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Inside this issue

ARTICLES• Genetic Testing for

Breast Cancer Risk

• Improved Patient Care: Working With Difficult Haematological Cytogenetics Referrals

CASE STUDY

• Potting Mix Can Be Harmful To Your Health

Incorporating:• Northern NSW• Hunter New England• Mid North Coast• Central Coast• Northern Sydney

SUMMER 2014

When I was younger, I used to read a story to my children called “Just 5 minutes peace”. The premise was that Mrs Elephant just wanted 5 minutes of peace and quiet from the demands of her three children.

Certainly, that nirvana of peace resonated with my wife and me. The

kids just thought it was a hoot! It seemed to encourage them to

pursue disruptive behaviours even more! Maybe, however, that was just an error of perception by my wife and me. Maybe, because we’d been shown this vision, it was more desirable, and its absence, although the same

as always, was more disconcerting and disappointing.

Even though I am older, and disappointingly, my kids no longer want

me to read to them, I still yearn for that “5 minutes peace”. That time when you can have space to “think” and not just “do”.

We are all so busy that the space for “5 minutes peace” seems to have vaporised. We are busy at work, at home, at play, at life. That “5 minutes peace” is so desirable and yet maybe so distant.

I’ve never thought that the “5 minutes of peace” I sought was an absence of all thought. I’ve always aspired to an ideal that that “5 minutes of peace” was an opportunity to reconnect with the world. To be more mindful. To be more aware. To be in the moment. To have an original thought.

This week, I was lucky enough to attend the retirement celebration for our colleague, Dr Stephen Graves, and last month a similar celebration for Irene Goodhew.

I was unable to attend the celebrations for Sandy Skinner, Russell Lane, Rita Castles, Bruno Conte, Clarence Fernandes, Malcolm King, Elaine Lewins, Mark Mackay, Dianne Nelson, Nicholas Radice, Patricia Smith and Helen Stewart. This group contributed 446 years of work life to public pathology in NSW. That contibution was remarkable, valuable and appreciated. All of these people contributed to the well being of our patients, and supporting our clients, and we are grateful for their efforts. They are all valued colleagues.

As I contemplate their retirement “destination”, I wonder if these colleagues would now be thinking “at last, 5 minutes of peace”. I do not believe so. If one believes their comments at their retirement celebrations, they were “celebrating” their work life. They spoke of the pleasure it gave them. They said they would miss the commitment and their colleagues.

The workplace that these colleagues entered, so many decades ago, has metamorphosed into a different environment. Participating in the change, whilst universally challenging, was endorsed by them as exciting, stimulating and satisfying. Change is now the norm. It is no less challenging, and the outcome no less rewarding, however.

I know it is difficult in our busy lives to find space for peace and thought. I hope, however, that we can all take the time to reflect on the valuable work we do, the people we help and the benefit that our work provides, so that we may be more mindful. We must all try to enjoy the journey as well as the destination.

Dr. Stephen Braye Director, Pathology North

FROM THE DIRECTOR

2Front Cover: Laila Daqiq, Hospital Scientist Cytogenetics Laboratory Pathology North Royal North Shore Hospital

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Genetic Testing For Breast Cancer RiskProfessor Rodney Scott - Molecular Genetics, Pathology North, John Hunter Hospital

rodney.scott@hnehealth.nsw.gov.au Phone: 02 4921 4974

Professor Rodney J. Scott obtained his PhD from the University of Western Australia in 1987. Since that time, he has been engaged in research directed at understanding how genetic change is associated with disease risk (especially changes associated with inherited predispositions to breast and colorectal cancer)

and outcomes, and has published over 400 research articles. His achievements were recognised by the award of Privat Dozent (DSc) in Cancer Genetics, by the University of Basel in 1997 and his Fellowship of the Royal College of Pathologists in 2005. More recently, he has been awarded his Fellowship of the Faculty of Science, Royal College of Pathologists of Australasia (2011).

