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East African Pathology Forum

May-Aug 2011 Vol. 2

www.pathcarekenya.com

Purpose,Values and Aspirations

Molecular Testing in Solid Tumours

Laboratory Diagnosis of Malaria

Lab Diagnosis of Brucellosis

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East African

East African

Dr Jane Mwangi Assistant Editor Benjamin Matheka

C.E.O/Chief Pathologist Dr Kiran Radia Coast Pathologist Dr Kishor Mandaliya Operations Manager Madhu Aggarwal Technical Manager Paul Okwach Financial Controller Charity Njuguna Marketing Manager Benjamin Matheka

From the Editor


Laboratory tests have become increasingly sophisticated in recent years and this has increased the importance of the laboratory in assisting clinicians in diagnosis and management of patients. Molecular pathology is rapidly developing and an ever-changing science. In this issue we have an article on molecular testing in solid tumours.We trust that you will find the information useful and that it will assist you in the understanding of the most modern techniques Pathcare has. Malaria continues to be a serious public health problem and our haematopathologist contributes a review on the laboratory diagnosis of malaria. The number of tests utilizing PCR-technology is growing at a spectacular rate. Brucellosis has for many years been a difficult disease to diagnose, confirm cure and predict relapses and has now also succumbed to the PCR wave. In this Forum we compare the use of conventional culture and serology for the diagnosis of brucellosis with the much more accurate and reproducible PCR-test. On the facing page you can read a statement about Pathcares Purpose, Values and Aspirations. This demonstrates the philosophy that underpins what we represent.

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laboratory tests in all the clinical settings viz. acute brucellosis, chronic infections, focal infections, follow-up of treatment and prediction of relapses. Laboratory tests Blood culture: These tests take up to 7 days to produce a result, have a sensitivity of 74% in the acute phase but the sensitivity is significantly reduced in the focal and chronic forms of the disease. Culture of the organism poses a high risk of contagion to the laboratory staffand therefore is not performed at our laboratory. Serology Serology tests have a poor sensitivity during the early stages of the disease due to low levels of antibodies. Serological methods lack specificity and titres often remain elevated for protracted periods after therapy even in cases of complete recovery. Less than 25% of patients show a significant rise in antibody titre during relapses. Serology has serious limitations, especially in chronic, relapsing and focal forms of disease.

Polymerase chain reaction (PCR) If buffy coat samples are used, PCR tests have been shown to have a 100% sensitivity and a 100% specificity in acute, chronic, relapsing and focal forms of disease. PCR tests can predict therapeutic failure and relapses with almost 100% accuracy. It is therefore the opinion of this laboratory that PCR tests should replace the current serology tests for the diagnosis of brucellosis. PCR testing can also be done after 6 weeks of effective treatment to ensure eradication of brucella and thereby prevent the high rate of recurrence.

Purpose, Values and Aspirations

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Molecular Testing in Solid Tumours

Laboratory Diagnosis of Malaria

Laboratory Diagnosis of Brucellosis

Pathcare Kenya Limited staff attending to members of the public at the Westgate Shopping Mall where they offered free testing for blood glucose and blood group

Pathcare staff speaking to a client on the services we offer after undertaking a free blood glucose and blood group test at the Westgate Shopping Mall

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East African

the laboratory diagnosis of brucellosis

Clinical background Brucellosis is a febrile bacterial disease acquired from domestic animals and encountered worldwide.Four Brucella species, each with its own animal reservoir, can cause human disease i.e. B. abortus (cattle), B. suis (swine), B.melitensis (sheep and goats) and B. canis (dogs). Of the four, B. melitensis is the commonest cause of symptomatic disease in humans. Humans are infected with the bacteria through: Contact with infected blood or tissue; Ingestion of contaminated meat or milk; Inhalation of contaminated aerosols.

Brucellosis is an occupational hazard among farmers, herders, veterenarians and abattoir workers. After entering the body, the bacteria spread via the bloodstream to the liver, spleen, lymph nodes and bone marrow where they multiply in macrophages, which undergo generalised hyperplasia. This may lead to lymphadenopathy and hepatosplenomegaly (B. melitensis and B. abortus) , the development of noncaseating granulomas in the liver, spleen, lymph nodes and bone marrow (B. abortus) or suppurative liver abscesses (B. suis) . The clinical symptoms of brucellosis are non-specific and several other febrile diseases may be mimicked. Features of acute disease are varied and onset may be insidious. Acute infection can be followed by chronic infection, relapses (undulating fever) or focal forms of infection. Features of chronic disease are vague and may persist or recur for years. The relapse rate after appropriate antibiotic therapy can be as high as 30%. It is therefore important to evaluate the performance of

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Written & Compiled by Dr Pierr Schoeman

