A Review on Complement Diagnostics

Volume 18, Issue 1, Jan 2023  (download full article in pdf)

Editorial note:

The complement system though commonly regarded as component of the innate immune system that protect our bodies from infection, it has increasingly evident that it has important roles in other immune surveillance and housekeeping functions, that it is involved in a wide and diverse range of clinical conditions. In this review, Dr Elaine Au provided an overview of the complement diagnostics and its clinical applications. We welcome any feedback or suggestions. Please direct them to Dr Elaine Au of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr Au Yuen Ling Elaine

Consultant, Division of Clinical Immunology, Department of Pathology, Queen Mary Hospital

The complement system not only as part of the innate immune system that contributes to the elimination of pathogens, and promote inflammation, it also modulates the adaptive immune response. Though its primary role is in host defense, it also serves an important role in clearance of apoptotic cells and immune complexes. Low or dysregulated activity in complement system has been described in a range of disease and pathological conditions.

The Complement system

The complement system comprises approximately 50 proteins, that are found in fluid phase or bound to cell surface (2). The central complement reaction involves the cleavage of C3 into C3b and C3a, which is promoted by the C3 convertase. Collectively, there are three activation pathways forming the C3 convertase. The classical pathway (CP) is triggered by the immune complexes, while the lectin pathway (LP) is triggered by the binding of mannan-binding lectin (MBL) or ficolins to carbohydrates or pathogen-associated molecular patterns. Both activation of CP and LP would lead to the formation of C4b2a as C3 convertase. On the other hand, in the alternative pathway (AP), there is a constant low-grade hydrolysis of C3, that binds factor B and cleaves factor D to generate a fluid phase C3 convertase, that is self- limited in healthy state. However, the AP will be activated and amplified through binding of the cleaved C3 to pathogens or altered tissues. Hence, AP helps to amplify complement activation initiated from CP and LP. The pathways converge in a common pathway to form the membrane attack complex (C5b-9). In addition, the cleavage of C3 and C5 generates C3a and C5 a, that act as anaphylatoxins, while the target bound C3 fragments (C3b, iC3b, C3d, g) facilitate phagocytosis.

The complement activation is delicately controlled by multiple soluble and membrane bound regulators. Factor H, C4b binding protein, the membrane proteins complement receptor 1 CR1 (CD35), decay acceleration factor (CD55), and membrane cofactor protein MCP (CD46), act as cofactors for plasma proteinase factor I, accelerating the decay of convertases. In addition, CD59 and C1 inhibitor regulate the C5b-9 complex and the C1 complex respectively.

Examples of complement diagnostics indications and associated disease conditions

A broad spectrum of clinical conditions is associated with complement deficiencies or its overactivation / dysregulation. The clinical consequences can be broadly categorized into three areas. 1) susceptibility to infection, 2) autoimmunity and 3) defects in controlling and limiting complement activation.

Infection susceptibility

In general, complement deficiencies are associated with increased risk of infections, especially encapsulated bacterial infections, most commonly Pneumococci, Hemophilus etc. In particular, individuals suffering from deficiencies in the terminal components (C5-C9) or properdin are susceptible to Neisseria infections. Hence, complement studies are indicated in the workup of young individuals suffering from recurrent infections (e.g. recurrent sinopulmonary infections, meningitis, etc), especially in recurrent infections caused by encapsulated bacteria. Nevertheless, primary component deficiency is rare, and most of these conditions are autosomal recessive (X-linked inheritance in properdin deficiency) (1).

Autoimmune diseases

Deficiency in early components of the CP, is frequently associated with lupus like autoimmune conditions. The associations range from 10% prevalence of lupus like conditions in C2 deficiency, to C1r/s (57% prevalence), C4 (75% prevalence) and C1q (90% prevalence) (2). These deficiencies can be confirmed in genetic studies and components measurement. Overall, primary deficiency is relatively uncommon. More often, lupus and other autoimmune immune complex diseases causes secondary complement components deficiency as consumption due to the immune complex activation. The component levels, e.g. C3 and C4 levels, are commonly employed in the workup and disease activity monitoring in these conditions. In some occasions, measuring autoantibodies, such as anti-C1q antibody in hypocomplementemic urticarial vasculitis syndrome (HUVS) and lupus, is useful for diagnosis and prognostication.

C3 nephropathy and Thrombotic microangiopathy (TMA)

Uncontrolled AP activation may result in a number of kidney diseases and systemic conditions. C3 glomerulopathy comprises C3 glumoerulonephritis (C3GN) and dense-deposit disease (DDD), is a pathological condition defined by predominant C3 accumulation, with absent or scantly immunoglobulin deposition. Atypical post infectious glomerulonephritis also falls in the continuum of C3 GN and DDD (3). In these conditions, underlying predisposition, be it genetic or acquired, may not be clinically evident until a triggering event, such as infection or pregnancy, that unfold the complement dysregulation. Besides genetic predisposition, presence of autoantibodies, e.g. C3 nephritic factor (C3 Nef), anti-factor H, have been observed in some patients. C3Nef are autoantibodies that bind to components of AP convertase, prolonging its functional half-life, leading to continuous C3 activation and consumption, with lowish CP and AP studies. Factor H has important role in the regulation of complement activation. In some patients, they are predisposed to the disease due to Factor H dysfunction caued by mutation or anti-Factor H. Useful workup for C3 nephropathy includes the complement pathways, components and activation products studies, testing for plasma cells disorders, determination of autoantibodies (C3 Nef, anti-factor H), along with gene panel (C3, CFH, CFI, CFB, CFHR1-5) testing (3).

aHUS is a primary TMA, that is characterized by uncontrolled AP activation, presenting with microangiopathic hemolytic anaemia, thrombocytopenia and acute renal failure. The dysregulated AP could be caused by mutations of complement regulators, most commonly factor H, and in around 6-10% of cases, by the presence of anti- factor H (4). Initial workup includes investigations to exclude other co-existing medical conditions associated with HUS or other forms of TMA. Similar to the workup of C3GN, checking the complement pathways, components and activation products, along with anti-factor H and genetic testing (C3, CFH, CGI, CFB, MCP, CFHR1-5, THBD, DGKE) are useful.

TMA leads to generalized endothelial dysfunction, that can progress to multiorgan injury. Apart from primary causes, some disease or medical conditions may predispose to TMA. In particular transplant associated TMA (TA-TMA) has been an important clinical entity, that carries high mortality and morbidity. Recent literature has shown that complement pathway dysregulation may play a role in the process. The pathogenesis in TA-TMA is complex, that multifactorial factors contribute to the endothelial injury and pathological process. Complications related to transplant, including GVHD or infections, may also stimulate the complement pathways. Complement blockage therapy, e.g. eculizumab, is useful in managing complex cases. After workup to exclude other potential differential diagnoses, risk assessment is important. Although not all patients with TA-TMA will have elevated sC5b-9, patients with elevation are at increased risk of death from TA-TMA (5). Hence, the activation product measurement has been used as risk stratification for consideration of complement blockade therapy (4,6).

Paroxysmal Noctural Hemoglobuinuria (PNH)

PNH is a rare acquired disorder, that patients suffered from hemolysis with acute exacerbations, leading to anaemia, bone marrow failure and increased risk of thrombosis. PNH arises from an expanded clonal proliferation of hematopoietic cells with somatic mutations of the X chromosomal gene PIG-A. Lack of PIG-A resulted in inability to bind GPI-anchored proteins, including the membrane bound complement regulators, DAF and CD59. As a result, cells having the mutation are susceptible to complement mediated intravascular haemolysis. Assessing the surface expression of CD55 and CD59 is helpful for the diagnosis.