In 1997 Professor Scott took up his appointment with the Hunter Area Pathology Service (now Pathology North) as Director of Molecular Medicine and became the Head of the Discipline of Medical Genetics at the University of Newcastle at the end of 1999. His current interests are in understanding genetic determinants associated with common diseases, but especially cancer risk and progression.

Clinical application

Approximately one in ten women will develop breast cancer at some time during their lives. Out of the women who do develop breast cancer, there is a small percentage who have inherited a genetic predisposition to disease. Women with a family history of early age of onset breast and/or ovarian cancer are at risk of having inherited a deleterious change in one of two genes, BRCA1 or BRCA2. It has recently been identified that women with a particular type of breast cancer are also likely to harbour a deleterious change in BRCA1 or BRCA2. The type of breast cancer is defined as the triple negative tumour since the tumour does not express the estrogen receptor, the progesterone receptor or the HER2 gene. It has recently been reported that up to 20% of patients with triple negative tumours harbour a germline mutation in either BRCA1 or BRCA2.

The aim of screening women for deleterious changes in BRCA1 or BRCA2 is to prevent women from developing disease by providing appropriate prophylactic measures to reduce risk. The actual risk of disease is significant with disease penetrance at approximately 70% by 70 years of age for breast cancer and approximately 35% for ovarian cancer. An equally important facet to screening is to identify women who do not harbour BRCA1 or BRCA2 mutations, since they can be treated similarly to the general population with respect to their disease risk.

When to request a test

Any woman who is concerned about her risk of breast cancer should be assessed with respect to her likelihood of being a member of a breast/ovarian cancer family. Women who report a number of first or second degree relatives who have been diagnosed with early onset breast and/or ovarian cancer should be referred to a Family Cancer Service to have their risk appropriately assessed. In the event a set of criteria are met, a blood sample will be taken and sent for genetic testing. The setting of criteria can deny some women access to publicly-funded BRCA1 or BRCA2 mutation screening, in which case, screening can occur but it must be funded by the patient.

Women who are diagnosed with triple negative breast cancer should consider having their family history assessed to determine if they too should be tested.

Sample required

Testing for BRCA1 and/or BRCA2 requires 20ml of EDTA blood sent to the Division of Molecular Medicine, Pathology North (Hunter) within 24 hours of collection. It does not need to be cooled but should not be placed in direct sunlight.

Reporting of results

There is a four week turnaround time for BRCA1 and BRCA2 screening if it is a mutation search. If the family mutation is already known the turn-around time is two weeks. All results are sent to the referring physician (usually a clinical geneticist specialising in cancer genetics).

Recommended treatment/disease management

There are a number of recommendations for women who harbour a germline mutation in BRCA1 or BRCA2. These should be discussed with each patient by their respective physician as they include both surgical (bilateral mastectomy, oophorectomy) and endocrinological options.

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Improved Patient Care: Working With Difficult Haematological Cytogenetics ReferralsNisha Singh, Senior Hospital Scientist, Pathology North, Northern Sydney

Nisha Singh is a Senior Cytogeneticist within the Genetics department based at Pathology North, Royal North Shore Hospital. She has been in her current post for 12 years and supervises a team of experienced staff. She has worked globally in the discipline of human genetics since 1985 and holds a BSc and MSc together with overseas

laboratory specialisation in genetics and MHGSA certification in Cytogenetics. She has recently completed a PhD by research through the Kolling Institute, University of Sydney in the subject of Cancer Genomics, her field of special interest.

Dr William Stevenson, Haematologist MB BS, BSc(Med), PhD, FRACP, FRCPA

Dr William Stevenson is a Haematologist at the Royal North Shore Hospital in Sydney where he supervises the Pathology North Cytogenetic and Molecular Haematology Laboratories. He was a graduate student at the Walter and Eliza Hall Institute of Medical Research in Melbourne and then had further training

as a Post-Doctoral Fellow within the Department of Leukemia at the M D Anderson Cancer Center in Houston, Texas. His interests include genetic diagnostics and novel medical therapies for Leukaemia.

