Published by PathWest Laboratory Medicine WAVolume 3, Number 1, April 2013

www.pathwest.com.au

PathWestNews Vol 3, No. 1 April 2013 1

PathWestNewsFremantle Hospital & Health Service n Queen Elizabeth II Medical Centre n Royal Perth Hospital n Women’s & Children’s Health Service

Department of HealthGovernment of Western AustraliaMetropolitan Health Service

INSIDE THIS ISSUE

Published by PathWest Laboratory Medicine WA

Department of HealthGovernment of Western AustraliaMetropolitan Health Service

INSIDE THIS ISSUEAncillary Testing for Chromosomal Aberrations Page 1in Cutaneous Melanoma.National Science Week Page 2New Building on site at QEII Page 3PathWest QEII Dematoppathology Page 4PathWest Doctors Forums Page 4

ANCIllAry TESTINg For CHroMoSoMAl ABErrATIoNS IN

CUTANEoUS MElANoMA

Dr Nathan Harvey*, Dr Ashleigh Murch^ and Dr Benjamin Wood*

*Anatomical Pathology, PathWest, QEII Medical Centre, Nedlands,^Cytogenetics, PathWest, King Edward Memorial Hospital

In 2009 there were just over 1000 cases of cutaneous melanoma diagnosed in Western Australia, accounting for approximately 10% of all cancers. It was the most common cancer amongst males aged between 15-39 years1. The pathological distinction of melanocytic naevi from cutaneous melanoma relies on histological examination, and this task is a common component of the everyday workload for many surgical pathologists. Indeed, in the US it is estimated that between 1 to 2 million biopsies a year may be performed for this reason alone2. While morphological criteria are relatively well established, in practice these are not always reliable as there are many lesions which are characterised by overlapping features. This is reflected in several studies demonstrating only moderate interobserver agreement for the diagnosis of melanocytic lesions,3,4 while others have reported that up to 11% of melanocytic diagnoses are significantly changed after specialist review5. Misdiagnosis of these lesions can lead to significant under- or over-treatment, and a false negative report involving a cutaneous melanoma remains the most common reason for malpractice claims against pathologists6. Thus, there exists a need for ancillary testing to aid in the distinction between benign and malignant melanocytic proliferations. Recent years have seen the advent of a method for utilising Fluorescent In-Situ Hybridisation (FISH) technology to identify chromosomal aberrations commonly associated with melanoma, providing pathologists with another tool to assist with difficult lesions.

Earlier studies utilising comparative genomic hybridisation demonstrated that both gains and losses of genetic material are common in melanomas, whereas they are rarely seen in benign naevi7. Out of a total of 132 melanomas and 54 benign naevi, 127 (96.2%) of the melanomas showed some form of copy number changes, compared with only 7 (13%) of the naevi7. These naevi were all Spitz naevi and had only one aberration each, involving an isolated gain of 11p in 6 out of the 7 cases. This particular aberration was not found in any of the melanomas. The most common losses seen in melanomas were from chromosomes 9p, 9q, 10q, 10p, 6q and 11q, while the most common gains were seen from chromosomes 6p, 1q, 7p, 7q, 8q, 17q and 20q. Subsequent work confirmed these findings, and demonstrated that distinct genetic alterations are found in different clinicopathological groups of melanomas8. Acral and mucosal melanomas have a much higher frequency of amplifications and losses than other types, although the actual types of aberrations differ between these two types. Melanomas from acral sites are significantly more likely to show an increase in chromosome 5p, for example8. Furthermore, melanomas occurring in non-chronically

sun damaged skin tend to be characterised by BRAF mutations and frequent losses of chromosome 10, whereas tumours from chronically sun damaged skin show less frequent mutations in BRAF and increased copy numbers of cyclin D1 (CCND1)9. Indeed, as our understanding of these differences improves it is feasible that a molecular classification of melanomas may eventually replace the current morphological system.

The potential for cytogenetic aberrations to provide a diagnostic tool for separating melanoma from benign proliferations was recognised early on. In an effort to develop a useful test, the more common chromosomal copy number changes identified in melanoma were analysed to determine combinations which would provide the highest sensitivity and specificity for melanoma2. FISH probes targeting 13 of these regions were developed and tested against a cohort of naevi and melanomas, eventually yielding a final probe set targeting 6p25 (RREB1), centromere 6, 6q23 (MYB) and 11q13 (CCND1). This probe set was then tested on a second cohort of naevi and melanomas to determine the cut off values which yielded the best discriminatory ability. This analysis resulted in an algorithm by which any of the following criteria would be considered evidence of melanoma2: Figure 1

Diagram: Low power (A), medium power (B) and high power (C) images of a difficult melanocytic lesion from the scapula of a 50 year old lady. Much of this particular lesion showed histological features of a compound naevus. However, the illustrated area shows more concerning features, such as an irregular growth pattern, confluent intraepidermal growth and cytological atypia. While the appearances were highly suspicious for a melanoma arising within a naevus, the possibility of “pseudomelanoma” change in a traumatised naevus was difficult to exclude histologically. FISH analysis (D) revealed numerous cells with an increase in 6p25 (RREB1) signals (red dots, arrowed cell). In this case the FISH analysis fulfilled 2 of the 4 criteria outlined in the text, supporting a diagnosis of melanoma.

