding for resistance to both first- and second-line drugs. This means full

drug resistance information within one working day. Being so versatile

means that this new approach has the potential to turn diagnostic

algorithms upside down in the sense that you don't need to know exactly

what you are looking for.

Some pathogens, e.g. HCV, need to be genotyped so that treating physicians

may have a better idea about the course of the treatment. In the case of HCV,

there are currently 6 main subtypes of which types 1 and 4 respond more

slowly to treatment than the others. Previously, it was painstaking work to

determine the subtype, now it can be done within a single working day.

Genotyping is also used to look for drug resistance mutations in a variety of

organisms, e.g. TB and HIV-1. For TB, it takes the form of two rounds of

DNA amplification followed by hybridization to filter strips in order to detect

mutations against the most frequently used first- and second-line drugs. The

TB bacillus is amenable to this approach since each drug-naïve isolate

develops de novo drug resistance in a predictable sequence. Only a few

genes are involved in the development of drug resistance and the mutations

have been characterized extensively. Using molecular methods, the drug

resistance profile of microscopy-positive sputum samples can be done within

one to two days instead of many weeks by conventional means.

HIV-1 is also prone to developing drug resistance mutations after long term

use of a specific regimen or where there is compliance or tolerability issues.

Since the majority of current drugs target the protease or the reverse

transcriptase genes, these 2 genes are amplified and their nucleotide

sequence determined. The sequence is then scanned against a database of

known mutations and a report is compiled which tells the requesting

physician which drugs not to use, as well as which drugs are still available.

Recently, a new test became available to identify drug resistance mutations

in the integrase gene to monitor resistance to the new class of integrase

inhibitors.

There are many exciting developments to come in the medium to long term

and a small number will be highlighted. New developments allow both an

increase in speed, e.g. the GeneXpert and depth of analysis, e.g. the Plex ID

system.

The GeneXpert (by Cepheid) is a fully automated, random-access system

capable of extracting nucleic acid, amplification of the target(s) and

generation of a report. Our main application of the system is for

hospitalised patients suspected of having TB, where time is of the

utmost essence. Not only does it identify the bacillus, it also

looks for resistance against rifampicin. In practice, this

system showed itself to be more sensitive than culture to

identify TB, with the added benefit of having an

answer within 2 hours and minimal operator input.

Unfortunately, this convenience comes at a

price, hence reserving the test for hospitalised

patients. If the cost of performing the test

does not come down, it is difficult to see

how it can be used as a first-line screening

for TB.

When it comes to depth of diagnosis,

the Plex ID system (by Abbott)

represents a totally new paradigm. It

is based on two steps: First, "general"

PCR is performed, for instance highly

conserved genome areas common to

all bacteria are used.


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isease

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Prim

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ukin

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NA

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ach

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isease

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gy

Colo

n

Haem

ato

logy

Meta

bolic

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rolo

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HL

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6D

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mu

tatio

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meth

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nsfe

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ken

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pu

latio

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s

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pu

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nsp

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phom

a

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cyto

sis

MD

S

MP

N

Myelo

ma

Solid

Tum

ours

Congenita

l

t(11;1

8),

t(14;1

8),

t(11;1

4),

3q27,

t(8;1

4)

and

varia

nts,

t(2;5

)

TC

Rre

arra

ngem

ent

IGH

Vre

arra

ngem

ent

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D816V

(CK

ITse

quencin

g)

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,5q31,5q33-q

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ha

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GFR

beta

FG

FR

1

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LW

515L

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4),

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,17p13.1

,t(1

4;1

6)

BR

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:

CO

LO

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NSC

LC

EG

FR

sequencin

g

Cyto

genetic

analy

sis

FIS

Hanaly

sis

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/neu

KRA

S

BRA

F

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MSI

IHC

HN

PC

C

Lym

phom

a

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cyto

sis

MD

S

MP

N

Myelo

ma

Solid

Tum

ours

Congenita

l

Cancer

Haem

ato

logy

t(8;2

1)

inv(1

6),

t(16;1

6)

t(15;1

7)

inv(3

),t(3

;3)

t(9;2

2)

t(v;1

1q23)

t(12;2

1)

t(1;1

9)

11q22.3

,12cen,13q14,13q34,1

7p13.1

t(9;2

2)

BC

RA

BL

copie

s,

BC

RA

BL

sequencin

g

(incase

ofim

atin

ibre

sistance)

AM

L

ALL

CLL

CM

L

Cancer

Haem

ato

logy

Fungal

Pneum

ocystis

jiroveci

Fungal

Para

siteM

ala

riasc

reen

Mala

riasp

ecie

sid

entific

atio

n

Para

site

Viru

sC

MV

quantita

tive

CM

Vqualita

tive

EB

V

Ente

roviru

s

HB

Vquantita

tive

HC

Vqualita

tive

HC

Vgenoty

pin

g

HIV

qualita

tive

HIV

quantita

tive

HIV

dru

gre

sistance

HP

Vhig

h-risk

scre

en

HSV

1and

2

Measle

sviru

s

Parv

oviru

sB

19

Resp

irato

ryviru

spanel

VZV

Viru

s

Mole

cula

rpath

olo

gy

isa

rapid

lydevelo

pin

gfie

ld.

We

will

regula

rlyupdate

our

listofavaila

ble

tests

whic

hyou

can

also

access

at

ww

w.p

ath

care

.co.za

.

Ple

ase

conta

ct

our

labora

tor y

at

02

1-5

96

34

00

or

mole

cula

r@path

care

.co.za

regard

ing

the

availa

bility

ofany

tests

whic

hdo

not

appear

on

the

list.

OTH

ER

Alp

ha-1

-antitry

psin

Apolip

opro

tien

E(A

po-E

)

Blo

om

syndro

me

Cystic

fibro

sis

Deafn

ess

Connexin

26

Epith

elia

lso

diu

mchannel

βch

ain

Fam

ilialH

yp

erc

ho

leste

rola

em

ia(F

ou

nd

er

varia

nts

)H

LA

B27

OTH

ER

vol. 3 no. 2 - pathcare south africa. 3 no. 2 2 3 12 14 molecular ... lyrics of gloria gaynor's...

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