Cytogenetics Laboratory, Royal North Shore Hospital: 02 992 64343

The role of cytogenetics analysis in the diagnosis, treatment and management of patients with leukaemia and lymphomas is ever expanding. Certain disease entities have proven challenging in the past due to biological factors and have produced sub optimal success rates by genetic analysis. This has hindered efficient diagnosis and management of patients. Local implementation of newer methodologies in both cell

culture and DNA based cytogenetics has resulted in clinically significant improvements.

Screenshot of FISH Analysis using Automated Spot Counting

Primary Myelofibrosis (PMF)

PMF is a sub type of the classical myeloproliferative neoplasms and is characterised by progressive bone marrow failure and a highly variable disease course. The addition of karyotyping data to the DIPPS Plus prognostic scoring system, highlights the role of cytogenetic studies in PMF that informs prognosis and therapeutic rationale. Bone marrow samples often fail to yield results due to the extensive bone marrow fibrosis that yields a “dry tap”. However, the peripheral blood of patients with PMF contains high numbers of clonal progenitor cells capable of spontaneous cell division and may be karyotyped successfully. Studies on 42 samples comparing the outcome of cytogenetics analysis between blood and bone marrow samples indicated an increased success rate, metaphase yield and a comparable abnormality detection rate by using peripheral blood. The study done on peripheral blood using a variety of genetic technologies, revealed novel pathogenic mechanisms in PMF and identified extended cohorts of patients with abnormalities carrying known prognostic information. This represents an improvement for patient care where a bone marrow biopsy may be avoided for ongoing monitoring.

Figure 1. Single nucleotide polymorphism micro array on two PMF samples showing clinically significant changes (Shaded region) : A.)Chromosome 9 :CNLOH 9p( usually accompanied by JAK2 mutation). B) Chromosome 17: top:

mosaic CNLOH17p, bottom: mosaic NF1 deleted region.(CNLOH: copy neutral loss of heterozygosity).

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“Double hit” lymphomas using paraffin embedded tissue

The double hit lymphomas are classified in the WHO guidelines as diffuse B cell lymphoma unclassifiable, with features intermediate between Burkitt lymphoma and diffuse large cell lymphoma (DLBCL). These “grey zone” lymphomas are highly aggressive with a particularly poor prognosis that are difficult to distinguish morphologically. A firm diagnosis is essential to direct appropriate patient treatment. The hallmarks that distinguish the double hit cases involve the presence of concurrent MYC and BCL2 gene rearrangements. Due to the limited amount of material that can be obtained from lymph node biopsies, the retrospective nature of testing and the quick turnaround time required, Fluorescent in situ hybridization (FISH) analysis for MYC and BCL2 rearrangement on paraffin embedded tissue sections has proven to be a reliable and accurate test for the large number of referrals to the laboratory. In addition, the laboratory also offers a service for the detection of clinically relevant abnormalities in paraffin sections from solid tumours.

Chronic lymphocytic leukaemia (CLL)

Patients with the del(p53) are resistant to standard CLL therapy and the identification of the chromosome 17p deletion is critical to direct appropriate treatment. It is also the inclusion criteria by which many patients are selected for current clinical trials in CLL. Due to the low proliferative potential of CLL cells and difficulties with obtaining representative karyotypes, targeted FISH analysis is the current gold standard for the detection of genetic abnormalities. Results in our laboratory using DSP/IL2 stimulation were in keeping with recent reports by other studies. This involved an abnormality detection rate of ~80%. In addition, karyotypes are able to verify borderline abnormal populations detected by FISH, minor abnormal clones and the detection of balanced translocations in CLL not observable by current DNA based methods. The latter has been found to be relatively common in this disease and a better understanding of these genetic changes may in future result in improved risk stratification for this group of patients.

Figure 2. CLL karyotype showing a reciprocal translocation t(13q;17q) and a sub microscopic del(p53). The del(p53) was verified by FISH analysis on representative metaphases from DSP30/IL2 cultures.

References

Singh et al. Polyploidy in myelofibrosis: analysis by cytogenetic and SNP array indicates association with advancing disease. Molecular Cytogenetics. 2013.