Inherited and Acquired C1 inhibitor deficiency

Hereditary angioedema (HAE) and acquired angioedema (AAE), are rare diseases caused by C1 inhibitor deficiency. As a result, unregulated bradykinin formation leads to angioedema. HAE is an autosomal dominant condition, with majority of cases suffered from reduced concentration (Type I) or less commonly, reduced function (Type II), of C1 inhibitor. Some patients may have similar clinical presentations as HAE cases, but as an acquired condition due to the presence of autoantibodies against C1 inhibitor. These patients usually presented at an older age, and may have underlying hematological malignancies or autoimmune conditions as predisposition. The diagnosis of HAE is based on C1 inhibitor and C4 measurement. It is important to include both antigenic and functional assays for C1 inhibitor, since around 15% of cases may have normal or elevated dysfunctional C1 inhibitor protein (Type II). Furthermore, serum C1q concentrations can be used to differentiate HAE from acquired angioedema (AAE) as the latter is characterized by decreased C1q antigen concentration and autoantibodies against C1-INH. Genetic analysis for SERPING1 variants status may also help in the workup.

Monitoring of Complement Regulatory Drugs

In recent years, drugs targeting complement activation has been in clinical use. Eculizumab is the first approved complement inhibitor, that it is a humanized monoclonal antibody that hinder C5 proteolytic activation, inhibit the generation of C5a and the initiation of the membrane attack complex C5b-9, through its binding to the C5. Eculizumab is approved in the treatment of PNH, aHUS and refractory myasthenia gravis. Complement studies, such as CH50/ AH 50, and activation products (sC5b-9), have been employed in the treatment monitoring (7). In some specialized laboratory, C5 function may also be tested. The best time to monitor the therapy is at trough, immediately before the next dose. With effective drug treatment, CH50/AH50 and C5 function will be low. The activation products will also be suppressed.

Complement assays

The assays used in complement assessment can be broadly divided into 1) screening assays of total functional complement activity, 2) quantification of individual components, 3) quantitation of activation products 4) detection of autoantibodies against the complement components 5) assessing cell surface expression or tissue deposition of complement proteins/ breakdown products, 6) genetic assays.

Apart from the rare primary component deficiency, complement is associated in a number of disease conditions (such as infections, sepsis, malignancy, immune complex diseases, etc) by activation via different pathways. When a component is activated in vivo, the component is taken up by receptors on leukocytes or Kupffer cells. This results in secondary deficiency as consumption. Note that in complement studies, some assays are sensitive to in-vitro activation. Consumption can also be an artifact from heat labile nature of the complement proteins combined with delayed freezing of specimen after sample collection. Overall, the specificity of single complement test is low. Assessing several markers of the pathways and careful interpretation of results as a whole, is useful. In some situations, complementary use of genetic tests may help in cases suspecting primary in nature.

Since EDTA is able to inhibit complement activation in vitro, it is commonly used for quantification of complement components, in particular for activation products. Since heparin and citrate are insufficient inhibitors of complement activation, these are not suitable. Serum, on the other hand, is used for complement function and autoantibodies assessment. Plasma and serum received for complement assays should be separated within 2 hours from collection and frozen at -70 degree Celsius (4). Careful attention to the pre analytical steps and storage is crucial in complement studies.

Screening assays for total functional complement activity

The main indication for total complement function screen is to detect complement deficiencies. Such deficiencies can be genetic (primary), acquired (secondary, e.g. to consumption after pathway activation), or as a consequence of treatment. These tests reflect the total amount of active complement component present in a freshly sampled serum, and reflect the potential of the serum sample to achieve full activation in vitro after addition of activator. The traditional assays used are CH50 and AH50, based on studying the lysis of antibody sensitized sheep erythrocytes (CH50 for the CP activity) and the lysis of untreated rabbit erythrocytes (AH50 for the AP activity). The lysis of erythrocytes correlates with the formation of the terminal membrane attack complex downstream of the pathways’ activation. The results are usually expressed as reciprocal dilutions of the sample required to produce 50% lysis. Besides the traditional assays, a variety of modified methods based on the hemolytic assay were done in different centers. The functional screen can also be tested by measuring the deposition of activation products (ELISA detecting C9 neoepitope generated in terminal complex formation) upon activation of the serum with immobilized complement activating substances on a microtiter plate. Targeted molecules for each pathway are coated in wells of the microtiter plates; Ig M for CP, mannan /acetylated bovine serum albumin for LP and LPS for AP. (8)

In general, the pathway screens may provide some hint to the underlying disease process. Absent/low AH50 with normal CH50 suggests alternative pathway component deficiency, while absent/low CH50 with normal AH 50 suggests early classical pathway components (C1, C2, C4) deficiency. Absent/low results in both AH50 and CH50 suggests a deficiency affecting common components (C3, C5, C6, C7, C8, C9) shared in both pathways or complement consumption. Further investigations, including quantitation of individual components, would be helpful. In the settings of multiple components deficiency, consumptive depletion is likely.

Quantitation of individual components

In cases where the screening assays indicating a complement deficiency, quantitation of individual components and interpreting the results as a profile is useful to further delineate the affected pathways and pathogenesis.

Measurement of complement components is commonly done by immunoprecipitation assays with polyclonal antibodies against the protein of choice, e.g. nephelometry and turbidimetry. Other assays, such as gel precipitation assays or enzyme immunoassays were also used. Overall, these assays are relatively robust, however, do not provide information on the conformation or activation status in vivo.

Quantitation of activation products

Abnormal total complement functional screen could be due to primary deficiency or deficiency secondary to consumptive loss. Measurement of individual components level is not able to distinguish between primary from secondary loss. On the other hand, in vivo complement activation in acute phase reaction may not always lead to low components measurement despite ongoing consumption. Hence, quantitation of activation products would be helpful in the assessment of complement activation. Among the activation products available for measurement, detection of the soluble form of the terminal complement complex (sC5b-9), is the most promising screen for complement activation. The terminal complex reflects the activation to the final stage of the three pathways. Moreover, sC5b-9 has a relatively long in vivo half-life (60 mins), compared to other activation products, and is more stable with respect to in vitro activation compared to early components fragments (1,4). Overall, these activation markers can be rapidly produced by complement activation in vitro, therefore, proper sample collection and handling is important.

Autoantibodies against complement components

Autoantibodies to complement components have been linked to a number of disease conditions. The pathogenesis is often caused by the dysregulation of complement activation, as in the case of C3NeF and anti-Factor H. Occasionally, it may be affecting non-complement pathway, as in the case of anti-C1 inhibitor related angioedema, that it is due to inefficient inhibition of the kallikrein-kinin system and bradykinin release (4).

Most often, these autoantibodies could be detected by enzyme immunoassays. Functional assays were also helpful in the assessment. For example, in C3 Nef detection, a hemolytic assay that utilizes unsensitized sheep erythrocytes, or assay detecting fluid-phase C3 conversion after incubation of patient serum with normal serum at 37degree Celsius, were commonly used for the C3 Nef activity detection (9).

Assessing cell surface expression or tissue deposition of complement proteins/ breakdown products Measuring complement components and activation products directly on cell surface provides valuable information for the workup. For example, examining the deposition of various complement components in the glomeruli and peritubular capillary is useful for glomerulopathies assessment. Furthermore, studying the expression of membrane bound regulators is also helpful in some conditions, such as the use of flow cytometry assessment of CD55 and CD59 on blood cells in the diagnosis of PNH.