East African

East African

References:
1. Malaria Working Party of the General Haematology Task Force of the British Committee for Standards in Haematology. The laboratory diagnosis of malaria. Clin Lab Haem 1997 (19) 165-170. 2. Hnscheid, T. Diagnosis of malaria: a review of alternatives to conventional microscopy. Clin Lab Haem 1999 (21) 235-245.
P . falciparum in thin blood film Gametocyte of P . falciparum in blood film Rapid diagnostic test

Molecular testing in anatomic pathology will almost certainly become critical for providing optimal patient care during the next 5 to 10years, as more assays are developed that provide valuablediagnostic,prognostic and therapeut ic informat ion for patient management. Traditionally pathologists have relied on thehaematoxylin-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 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. Molecular Pathology of Colorectal Adenocarcinoma: 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).

Figure 1:

3. http://www.wpro.who.int/sites/rdt/ documents/The Use of Malaria Rapid Diagnostic Tests. 4. http://www.wpro.who.int/sites/rdt/ documents/ Malaria Diagnosis: New perspectives: WHO 2000. 5. Perandin F. et al. Development of real-time PCR assay for detection of Plasmodium falciparum, Plasmodium vivax, and Plasmodium ovale for routine clinical diagnosis. J Clin Microbiol 2004 (42) 1214-1219. 6. http://rbm.who.int/wmr2005.Word Malaria report 2005. Prepared by: Roll Back Malaria. World Health Organization. UNICEF.

Acridine orange, a fluorescent dye, has affinity for nucleic acids. The quantitative buffy coat method (QBC) uses acridine orange and a concentration technique to enhance detection of the malaria parasite. An acridine orange coated capillary filled with 50-100 l blood is centrifuged and the buffy layer is examined under a fluorescent light microscope for the presence of red cells containing parasites. The sensitivity of the QBC method has been reported to be between 55% to greater than 90% (2).

Fluorescence based microscopy tests:

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). 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 MLH1 . 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 MLH1, MSH2, MSH6 or PMS2 . 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 MLH1 . CIMP testing can be done to detect abnormal DNA methylation (See Table 1). Table 1 Breakdown of the molecular pathogenesis of colorectal cancers.

Real time PCR has recently been adapted to detect all four human malaria parasites with 100% sensitivity and specificity and allow quantification (5). The real time PCR assay offers a practical and clinically acceptable alternative for rapid and accurate diagnosis of malaria infestations in patients presenting with symptoms of this disease and should in future, when suitable commercial kits are available, be incorporated into diagnostic algorithms as an adjunct to microscopic and immunological tests. In a disease like malaria where misdiagnosis results in significant morbidity and mortality and taking into account the limitations of the diagnostic tests, the following is suggested as a decision chart for treatment:

New tests

SUSPECTED CLINICAL CASES


Microscopy / RDT Positive Nonfalciparum Treatment protocol High suspicion of malaria Treat while excluding other disease Negative Look for other disease Review and repeat smears Refer

2HNPCC 15MSI-H
methylation, CIMP+

P . falciparum

Severe malaria
85MSS/MSI-L
chromosomal instability

Uncomplicated malaria Treatment protocol

Treatment protocol

Written and Compiled by Dr LindaSteyn

East African

East African

the laboratory diagnosis of malaria

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) Table 2 Molecular Pathologic Classification of Colorectal Cancer
Microsatellite 1 2 CIMP high CIMP high CIMP low CIMP negative CIMP negative Partial Methylation MSI-H MSS/MSI-L MSS/MSI-L MSS MSI-H Chromosomal Status Stable diploid Stable diploid Unstable diploid Unstable diploid Stable diploid Adenoma Adenoma Precursor Proportion 12% 8% 20% 57% 3%

At the end of 2004 3.2 billion people lived in areas at risk of malaria t r a n s m i s s i o n . An estimated 350-500 million clinical malaria episodes occur annually, most caused by P.falciparum and P. vivax. Falciparum malaria causes more than 1million deaths each year.About 60% of the malaria cases worldwide, about 75% of global falciparum malaria cases and 80% of malaria deaths occur in Africa south of the Sahara (6). Misdiagnosis of malaria results in significant morbidity and mortality. Rapid and accurate detection, speciation and enumeration of malaria parasites has an important role in addressing this and in promoting rational use of increasingly costly drugs. The tests that are available today include microscopy (thick smear, thin smear, fluorescence microscopy), antigen detection and detection of parasites with the polymerase chain reaction (PCR). At PathCare we follow guidelines for diagnosis of malaria as prepared by the Malaria Working Party of The General Haematology Task Force of the British Committee for Standards in Haematology. The recommendations state the following - both thick and thin blood films should be examined and microscopy may be supplemented by an immunological or fluorescence based method (1).