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PathWestNewsFremantle Hospital & Health Service n Queen Elizabeth II Medical Centre n Royal Perth Hospital n Women’s & Children’s Health Service

a. More than 29% of nuclei contain greater than 2 6p25 signals. b. More than 55% of nuclei contain more 6p25 than centromere 6 signals. c. More than 40% of nuclei contain fewer 6q23 than centromere 6 signals. d. More than 38% of nuclei contain greater than 2 signals for11q13.

This algorithm was subsequently tested on a third cohort, where it correctly identified 72/83 melanomas and 82/86 naevi (sensitivity 86.7%, specificity 95.4%). Finally the algorithm was tested on a group of 27 histologically ambiguous tumours, 6 of which had subsequently shown development of metastases. All 6 of these proven melanomas were positive by the FISH algorithm2.

Increasing experience with the FISH probe set has demonstrated that 80% of melanomas show either a gain of 6p25 or a loss of 6q23, indicating that cytogenetic abnormalities of chromosome 6 occur with high frequency in all subtypes of melanoma. This is thought to be related to an isochromosome where the loss of one arm is replaced by an identical copy of the remaining arm10. In addition, the presence of increased copies of 11q13 is seen more frequently in melanomas from chronically sun damaged skin, correlating with the previously identified increase in cyclin D1 mutations in this subgroup9,10. Subsequent studies have demonstrated the usefulness of this FISH probe set in superficial melanocytic neoplasms with pagetoid melanocytosis11, as well as in Spitzoid melanomas where the sensitivity is improved by the addition of a probe against 9p2112. At PathWest we have recently begun using this FISH probe set to help us interpret difficult melanocytic lesions. An example of one of our cases is illustrated in Figure 1.

Thus, cytogenetic analysis of melanocytic neoplasms using FISH is emerging as a useful adjunctive test. Other techniques such as array-based comparative genomic hybridisation may also prove useful, and have the advantage of being able to detect a much broader range of mutations. However the requirement for larger amounts of DNA means that this technique may have limited usefulness in more superficial lesions. As with all adjunctive tests, correlation with the morphological appearances and clinical scenario are critical for correct interpretation.

References:[1] Threlfall TJ, Thompson JR (2010). Cancer incidence and mortality in Western Australia, 2009. Department of Health, Western Australia, Perth. Statistical Series Number 91[2] Gerami P, Jewell SS, Morrison LE, Blondin B, Schulz J, Ruffalo T, et al. Fluorescence in situ hybridization (FISH) as an ancillary diagnostic tool in the diagnosis of melanoma. Am J Surg Pathol. 2009; 33: 1146-56.[3] Farmer ER, Gonin R, Hanna MP. Discordance in the histopathologic diagnosis of melanoma and melanocytic nevi between expert pathologists. Hum Pathol. 1996; 27: 528-31.[4] Corona R, Mele A, Amini M, De Rosa G, Coppola G, Piccardi P, et al. Interobserver variability on the histopathologic diagnosis of cutaneous melanoma and other pigmented skin lesions. J Clin Oncol. 1996; 14: 1218-23.[5] McGinnis KS, Lessin SR, Elder DE, Guerry Dt, Schuchter L, Ming M, et al. Pathology review of cases presenting to a multidisciplinary pigmented lesion clinic. Arch Dermatol. 2002; 138: 617-21.