Genetic assays

With the advances in molecular diagnostics, complementary use of molecular diagnostics with traditional assays, has been increasingly employed in cases suspecting primary deficiency of complement factors or regulators. For example, gene panels study has been recommended in the workup of aHUS and C3 glomerulonephritis (3, 10-11).

Conclusion

With the vast and constantly growing knowledge in various disease process, along with expanding indications and emerging treatment options in complement mediated disorders, the application of complement diagnostics has been broadened and not limited to diagnosing rare primary genetic entities only. However, many of these assays remains highly subspecialized with limited availability, lack of standardization and complex interpretations. Careful standardization and close international collaborations and experience sharing, would be important for both the laboratory development and clinical applications in the field.

 

References

1. Kirschfink M, Mollnes TE. Modern complement analysis. Clin Diagn Lab Immunol. 2003 Nov;10(6):982-9.
2. Pickering MC, Botto M, Taylor PR, Lachmann PJ, Walport MJ. Systemic lupus erythematosus, complement deficiency, and apoptosis. Adv Immunol. 2000;76:227-324.
3. Angioi A, Fervenza FC, Sethi S, Zhang Y, Smith RJ, Murray D, Van Praet J, Pani A, De Vriese AS. Diagnosis of complement alternative pathway disorders. Kidney Int. 2016 Feb;89(2):278-88.
4. Ekdahl KN, Persson B, Mohlin C, Sandholm K, Skattum L, Nilsson B. Interpretation of Serological Complement Biomarkers in Disease. Front Immunol. 2018 Oct 24;9:2237.
5. Jodele S, Davies SM, Lane A, Khoury J, Dandoy C, Goebel J, Myers K, Grimley M, Bleesing J, El-Bietar J, Wallace G, Chima RS, Paff Z, Laskin BL. Diagnostic and risk criteria for HSCT-associated thrombotic microangiopathy: a study in children and young adults. Blood. 2014 Jul 24;124(4):645-53.
6. Jodele S, Dandoy CE, Lane A, Laskin BL, Teusink-Cross A, Myers KC, Wallace G, Nelson A, Bleesing J, Chima RS, Hirsch R, Ryan TD, Benoit S, Mizuno K, Warren M, Davies SM. Complement blockade for TA-TMA: lessons learned from a large pediatric cohort treated with eculizumab. Blood. 2020 Mar 26;135(13):1049-1057.
7. Ricklin D, Barratt-Due A, Mollnes TE. Complement in clinical medicine: Clinical trials, case reports and therapy monitoring. Mol Immunol. 2017 Sep;89:10-21.
8. Mollnes TE, Lea T, Frøland SS, Harboe M. Quantification of the terminal complement complex in human plasma by an enzyme-linked immunosorbent assay based on monoclonal antibodies against a neoantigen of the complex. Scand J Immunol. 1985 Aug;22(2):197-202.
9. Nilsson B, Ekdahl KN. Complement diagnostics: concepts, indications, and practical guidelines. Clin Dev Immunol. 2012;2012:962702.
10. Goodship TH, Cook HT, Fakhouri F, Fervenza FC, Frémeaux-Bacchi V, Kavanagh D, Nester CM, Noris M, Pickering MC, Rodríguez de Córdoba S, Roumenina LT, Sethi S, Smith RJ; Conference Participants. Atypical hemolytic uremic syndrome and C3 glomerulopathy: conclusions from a "Kidney Disease: Improving Global Outcomes" (KDIGO) Controversies Conference. Kidney Int. 2017 Mar;91(3):539-551.
11. Loirat C, Fakhouri F, Ariceta G, Besbas N, Bitzan M, Bjerre A, Coppo R, Emma F, Johnson S, Karpman D, Landau D, Langman CB, Lapeyraque AL, Licht C, Nester C, Pecoraro C, Riedl M, van de Kar NC, Van de Walle J, Vivarelli M, Frémeaux-Bacchi V; HUS International. An international consensus approach to the management of atypical hemolytic uremic syndrome in children. Pediatr Nephrol. 2016 Jan;31(1):15-39.

Trainee Presentation Session 2022

Venue: Pao Yue Kong Auditorium, Hong Kong Academy of Medicine Jockey Club Building

Time: 26 Nov 2022 12:00 pm - 16:00 pm

Detailed schedule in pdf

2022 TPS Abstract Book in pdf

Introduction

Dr. YAU Tsz Wai Derek

12:00 - 12:10

Platform Oral Presentation

12:10 - 13:40

Dr. CHAN Aden Ka-yin

Combinations of single gene biomarkers can precisely stratify 1,028 adult gliomas for prognostication

Dr. LI Wai Yan Jamilla

Unsupervised Machine Learning for Flow Cytometric Data Analysis in T-lymphoblastic leukaemia Measurable Residual Disease (MRD) Monitoring

Dr. LEE Chung Ho Anson

Direct Detection of Extended-spectrum b-lactamases (CTX-M) from Blood Cultures by NG-Test CTX-M MULTI Immunochromatographic Assay

Dr. LAM Tony

A case of hypertrophic cardiomyopathy presenting with sudden death at age of 55 days

Dr. TSEUNG Sik-Bit Jeremiah

Tiletamine as an emerging ketamine analogue of abuse: case series of acute poisonings in local Hong Kong population

Dr. LOONG Chi Wang Thomson

Paediatric type mesothelioma with ALK translocation: A case report

Poster Presentation

13:40 - 15:25

Dr. LING Cheuk Nam

Lipoprotein glomerulopathy with ApoE Kyoto mutation in an asymptomatic Chinese male patient: a case report

Download poster in pdf

Dr. TSANG Cheuk Ho

Primary T-cell lymphoma of the central nervous system mimicking a brain abscess

Download poster in pdf

Dr. TSANG Chui San Zara

Multiple chorangiomas of placenta: a case report

Download poster in pdf

Dr. LUNG Chee Heng Cheryl

Intracranial mesenchymal tumour: case report of a rare intracranial tumour with angiomatoid fibrous histiocytoma-like features and FET::CREB fusion

Download poster in pdf

Dr. LAU Cheuk Hei Derek

Extragastrointestinal GIST present as vaginal mass in a 57 year old female: a case report

Download poster in pdf

-- Break --

Dr. FONG Nga Yee Katie

A case report of Macrophage Activation Syndrome associated with adult-onset Still's disease

Download poster in pdf

Dr. YIU Sze Wan Rachel

Rewriting the future of newborns with a newly treatable rare disease

Download poster in pdf

Dr. HO Tin Wai Elson

EBV-negative fibrin-associated large B-cell lymphoma arising in thyroid hyperplastic nodule

Download poster in pdf

Dr. LI Yuk Wah

A Case of Atypical Kawasaki Disease: Sudden Death of a 9-Year-Old Child with Ruptured Coronary Artery Aneurysm

Download poster in pdf

Dr. HO Cheuk Lam

Mesonephric remnants with epididymis-like morphology in a postmenopausal woman with endometrial carcinoma - A case report and review of the literature

Download poster in pdf

Dr. LEUNG HO Wai

Reliability of the nonalcoholic steatohepatitis clinical research network and steatosis activity fibrosis histological scoring systems

Download poster in pdf

Dr. LI Xin

Risk factors for slow viral decline in COVID-19 patients during the 2022 Omicron wave