Best et al. The novel Hsp-90 inhibitor SNX7081 is significantly more potent than 17-AAG against primary CLL cells and a range of haematological cell lines, irrespective of lesions in the TP53 pathway. British Journal of Haematology, 2010.

Singh et al. The role of FISH to identify MYC and BCL2 rearrangements in high grade non Hodgkin Lymphoma. HSANZ, 2009.

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Case Study

Potting Mix Can Be Harmful To Your Health, Pathology North, Kempsey Hospital

Colleen Allen, Scientist, Kempsey Hospital

Colleen Allen started her career in Pathology as a final year university student setting up Microbiology samples on weekends at Blacktown Hospital in 1976. This was followed by 4 years employed in the Blacktown Pathology laboratory. In 1981 she moved onto a private pathology laboratory at Emu plains as a Senior Scientist in Microbiology.

In 1989 she commenced at ICPMR at Westmead as a Senior Scientist in Charge of Enteric/Respiratory Laboratories and Media Production. In 1993, she became a NATA assessor in the field of Microbiology.

In 1995, with a young family and the lure of a “Sea Change”, Colleen commenced working at Port Macquarie Base Hospital, Pathology Laboratory. She is currently working full-time for Pathology North at Kempsey District Hospital. “Her Culture is Your Bacteria” and Microbiology is still her passion.

A 55 year old woman punctured her left forearm with a pair of long-nosed scissors whilst opening a bag of potting mix. The scissors, which were coated with potting mix, caused a deep laceration in her forearm.

She developed cellulitis and was treated with Flucloxacillin, Clindamycin and Augmentin over a period of 3 weeks.

The wound healed well until 2 months later when the cellulitis reappeared. The patient was treated with Clindamycin for 7 days. After covering the wound with magnoplasm paste for a week, the wound began oozing pus. A swab was taken and the initial gram showed numerous pus cells and no organism seen. After 48 hours the blood plate grew a hazy growth of small white “waxy-looking” colonies.

The gram stain showed numerous tiny gram-positive rods with elbow shapes and beading.

A Ziehl-Neelsen stain showed the presence of acid fast bacilli. The organism was sent to a referring laboratory where it was identified as Mycobacterium fortuitum. The patient was subsequently treated with Moxifloxacin over a 4-month period.

Mycobacterium fortuitum is a rapidly growing organism ubiquitous in nature and can be found in soil, dust, mulch, compost, water and potting mix.

Simple precautions should be followed when handling potting mix. Wear gloves, wear a face mask to avoid inhaling aerosols, keep mixture damp when in use and wash hands after handling, especially before drinking or eating.

Happy Gardening!

Thank you to the staff member who allowed her story to be published.

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Blood Agar Culture of M. fortuitum

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Know Pathology Know Healthcare Know Pathology Know Healthcare, (www.knowpathology.com.au) is the national initiative to educate the public on the value of pathology to healthcare. The campaign was launched in June at Parliament House, Canberra, where Former Australian of the Year Cathy Freeman OAM told attendees and ABC News Breakfast how she relies on pathology to help manage her Type 2 Diabetes.

Since June, Federal politicians around Australia have been touring their local pathology laboratories to learn more about the 25,000 skilled professionals working behind the scenes in the engine room of healthcare.

Collection staff across Australia have begun to engage their patients on the value of pathology and are achieving great results.

Pathology North collectors have also begun training to replicate the success of the campaign seen in collection centres in Queensland, Victoria and South Australia.

Please visit the Know Pathology Know Healthcare website and register your support.

(Link to TV clip: http://vimeo.com/londonagency/review/98604504/b37dba7870)

Cathy Freeman at launch

International Pathology Day - Wednesday 5 November 2014Pathology North celebrated International Pathology Day across the organisation by setting up stalls for patients and clinicians, and offering Laboratory open days.

Pathology North is committed to providing the highest quality service to doctors and their patients. International Pathology Day was an opportunity to celebrate.