[6] Troxel DB. Trends in pathology malpractice claims. Am J Surg Pathol. 36: e1-5.[7] Bastian BC, LeBoit PE, Hamm H, Brocker EB, Pinkel D. Chromosomal gains and losses in primary cutaneous melanomas detected by comparative genomic hybridization. Cancer Res. 1998; 58: 2170-5.[8] Bastian BC, Olshen AB, LeBoit PE, Pinkel D. Classifying melanocytic tumors based on DNA copy number changes. Am J Pathol. 2003; 163: 1765-70.[9] Curtin JA, Fridlyand J, Kageshita T, Patel HN, Busam KJ, Kutzner H, et al. Distinct sets of genetic alterations in melanoma. N Engl J Med. 2005; 353: 2135-47.[10] Gerami P, Mafee M, Lurtsbarapa T, Guitart J, Haghighat Z, Newman M. Sensitivity of fluorescence in situ hybridization for melanoma diagnosis using RREB1, MYB, Cep6, and 11q13 probes in melanoma subtypes. Arch Dermatol. 2010; 146: 273-8.[11] Gerami P, Barnhill RL, Beilfuss BA, LeBoit P, Schneider P, Guitart J. Superficial melanocytic neoplasms with pagetoid melanocytosis: a study of interobserver concordance and correlation with FISH. Am J Surg Pathol. 2010; 34: 816-21.[12] Gammon B, Beilfuss B, Guitart J, Gerami P. Enhanced detection of spitzoid melanomas using fluorescence in situ hybridization with 9p21 as an adjunctive probe. Am J Surg Pathol. 2012; 36: 81-8.

For further information, please contact Dr Nathan Harvey, Anatomical Pathology, PathWest, by email nathan.harvey@health.wa.gov.au or telephone (08) 9346 2160; Dr Benjamin Wood, Anatomical Pathology, PathWest by email benjamin.wood@health.wa.gov.au or telephone (08) 93461862; Dr Ashleigh Murch, Cytogenetics, PathWest by email ashleigh.murch@health.wa.gov.au or telephone (08) 9340 2763.

NATIoNAl SCIENCE WEEk CHAllENgE

The feature on ABC’s science magazine, Catalyst, was the last stage of a long journey. Preparations began a year ago when we drafted our proposal for a 1000 km pathology road show in the Kimberley. As soon as we’d been given the go-ahead by the organisers of National Science Week, we tapped into the Heath Department’s mosquito surveillance programme and joined a mosquito-trapping team working out of Kununurra during the Easter Holiday weekend. We used the opportunity to run our set of PCR assays with our portable equipment in the Kununurra PathWest lab. Though this was a first for the Kununurra lab, we stopped short of trying out our equipment on the road. We had to hold fire until National Science Week in August.

Picture: The PathWest National Science Week Lab Without Walls set up.

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Fremantle Hospital & Health Service n Queen Elizabeth II Medical Centre n Royal Perth Hospital n Women’s & Children’s Health Service

PathWestNewsThe road show started in Broome with three days of public science activities, overnight mosquito trapping and follow-on PCR work. None of the trapped mosquito samples contained evidence of Ross River virus, Murray Valley Encephalitis virus, Barmah Forest virus or Kunjin virus, as expected during the long dry season.

After a day in Derby we headed off along the Gibb River Road, stopping at 100km intervals to take melioidosis soil samples.

Each evening we ran our PCR assays and set mosquito traps. After overnighting at Drysdale Station we met up with the Catalyst film crew, who spent two days following our progress towards the eastern end of the Gibb.

The feature that went to air on November 1st only told the melioidosis part of the story, showing how the work we’re doing is adding evidence that the infection originated in northern Australia. There is much more to the Lab Without Walls than you can squeeze into a 10 minute TV feature. On this occasion the PathWest team were able to detect evidence of Ross River virus in mosquitoes trapped around wetlands in the East Kimberley. This is a significant, if small, piece in the jigsaw of mosquito-borne virus infection in northern WA. We also detected the bacteria that cause melioidosis (Burkholderia pseudomallei) in several places near the junction of the Gibb River and Kulumburu roads. These isolates are now undergoing detailed molecular analysis including whole genome sequencing, which we can’t yet run with deployable equipment.

The road show proved a good opportunity to try out new approaches to support public and environmental health in an economically dynamic part of our state. The logistic challenge of putting advanced microbiology methods into action in remote places has taught us some valuable lessons. We now have a better grasp of the technology improvements needed to use these methods to strengthen clinical laboratory services in regional hospital pathology laboratories. Looking forwards, the Lab Without Walls will continue to work on pathology capability building for places that currently lack the benefits of molecular and other emerging test technology.

You can find out more about the Lab Without Walls, melioidosis, arbovirus infections and our other fieldwork projects from the Micrognome website: http://micrognome.priobe.net

For further information, please contact Dr Tim Inglis, Consultant Microbiologist, PathWest, by email tim.inglis@health.wa.gov.au or telephone (08) 9346 3461

NEW BUIlDINg oN SITE AT QEII

PathWest at the QEII site has commenced relocating to the purpose built state-of-the-art pathology facility in PP Block. This is an iconic building with its distinctive façade of red blood cells and the entrance “heart beat”.

The construction of PP block is part of NMHS redevelopment plans as recommended by the Clinical Services Plan 2005-2015.