Download poster in pdf

Dr. MOK Ka Kin

A case of Coronary Fibromuscular dysplasia

Download poster in pdf

Dr. WONG Yuen Sze Sivia

Carcinosarcoma of gallbladder: a case report

Download poster in pdf

Dr. CHEUNG Yee Ting

Glycerol intoxication mimicking toxic alcohol ingestion: A case report

Download poster in pdf

Dr. WONG Ho Yan

Sudden/Unattended deaths due to diabetic ketoacidosis in Hong Kong

Download poster in pdf

Dr. WONG Tak Siu

Tenosynovial giant cell tumor of temporomandibular joint – A case report with literature review

Download poster in pdf

Dr. YEUNG Ka Pik Vivian

Mast Cell Leukaemia: A Case Report

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Dr. YUEN Wing Nam

Endometrial Biopsy with Non-neoplastic Signet-Ring Cells: Potential Pitfall in Diagnosis

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Judges Meeting and Words from Judges

15:25 - 15:40

Announcement of Prize and Certificate Presentation

15:40 - 15:55

Results of College Exam 2022 (Council Meeting 28 September 2022)

 

Fellowship Assessment:

 

CANDIDATE NO.	RESULT
E22201	PASS
E22202	FAIL
E22203	PASS
E22204	PASS
E22205	PASS
E22206	FAIL
E22207	FAIL
E22208	PASS
E22209	PASS
E22210	PASS
E22211	PASS
E22212	PASS
E22213	PASS
E22214	PASS
E22215	PASS
E22216	FAIL
E22217	PASS
E22218	PASS
E22219	PASS
E22220	FAIL
E22221	PASS
E22301	PASS
E22302	FAIL
E22303	PASS
E22304	PASS
E22305	PASS
E22306	PASS

 

Membership Examination:

 

CANDIDATE NO.	RESULT
E22101	PASS
E22102	FAIL
E22103	PASS
E22104	PASS
E22105	FAIL
E22106	PASS
E22107	FAIL
E22108	PASS
E22109	FAIL
E22110	PASS
E22111	FAIL
E22112	PASS
E22113	PASS
E22114	FAIL
E22115	PASS
E22116	FAIL
E22117	PASS
E22118	PASS

 

 

 

Volume 31, Issue 1 (click here to download the full pdf version)

Message from the President

It gave me great pleasure in writing this message, as the College Newsletter always has a special place in my heart.

It seems like yesterday when I first joined the College Council and took up the College Newsletter Chief Editor position in 2004. It has come a long way for the College newsletter to evolve from a black-and-white hard copy, to the current colourful electronic soft copy. I still remember the days when the Editorial Board meeting took place in a casual atmosphere in a restaurant in Admiralty, how the name “Pathologue” was proposed by the scholarly Dr LOO Ka Tai, and how the various new ideas were born through members of the Editorial Board. I am happy to see the continuous development of the newsletter over the subsequent years.

The COVID-19 pandemic has revolutionalized the practice of medicine. Various College meetings have been conducted via teleconferencing, and such practice has extended to the participation by External Examiners in various College examinations, and to Trainee Presentation Session in form of a hybrid mode. Later this year, I shall also join the International Liaison of Pathology Presidents (ILPP) meeting in Chicago via teleconferencing. The world is changing fast, and our College will adapt to the change. The COVID-19 pandemic has pushed us to embrace technology in our daily practice: teleconferencing, web-based seminars, the possibility of telepathology, just to name a few. Our College shall keep a close look at the latest outbreak situation and the related policy, and adjust our practice accordingly.

We managed to host our College Conferment Ceremony in 2021 despite the COVID-19 outbreak, taking various infection control precautions. For this year, our Annual General Meeting and Conferment Ceremony have been tentatively planned for 26 November 2022 (Saturday): please mark your diary, and we look forward to seeing you all.

Our profession has been facing manpower shortage for some time. The situation has been escalating, with the expansion of services in various pathology specialties and the attrition subsequent to various reasons. Together with the Academy, our College shall aim to uphold the professional standards while considering different options to address the matter.

We are pleased to see an increasing number of trainees and young Fellows in our College. The recent establishment of the Young Fellows Chapter in the Academy and in our College has provided an excellent opportunity to engage our younger generation in Academy’s and College’s activities. Young Fellows have brought in new ideas and have helped to organize various activities, and they are the future of our College.

After several years of preparation, our College rolled out the Genetic and Genomic Pathology training programme last year. More and more trainees have now registered for this programme, and we hope many new Fellows will complete this programme in the near future to cater for the needs of this rapidly expanding field.

The establishment of the Genetic and Genomic Pathology programme has brought our College “Into a New Era”, and it happens that the year 2021 marked the 30th Anniversary of our College. An anniversary book is currently under preparation, and I thank the team from the Professional & General Affairs Committee for their hard work. I also thank all the previous Presidents for their contributions, and this book will certainly become a valuable item of memorabilia.

As College President, I attended the 4th AMM-AMS-HKAM Tripartite Congress & the 55th Singapore-Malaysia Congress of Medicine on 22-24 July 2022 in Singapore virtually. A summary of the event is included in this newsletter.

There are challenges ahead, and let’s ride out the storm together. After all, tomorrow is another day.

Dr. CHAN Chak Lam, Alexander
President

July 2022

Molecular diagnostics for breast cancer

Volume 17, Issue 2, July 2022  (download full article in pdf)

Editorial note:

New molecular techniques have contributed to the ever-expanding armamentarium for breast cancer diagnosis, treatment and prognostication. Since the molecular classification of breast cancer was established, pathologists have been using immunohistochemistry and DNA sequencing techniques to routinely grade and subtype breast cancer. RNA expression profiling using various platforms such as microarrays, quantitative PCR and Nanostring has also been used to guide patient treatment in early diseases. This topical update provides a concise review on the current diagnostic and prognostic modalities in breast cancer management. We welcome any feedback or suggestions. Please direct them to Dr. Alvin Cheung of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. Alvin Ho-Kwan Cheung¹ and Dr. Karen Ka-Wan Yuen²

1. Department of Anatomical and Cellular Pathology, Prince of Wales Hospital, the Chinese University of Hong Kong
 
2. Department of Clinical Pathology, North District Hospital

Introduction

Since the seminal report on breast cancer classification in 2000[1], increased understanding in the molecular biology of breast cancer has led to numerous immunohistochemical markers and molecular panels used as adjunct biomarkers. These biomarkers mainly serve the following purposes: As prognostic markers, to gauge the likelihood of a clinical event, disease recurrence or progression; as predictive markers, to assess the likelihood of favourable or unfavourable effect from exposure to a medical product or a therapeutic agent[2]. In this review, the classical biomarkers of breast cancer will be briefly discussed, followed by a more detailed elaboration of molecular panels which are based on DNA alterations and gene expression levels. 

Hormonal receptor and proliferative index markers

Unlike other cancers, the molecular classification of breast cancer (luminal A/B, HER2 positive, and basal-like cancers) have been translated well to the clinic[3], and immunohistochemical markers have been established to facilitate such classification without resorting to molecular methods[4, 5]. Some authorities believe that normal-like breast cancer are an artifact of contamination by normal cells[6, 7]. The Estrogen Receptor (ER) and Progesterone Receptor (PR) are predictive biomarkers for endocrine therapy[8]. ER or PR-expressing tumours tend to have a better outcome than those lacking the receptors.