Distribution of malaria

Anopheles mosquito

Malaria parasite in thick blood film

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The thick film is used for the detection of the parasites and the thin film is used for species identification. When a minimum of 200 oil immersion fields (x100 objective) is viewed on a thick film, approximately 0.50 l of blood is examined. The detection limit of a thick film is 5-20 parasites per l. When 100 oil immersion fields (x100 objective) of a thin blood film are viewed, 0.005 l of blood is examined. The detection limit of a thin blood film is 50-200 parasites per l (2).

Thick film and thin films:

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

Immunological tests for malaria are also known as rapid diagnostic tests (RDTs). These tests are based on the detection of antigens derived from malaria parasites in lysed blood, using immunochromatographic methods. The three antigens that are commonly detected are Plasmodium falciparum histidine-rich protein 2 (HRP2), a panspecific malaria aldolase and parasitespecific lactate dehydrogenase. The RDT currently in use at Pathcare detects the HRP2 of P . falciparum and a panspecific plasmodium aldolase. . falciparum at RDTs generally achieve a sensitivity of >90% in the detection of P densities above 100 parasites per l blood (4) and the specificity of RDT's is uniformly high (mostly >90%). Limitation of the tests include limited species identification, false positive results which have been reported in patients with rheumatoid factor in kits testing for the HRP2 antigen (4), and HRP2 tests remaining positive for 7-14 days following chemotherapy in a proportion of individuals, even though these patients no longer have symptoms or parasitaemia (as assessed by blood smears), thus limiting the use of the RDT's in follow-up of treatment (2). Kits that incorporate testing for both the panmalaria aldolase and HRP2, to . facilparum and nonfalciparum species, cannot differentiate detect both P . vivax, P . ovale and P . malariae, between P nor can they distinguish pure P . fa lcipa r um infections from mixed infections that include P . fa lcipa r um (4)

Immunological tests:

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 HNPCCLynch syndrome; however the potential benefit over colonoscopic surveillance has not been studied. KRAS Mutation testing in Colorectal Cancer 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 KRAS gene (see Figure 2 ). Because of this compelling data the determination ofKRAS 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.

Recent studies have shown that BRAF status, EGFR amplification and expression of PTEN were associated with outcome measures in KRAS wild-type patients treated with a cetuximab-based regimen. Subsequent studies will be required to confirm the clinical utility of these markers. Figure 2

Sequence electrophoretogram of the PCR product of BRAF exon 15 showing two overlapping peaks at position 1799, which is diagnostic of a T A mutation at this position. Molecular Pathology of Breast Tumours Approximately 5% to 10% of breast cancers are caused by mutations in high penetrance breast cancer susceptibility genes and includeBRCA1 and BRCA2 . These genes confer a high risk of breast and ovarian cancer. Two genes associated with rare cancer syndromes, P53 and PTEN also confer a very high risk of breast cancer. BRCA1 associated breast cancers have distinct morphology, being more often medullary-like, triple negative and showing a basal phenotype. BRCA2 and BRCAX cancers are a heterogenous group without a specific phenotype. At present the role of BRCA1 andBRCA2 in DNA repair is being exploited to develop novel therapies (Poly-ADP ribose polymerase inhibitors). A number ofl ow to moderate penetrant genes /loci 9FGFR2, TNRC9, MAP3K and LSP1 have also been identified but their role and contribution in breast cancer development is still under investigation.

Interpretation of RDT's:

CONTROL LINE POSITIVE HRP2 line only positive P . falciparum infection or P . falciparum infection plus infection with non-falciparum species of such low levels that the aldolase antigen does not test positive HRP2 and aldolase lines positive P . falciparum or mixed infection with P . falciparum, vivax, ovale, malariae Aldolase line only positive Non-falciparum infection

Written & Compiled by Dr Illse Louw

East African

East African

One of the most common applications of molecular pathology in solid tumours is to test for amplification of HER2 gene (See Figure 3). HER2 positive breast cancer is significantly correlated with several unfavourable pathologic tumour characteristics. Herceptin was developed as a biologic targeted therapeutic against theHER2 receptor. Figure 3 HER2 -positive breast cancer.

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. Oligodendroglioma and loss of 1p and 19q 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. Figure 4

Molecular Haematopathology 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. Conclusion: Summary of some of the commonly used molecular tests in Solid Tumours:

Molecular Pathology of Soft Tissue Tumours 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.

C
A: HER2 immunostain demonstrates intense membrane staining of all tumour cells in a chicken-wire pattern indicating HER2 protein overexpression (3+).

ORGAN
COLORECTUM COLORECTUM BREAST BREAST

TEST

APPLICATION

B
(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.

BREAST BREAST BREAST HAEMATOPATHOLOGY HAEMATOPATHOLOGY Mammaprint Prognosis

Diagnostic

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. KRAS 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

B: FISH assay using a dual probe system. Red signals denote HER2 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 HER2 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