The expression of Human Epidermal growth factor Receptor 2 (HER2) defines the molecular basis of the “HER2-positive” group of cancer. They account for slightly less than 20% of breast cancers, and have a worse prognosis compared to ER+/PR+/HER2- cancers[9]. It serves as a therapeutic target for trastuzumab and pertuzumab. While ER and PR are routinely detected by immunohistochemistry (IHC), HER2 expression can be detected by IHC, Dual in situ hybridization (DISH) or Fluorescence in situ hybridization (FISH)[10].

 

The proliferation marker Ki-67 serves as a useful adjunct investigation in the grading of breast cancer[11]. Calculated as the percentage of nuclear staining in cancer cells, the prognosis is said to be better when Ki-67 is 30% for early disease[12].

Molecular tests at the DNA level

Some genetic aberrations in breast cancer are worth mentioning because they may be susceptible to targeted therapy and can predict treatment response. PIK3CA mutation occurs in about 36% of breast cancer[13, 14]. In advanced or metastatic hormonal receptor-positive cancer, or in patients with disease progression on endocrine-based regimen, combination therapy with the PI3K inhibitor, alpelisib, together with fulvestrant may be a treatment option if there is PIK3CA mutation[15]. The mutation can be detected by the companion diagnostic kit Therascreen, with Sanger sequencing, or with next generation sequencing. In secretory carcinoma, NTRK fusion is targetable by larotrectinib or entrectinib[16]. The presence of translocation can be detected with immunohistochemistry, next generation sequencing (NGS), Reverse-transcriptase (RT)-PCR or FISH. In non-secretory type breast cancers, NTRK fusion is very rare[17], such that the routine testing of this gene is unnecessary. For triple-negative breast cancer, BRCA aberrations can be present in about 6.5-34% of the cases[18]. The BRCA proteins constitute a part of the homologous recombination repair pathway. They are encoded by relatively large genes, with BRCA1 being present on chromosome17q21, having 23 exons; and BRCA2 on chromosome 13q13.1, having 27 exons. The incidence of aberrations is markedly higher among Ashkenzi Jews (2.5%) than the general population (0.1%)[19]. BRCA-mutated tumour highly depends on PARP, another DNA repair protein, to maintain the tumour genome integrity. Therefore, PARP inhibitor therapy are useful in BRCA-mutated tumours, and this treatment approach is termed a “synthetic lethality”[20].  Due to the size of these genes, NGS would be the preferred detection platform, while multiplex ligation-dependent probe amplification (MLPA) is also suitable[21]. 

 

For other advanced cancer or triple negative breast cancer, immune checkpoint inhibitor may be indicated in some patients. Besides testing for PD-L1 expression by the companion diagnostic kits for atezolizumab and pembrolizumab, some data support the testing for microsatellite instability (MSI) and tumour mutation burden (TMB) as well[22]. MSI-high breast cancer may be treated with pembrolizumab, as are tumours with high TMB as assessed by the FoundationOne companion diagnostic or other NGS platforms[23].

Molecular tests at the RNA expression level

Oncotype Dx

Oncotype Dx was launched in year 2004. It involves mRNA extraction from formalin-fixed paraffin-embedded (FFPE) tissues[24]. The detection panel includes 21 genes (16 cancer-related genes and 5 reference genes), and the detection platform is by quantitative-PCR (qPCR). The test had been studied in several trials, including the NASBP trial (National Surgical Adjuvant Breast and Bowel Project)[25], TAILORx trial (including 10273 women), and TxPONDER trial (5018 women)[26]. The test generates a recurrence score (RS) in the range of 0-100. In the TAILORx trial, patients of age >50 years had a substantial benefit from chemotherapy when RS >=26, whereas younger patients may be benefited when RS >=16.

MammaPrint

MammaPrint was launched in 2007. It consists of a 70-gene microarray, which accepts both fresh frozen or FFPE tissue for testing. It categorizes patients into “High risk” or “Low risk”. The MINDACT trial included 6693 patients and the RASTER trial included 427 patients for this test[27, 28]. There are some preliminary data to suggest systemic treatment can be recommended for the patients in the “High risk” group.

Blueprint

The Blueprint assay was developed by the same company as Mammaprint, and the test can be used together with MammaPrint. It involves a 80-gene panel, and serves to categorize tumour into luminal-A, luminal-B, HER2, or basal subtypes. Although this may overlap with the objective of IHC study described above, one important difference is that the luminal A and B groups can be associated with a different chemosensitivity and prognosis according to the Blueprint schema. Particularly, in the luminal B, Her2, and basal subtypes, chemotherapy can be beneficial to some patients with an improved survival. In contrast, for the luminal A group, the benefit for chemotherapy is not pronounced[29].

Prosigna (PAM50)

The Prosigna assay was launched in 2013. Following RNA extraction from FFPE tissue, the expression of a panel of 50 genes are detected by the NanoString “nCounter” platform[30]. This test is indicated for post-menopausal patients. Two large trials were conducted, including The ABCSG-8 study (Austrian Breast and Colorectal Cancer Study Group 8) and TransATAC study (translational arm of the anastrozole or tamoxifen alone or combined)[31]. While a scoring scheme of 0-100 is used, the risk stratification is different depending on the lymph node status. For node-negative cancers, they are classified as low (0-40), intermediate (41-60), or high (61-100) risk; as for node-positive cancers, they are classified as low (0-40) or high (41-100) risk. The suggested treatment for low risk disease is hormonal therapy alone, while for high risk disease, chemotherapy in addition to hormonal therapy may be beneficial.

The Breast Cancer Index

The Breast Cancer Index was launched in 2008[32]. As an RT-PCR assay on FFPE tissue, it features a 11-gene panel with two major testing endpoints: Whether there is a benefit of extended endocrine therapy (for 5 years), and the risk of recurrence 5 to 10 years after diagnosis. The ratio of expression between estrogen signaling pathway genes HOXB13 and IL17BR (H/I ratio) is an important parameter, as in the MA.17 trial, high H/I indicated a higher risk of late recurrence and a benefit from extended letrozole therapy. Another trial, the aTTom study, included H/I high patients for an extended therapy and found up to 15% reduction in recurrence risk[33, 34]. The test results for the Breast Cancer Index are simple enough to be interpreted even by patients, with “Yes” and “No” to the question of whether extended endocrine therapy is beneficial, and recurrence risk in percent to report the chance of late distant recurrence.

Comparisons between test modalities

When the included genes are compared, it is noted that the Oncotype Dx and PAM50 panels have the most overlap. 11 genes are in common for Oncotype Dx and PAM50, for example BCL2, CCNB1, MMP11, which are markers for apoptosis, cell cycle, and tumour invasiveness[35]. Interestingly, for the 70 genes included in Mammaprint, only one gene, SCUBE2, overlaps with Oncotype DX, and two genes, MELK and ORC6L, overlap with Prosigna PAM50[35]. It remains to be studied whether the results in one test can be correlated with another test, but some key differences are still worth to be noted. For hormonal receptor positive stage I-II invasive breast cancer, all the tests have some use for prognostication. However, concerning whether chemotherapy is recommended, only Oncotype DX has an established predictive value, while there is insufficient evidence for Mammaprint, Blueprint and Prosigna [36]. The Breast Cancer Index has predictive value for extended endocrine therapy. Some efforts have also been taken to translate some of these tests to hormonal receptor positive DCIS. Oncotype DX DCIS and DCISionRT have some use in patient prognostication, however, both tests have insufficient evidence to guide chemotherapy[37, 38].

For the regulatory status, Oncotype DX has been included in the NCCN/ASCO guidelines for the management of breast cancer patients. As for Mammaprint and Prosigna, these kits have been FDA-cleared for specific clinical settings. Logistically, both Oncotype DX and Mammaprint require end users to deliver specimens to a central laboratory for testing. Prosigna is available in a kit format for local laboratories to perform the test.

Conclusions and future perspectives

Unlike most other cancer types, RNA expression profiling has found remarkable translational use in breast cancer treatment. This can be attributed to an increased understanding of molecular classifications, hormonal receptor functions and breast cancer biology. While panels including other RNA expression signatures can be expected to emerge, it is important to understand the indications and differences for each testing system, as an increased number of testing options can be confusing to patients, while contradicting results among platforms can complicate the interpretation. Because RNA expression level has an inherent variability among patients, the subgrouping of patients into risk groups may not be ideal, and some patients may be placed in the wrong group using only one particular panel. Hopefully, with further elucidation of the breast cancer genome, novel molecular targets based on DNA alterations can be uncovered, as the presence of a particular mutation or translocation is a more consistent marker of susceptibility to targeted therapy. As we enter the era of personalized medicine, histologic assessments, immunohistochemical studies such as hormonal receptors and PD-L1 status, and molecular diagnostics can be expected to go hand in hand in the formulation of management plans and prognostication in breast cancer patients.

Reference

1. Perou CM, Sørlie T, Eisen MB, Van De Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. nature. 2000;406(6797):747-52.
2. Califf RM. Biomarker definitions and their applications. Experimental Biology and Medicine. 2018;243(3):213-21.
3. Sotiriou C, Pusztai L. Gene-expression signatures in breast cancer. New England Journal of Medicine. 2009;360(8):790-800.
4. Goldhirsch A, Winer EP, Coates A, Gelber R, Piccart-Gebhart M, Thürlimann B, et al. Personalizing the treatment of women with early breast cancer: highlights of the St Gallen International Expert Consensus on the Primary Therapy of Early Breast Cancer 2013. Annals of oncology. 2013;24(9):2206-23.
5. Cheang MC, Martin M, Nielsen TO, Prat A, Voduc D, Rodriguez‐Lescure A, et al. Defining breast cancer intrinsic subtypes by quantitative receptor expression. The oncologist. 2015;20(5):474-82.
6. Tsang J, Tse GM. Molecular classification of breast cancer. Advances in anatomic pathology. 2020;27(1):27-35.
7. Peppercorn J, Perou CM, Carey LA. Molecular subtypes in breast cancer evaluation and management: divide and conquer. Breast Cancer. 2007:125-42.
8. Prat A, Baselga J. The role of hormonal therapy in the management of hormonal-receptor-positive breast cancer with co-expression of HER2. Nature Clinical Practice Oncology. 2008;5(9):531-42.
9. Cooke T, Reeves J, Lanigan A, Stanton P. HER2 as a prognostic and predictive marker for breast cancer. Annals of oncology. 2001;12:S23-S8.
10. Kurozumi S, Padilla M, Kurosumi M, Matsumoto H, Inoue K, Horiguchi J, et al. HER2 intratumoral heterogeneity analyses by concurrent HER2 gene and protein assessment for the prognosis of HER2 negative invasive breast cancer patients. Breast cancer research and treatment. 2016;158(1):99-111.
11. Cheang MC, Chia SK, Voduc D, Gao D, Leung S, Snider J, et al. Ki67 index, HER2 status, and prognosis of patients with luminal B breast cancer. JNCI: Journal of the National Cancer Institute. 2009;101(10):736-50.
12. Nielsen TO, Leung SCY, Rimm DL, Dodson A, Acs B, Badve S, et al. Assessment of Ki67 in breast cancer: updated recommendations from the international Ki67 in breast cancer working group. JNCI: Journal of the National Cancer Institute. 2021;113(7):808-19.
13. Anderson EJ, Mollon LE, Dean JL, Warholak TL, Aizer A, Platt EA, et al. A systematic review of the prevalence and diagnostic workup of PIK3CA mutations in HR+/HER2–metastatic breast cancer. International Journal of Breast Cancer. 2020;2020.
14. Deng L, Zhu X, Sun Y, Wang J, Zhong X, Li J, et al. Prevalence and prognostic role of PIK3CA/AKT1 mutations in Chinese breast cancer patients. Cancer Research and Treatment: Official Journal of Korean Cancer Association. 2019;51(1):128.
15. André F, Ciruelos E, Juric D, Loibl S, Campone M, Mayer I, et al. Alpelisib plus fulvestrant for PIK3CA-mutated, hormone receptor-positive, human epidermal growth factor receptor-2–negative advanced breast cancer: final overall survival results from SOLAR-1. Annals of Oncology. 2021;32(2):208-17.
16. Ricciuti B, Genova C, Crinò L, Libra M, Leonardi GC. Antitumor activity of larotrectinib in tumors harboring NTRK gene fusions: a short review on the current evidence. OncoTargets and therapy. 2019;12:3171.
17. Remoué A, Conan‐Charlet V, Bourhis A, Flahec GL, Lambros L, Marcorelles P, et al. Non‐secretory breast carcinomas lack NTRK rearrangements and TRK protein expression. Pathology International. 2019;69(2):94-6.
18. Hartman AR, Kaldate RR, Sailer LM, Painter L, Grier CE, Endsley RR, et al. Prevalence of BRCA mutations in an unselected population of triple‐negative breast cancer. Cancer. 2012;118(11):2787-95.
19. Warner E, Foulkes W, Goodwin P, Meschino W, Blondal J, Paterson C, et al. Prevalence and penetrance of BRCA1 and BRCA2 gene mutations in unselected Ashkenazi Jewish women with breast cancer. Journal of the National Cancer Institute. 1999;91(14):1241-7.
20. Cong K, Peng M, Kousholt AN, Lee WTC, Lee S, Nayak S, et al. Replication gaps are a key determinant of PARP inhibitor synthetic lethality with BRCA deficiency. Molecular cell. 2021;81(15):3128-44. e7.
21. Wallace AJ. New challenges for BRCA testing: a view from the diagnostic laboratory. European Journal of Human Genetics. 2016;24(1):S10-S8.
22. Rozenblit M, Huang R, Danziger N, Hegde P, Alexander B, Ramkissoon S, et al. Comparison of PD-L1 protein expression between primary tumors and metastatic lesions in triple negative breast cancers. Journal for Immunotherapy of Cancer. 2020;8(2).
23. Sivapiragasam A, Ashok Kumar P, Sokol ES, Albacker LA, Killian JK, Ramkissoon SH, et al. Predictive biomarkers for immune checkpoint inhibitors in metastatic breast cancer. Cancer medicine. 2021;10(1):53-61.
24. Carlson JJ, Roth JA. The impact of the Oncotype Dx breast cancer assay in clinical practice: a systematic review and meta-analysis. Breast cancer research and treatment. 2013;141(1):13-22.
25. McVeigh TP, Hughes LM, Miller N, Sheehan M, Keane M, Sweeney KJ, et al. The impact of Oncotype DX testing on breast cancer management and chemotherapy prescribing patterns in a tertiary referral centre. European Journal of Cancer. 2014;50(16):2763-70.
26. Lafitte E, Sabatier R. Genomic/transcriptomic signatures in breast cancer. A review of three prospective studies. Innovations & Thérapeutiques en Oncologie. 2022;8(1):5-11.
27. Metzger O, Cardoso F, Poncet C, Desmedt C, Linn S, Wesseling J, et al. Clinical utility of MammaPrint testing in invasive lobular carcinoma: results from the MINDACT phase III trial. European Journal of Cancer. 2020;138:S5-S6.
28. Drukker CA, Bueno‐de‐Mesquita J, Retèl VP, van Harten WH, van Tinteren H, Wesseling J, et al. A prospective evaluation of a breast cancer prognosis signature in the observational RASTER study. International journal of cancer. 2013;133(4):929-36.
29. Mittempergher L, Delahaye LJ, Witteveen AT, Snel MH, Mee S, Chan BY, et al. Performance characteristics of the BluePrint® breast cancer diagnostic test. Translational oncology. 2020;13(4):100756.
30. Wallden B, Storhoff J, Nielsen T, Dowidar N, Schaper C, Ferree S, et al. Development and verification of the PAM50-based Prosigna breast cancer gene signature assay. BMC medical genomics. 2015;8(1):1-14.
31. Lænkholm A-V, Jensen M-B, Eriksen JO, Roslind A, Buckingham W, Ferree S, et al. Population-based study of Prosigna-PAM50 and outcome among postmenopausal women with estrogen receptor-positive and HER2-negative operable invasive lobular or ductal breast cancer. Clinical breast cancer. 2020;20(4):e423-e32.
32. Ma X-J, Salunga R, Dahiya S, Wang W, Carney E, Durbecq V, et al. A five-gene molecular grade index and HOXB13: IL17BR are complementary prognostic factors in early stage breast cancer. Clinical cancer research. 2008;14(9):2601-8.
33. Noordhoek I, Treuner K, Putter H, Zhang Y, Wong J, Kranenbarg EM-K, et al. Breast cancer index predicts extended endocrine benefit to individualize selection of patients with HR+ early-stage breast cancer for 10 years of endocrine therapy. Clinical Cancer Research. 2021;27(1):311-9.
34. Bartlett J, Sgroi D, Treuner K, Zhang Y, Ahmed I, Piper T, et al. Breast Cancer Index and prediction of benefit from extended endocrine therapy in breast cancer patients treated in the Adjuvant Tamoxifen—To Offer More?(aTTom) trial. Annals of Oncology.
2019;30(11):1776-83.
35. Pennock ND, Jindal S, Horton W, Sun D, Narasimhan J, Carbone L, et al. RNA-seq from archival FFPE breast cancer samples: molecular pathway fidelity and novel discovery. BMC medical genomics. 2019;12(1):1-18.
36. Duffy M, Harbeck N, Nap M, Molina R, Nicolini A, Senkus E, et al. Clinical use of biomarkers in breast cancer: Updated guidelines from the European Group on Tumor Markers (EGTM). European journal of cancer. 2017;75:284-98.
37. Nofech-Mozes S, Hanna W, Rakovitch E. Molecular evaluation of breast ductal carcinoma in situ with oncotype DX DCIS. The American journal of pathology. 2019;189(5):975-80.
38. Shah C, Bremer T, Cox C, Whitworth P, Patel R, Patel A, et al. The Clinical Utility of DCISionRT® on Radiation Therapy Decision Making in Patients with Ductal Carcinoma In Situ Following Breast-Conserving Surgery. Annals of surgical oncology. 2021;28(11):5974-84.

Results of College Written Exam 2022 (Council Meeting 20 July 2022)

Results of College Written Exam 2022 in Chemical Pathology, Clinical Microbiology & Infection and Haematology

CANDIDATE NO.	RESULT
E22213	PASS
E22214	PASS
E22215	PASS
E22216	FAIL
E22217	PASS
E22218	PASS
E22219	PASS
E22118	PASS

Passed candidates can proceed further in Fellowship Assessment / Membership Examination in 2022.

Measurable residual disease (MRD) for Haematological Malignancy

Volume 17, Issue 1, January 2022  (download full article in pdf)

Editorial note:

Measurable residual disease (MRD) monitoring has emerged as an important indicator for risk stratification and treatment planning in patients with haematological malignancies. In the past decade, various techniques in measuring MRD have become available in Hong Kong. In this Topical Update, Dr. YIP Sze-fai provides an overview of the current techniques available for MRD monitoring. We welcome any feedback or suggestions. Please direct them to Dr. Alvin IP of Education Committee, the Hong Kong College of Pathologists. Opinions expressed are those of the authors or named individuals, and are not necessarily those of the Hong Kong College of Pathologists.

Dr. YIP Sze-fai

Consultant Haematologist, Department of Clinical Pathology, Tuen Mun Hospital

Introduction

Measurable residual disease (MRD) describes the application of assays for detection of submicroscopic level of residual disease burden which cannot be detected by morphology. Numerous studies have observed the association of MRD level and disease prognosis. It provides an objective parameter on the tumor burden, and guide stratified treatment including the application of haemopoietic stem cell transplantation (HSCT). Its ability to monitor disease and to detect molecular relapse enables preemptive therapy to prevent frank disease relapse [1]. For all these reasons, we see an increasing use of MRD in the field of haematological malignancy.

Different technologies are used for MRD measurement

1. Multiparametric flow cytometry (MFC)

MFC is commonly used for MRD detection in acute leukaemias. At diagnosis, the leukaemia-associated immunophenotype (LAIP) of the blasts can be determined by using a multitude of fluorochrome-labeled monoclonal antibodies against different cellular markers that aids identification of the leukaemic population as well as detecting the aberrant cellular marker expression. If the LAIP was not determined at diagnosis, a different-from-normal (DfN) approach can be used to detect the abnormal cells, as well as detecting any new or disappearance of known phenotypic aberrancies [1,2]. With technological advancement, more fluorochromes are available and 8 to 12-colour panels are commonly used. Flow cytometry has the advantage of a short turnaround time which can provide timely results for clinical decision making. The sensitivity of MRD detection is at the level of 10^-4 to 10^-5.

2. Next generation flow (NGF) for plasma cell myeloma

Novel Euroflow-based next generation flow (NGF) approach is being developed for highly sensitive and standardized MRD detection, primarily in plasma cell myeloma, using an optimized 2-tube 8-color antibody panel [3]. The NGF approach uses tools and procedures that are developed by the EuroFlow Consortium for a standardized sample preparation, antibody panel (including the type of antibody and fluorochrome), and automatic identification of plasma cells against reference databases of normal and patient BM using Infinicyt software. The sensitivity of MRD detection is close to 10^-6.

3. Quantitative polymerase chain reaction (qPCR) technique

a. Detection of leukaemia-specific fusion transcript

The MRD can be measured by detecting the amount of leukaemia-specific fusion transcripts present. The classical example is BCR-ABL1 fusion in chronic myeloid leukaemia (CML). The sensitivity is higher than that of flow cytometry, reaching the level of 10^-4 to 10^-6. The test is relatively easy to be performed in hospital service laboratory. The MRD is represented in a ratio of normalized copy number of the fusion transcript and the control gene transcript (e.g. ABL1). For CML monitoring, an international scale (IS) ratio is developed for standardization of results among different laboratories [4]. Yet, this method is limited to cases with targetable fusion transcripts available for detection.

b. Allele-specific oligonucleotide (ASO) qPCR for immunoglobulin (IG) or T cell receptor (TCR) gene rearrangement

ASO qPCR can be employed to detect the disease-specific sequence of rearranged IG gene or TCR gene in the sample. The sensitivity of this method is 10-4 to 10-5. It is applicable to most of the cases of acute lymphoblastic leukemia (ALL) and plasma cell myeloma as long as a disease-specific rearrangement can be determined by sequencing. Patient-specific primers would need to be designed for each case. It has a disadvantage that if there is a clonal evolution, the disease-specific rearrangement can be lost and a false-negative result can be generated.

4. Digital droplet polymerase chain reaction (ddPCR)

In ddPCR, the sample is compartmentalized into very large number of separate small volume reactions. As a result, either zero or one target molecule could be detected inside any individual reaction. Thermal cycling would be performed to endpoint using same primer and probes as qPCR. Any target-containing compartments will become brightly fluorescent while compartments without targets will have only background fluorescence. Total number of ‘positive’ reactions is equal to the number of original target molecules in the entire volume, and the total number of reactions multiplied by the individual reaction volume equals the total volume assayed. Therefore, ddPCR provides an absolute quantification of the target molecules. The ddPCR has the advantage of very high sensitivity of ~10^-6, does not require a standard curve unlike qPCR, and is tolerant to PCR inhibitors due to small partition volume. The application of ddPCR includes monitoring of NPM1 and ASO IG or TCR gene rearrangement [5,6].

5. Next generation sequencing (NGS)

NGS is a robust method to perform multiple sequencing in parallel which can also be used for MRD detection apart from the detection of mutations that are of diagnostic, prognostic and therapeutic importance. For MRD detection, the LymphoTrack platform can be used to detect disease-specific IG or TCR gene rearrangements. The sensitivity of the method can be up to 10^-5 or higher [7]. A diagnostic sample would be required for identification of the disease-specific rearrangement. However, this method is also capable of detecting clonal evolution.

Reference

1. Schuurhuis GJ, Heuser M, Freeman S, et al. Minimal/measurable residual disease in AML: a consensus document from the European LeukemiaNet MRD Working Party. Blood. 2018 Mar 22;131(12):1275-1291. doi: 10.1182/blood-2017-09-801498.
2. Baer MR, Stewart CC, Dodge RK, et al. High frequency of immunophenotype changes in acute myeloid leukemia at relapse: implications for residual disease detection (Cancer and Leukemia Group B Study 8361). Blood. 2001 Jun 1;97(11):3574-80. doi: 10.1182/blood.v97.11.3574.
3. Flores-Montero J, Sanoja-Flores L, Paiva B, et al. Next Generation Flow for highly sensitive and standardized detection of minimal residual disease in multiple myeloma. Leukemia. 2017 Oct;31(10):2094-2103. doi: 10.1038/leu.2017.29.
4. Hughes T, Deininger M, Hochhaus A, et al. Monitoring CML patients responding to treatment with tyrosine kinase inhibitors: review and 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. doi: 10.1182/blood-2006-01-0092.
5. Bill M, Grimm J, Jentzsch M, et al. Digital droplet PCR-based absolute quantification of pre-transplant NPM1 mutation burden predicts relapse in acute myeloid leukemia patients. Ann Hematol. 2018 Oct;97(10):1757-1765. doi: 10.1007/s00277-018-3373-y. Epub 2018 May 22. PMID: 29785446.
6. Takamatsu H, Wee RK, Zaimoku Y, et al. A comparison of minimal residual disease detection in autografts among ASO-qPCR, droplet digital PCR, and next-generation sequencing in patients with multiple myeloma who underwent autologous stem cell transplantation. Br J Haematol. 2018 Nov;183(4):664-668. doi: 10.1111/bjh.15002. Epub 2017 Dec 22. PMID: 29270982.
7. Yao Q, Bai Y, Orfao A, Chim CS. Standardized Minimal Residual Disease Detection by Next-Generation Sequencing in Multiple Myeloma. Front Oncol. 2019 Jun 6;9:449. doi: 10.3389/fonc.2019.00449.

Volume 30, Issue 1 (click here to download the full pdf version)

Message from the President

In this issue of the College Newsletter, I am delighted to announce that Professor Dennis LO, Professor Joseph Sriyal Malik PEIRIS and Professor Kwok Yung YUEN (in the order of their surnames) have received the prestigious Royal Medal of The Royal Society as well as the Future Science Prize 2021 in life sciences for their contributions in the discoveries of cell-free DNA for noninvasive prenatal diagnosis and zoonotic transfer of coronavirus to human causing Severe Acute Respiratory Syndrome (SARS) in 2003, respectively. My heartfelt congratulations!

Under the new normal of COVID-19, all examinations in 2021 were still conducted using video conferencing technology. I would also like to thank all external and local examiners for their hard work and to congratulate all successful candidates in the College Membership Examination and Fellowship Assessment. They have added new workforce to the diagnostic services in Hong Kong.

Another issue of postgraduate education under the new normal is how to receive training without a face-to-face arrangement. The Academy recently held a Medical Education Conference to discuss the way forward on e-learning, competency-based training and continuous workplace-based assessment. Both educators and students need to acquire new skills and improve their resilience to training progress.

Dr Derek HUNG and Professor KY YUEN wrote a Topical Update in this issue of newsletter titled “Diagnosis of COVID-19”. It is a timely topic to review all the diagnostic modalities to combat COVID-19.

Last but not least, our College, jointly with Academy, has prepared promotional materials for COVID-19 vaccination including posters and videos. They are uploaded to the COVID corner of our College website and Facebook. Please feel free to share them with your friends and family.

Finally, allow me to wish you all ‘good health’ going forward !

Dr. CHAN Ho Ming
President

October 2021

Volume 29, Issue 2 (click here to download the full pdf version)

Message from the President

In this issue of the College Newsletter, I am delighted to announce that the Genetic and Genomic Pathology (GGPath) Training Programme and all successful First Fellows of GGPath have been fully accredited by The Hong Kong Academy of Medicine and this new specialty has been listed as one of the sixty-three specialties in the Specialist Register of The Medical Council of Hong Kong. Therefore, College would like to remind all concerned Fellows about the proper quotation of this new qualification. Also, I would like to thank the joint effort of all members of GGPath Training Programme Working Group, GGPath First Fellow Assessment Team and the newly established GGPath Specialty Board for their hard work. Hopefully, our College can enroll the first batch of GGPath trainees very soon.

As President of the College, I participated in Academy of Medicine’s 27th Annual General Meeting held last December. The Sir David Todd Orator 2020 was Professor Francis CHAN with his talk titled ‘Being a Leader’. The Conferment Ceremony was conducted in the Academy Building in a very unusual way due to precautionary measures for COVID-19 with all the Fellowship recipients joining via video conferencing. Each College President announced his/her new Academy Fellows on screen.

I would like to send my heartfelt congratulations to Dr FOO Ka Chung, our new Distinguished Young Fellow, who also wrote a Topical Update in this issue of newsletter as well as all new Fellows and Members who were successful in College Examinations 2020 conducted via video conferencing during COVID-19 pandemic. I would also take this chance to thank our External Examiners, Chief Examiners, Deputy Chief Examiner and Local Examiners for their tremendous effort in making it possible for the College Examinations to be conducted as scheduled.

In the Out of the Whitecoat article, Dr LEE Kam Cheong shared with us about his fascinating bird-watching stories.

Professor Paul CHAN was recently interviewed by ‘PathWay’ of The Royal College of Pathologists of Australasia to share how Hong Kong managed COVID-19.

Finally, allow me to wish you all ‘good health’ going forward !

Dr. CHAN Ho Ming
President

March 2021