The Clinical Services Framework 2005-2015 outlined the need to reorganise public hospital services in the metropolitan area. This required an expansion and reorganisation of tertiary level pathology services on the QEII Medical Centre site (QEII). In particular this allowed the aggregation of state-wide Clinical Genetics diagnostic services into a single laboratory. Furthermore expanded high containment facilities were required to support the safe examination of potentially high risk clinical isolates such as multi drug-resistant TB, pandemic influenza, SARS and other high risk infectious agents, and for the safe investigation of suspected bioterrorist materials. The new pathology building will also accommodate paediatric pathology when the new Children’s Hospital is completed. It will also ensure that the laboratories conform to best practice in laboratory design.

This transition will be staged to allow minimal disruption to the delivery of services, while moving equipment and staff as quickly as possible. The new facility in PP block is less than 100 metres from existing PathWest locations on the QEII site.

The transfer of services on a department-by-department basis commenced in February 2013 and is expected to be fully completed

by June 2013.

The new PathWest building at QEII, showing the “heartbeat” entrance.

Picture: Dr Tim Inglis collecting samples

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PathWestNewsFremantle Hospital & Health Service n Queen Elizabeth II Medical Centre n Royal Perth Hospital n Women’s & Children’s Health Service

PATHWEST QEII DErMAToPATHology

PathWest QEII Dermatopathology is a newly established subspecialist clinical and academic Dermatopathology service based at the QEII Medical Centre. The Dermatopathology Group is comprised of Dr Benjamin A. Wood FRCPA (Lead Pathologist), Dr Nathan T. Harvey FRCPA and Dr Tamazin N. Leecy FRCPA. The service has the following goals:

• To provide an exceptional and timely diagnostic service to patients with skin disease and their treating clinicians.

• To conduct research in diagnostic dermatopathology and provide a histopathological basis for clinical and basic science research in skin disease.

• To contribute to the post-graduate education of practitioners and trainees in Pathology, Dermatology, General Practice and other specialities dealing with skin disease.

• To provide a consultative service for pathologists and clinicians dealing with unusual skin diseases.

• To provide a referral service for complex cases in which subspecialist opinion, cytogenetic, molecular or other ancillary testing may be of value.

In general, we provide a histopathology report within 2-3 business days of specimen receipt. This time-frame may be extended in more complex cases requiring immunohistochemical, molecular or other adjunctive testing, but in these cases a preliminary report will be provided. Rapid results (<24 hours) can be provided in clinically urgent cases by contact with the Dermatopathology Group.

As an academic unit, we are available to participate in clinical and basic research studies requiring dermatopathology input. We also have a number of research opportunities available for interested clinicians and trainees which we are happy to discuss.

The PathWest QEII Dematopathology staff consists of:

Nathan T. Harvey BSc MBBS (Hons) FRCPA

After completing a Science degree with a major in biochemistry, Nathan studied Medicine at the University of Adelaide. Following his intern year, he spent 4 years as a full time researcher in the field of embryonic stem cells. Nathan’s interest in cellular and molecular biology led him to train in Anatomical Pathology at the Institute of Medical and Veterinary Science in Adelaide. Nathan joined PathWest as a Consultant Pathologist in 2010 and soon thereafter joined the QEII Dermatopathology group. He continues to maintain an interest in the molecular biology of skin lesions, with ongoing research projects in collaboration with the University of Western Australia.

Tamazin N. Leecy MBBS (Hons) FRCPA

Tamazin graduated in Medicine with Honours from the University of Western Australia in 2000. After training in Anatomical Pathology in Sydney, she became a consultant Pathologist at Royal Prince Alfred Hospital and the Sydney Melanoma Unit and conducted research at the Garvan Institute. Tamazin returned to Western Australia and joined PathWest and the QEII Dermatopathology Group in 2012. She enjoys many aspects of clinical dermatopathological practice.

Benjamin A. Wood BMed FRCPA

A graduate of the University of Newcastle and trained in Anatomical Pathology with the Hunter Area Pathology Service, Benjamin joined PathWest as a Consultant Pathologist and the Universtiy of Western Australia as Clinical Senior Lecturer in 2006. In the role of Lead Pathologist, he established the PathWest QEII Dermatopathology Group in 2010. In 2011, Benjamin spent a sabbatical period as a post-doctoral fellow at University of California San Fransisco Dermatopathology. He has wide diagnostic and research interests in dermatopathology, including cutaneous adnexal neoplasms, melanocytic lesions and the role of digital microscopy in dermatopathology.

To refer cases, please use one of the specially labelled request forms or mark a standard request form “Attention QEII Dermatopathology Group”.

For further information, please contact Dr Benjamin A. Wood by email benjamin.wood@health.wa.gov.au or telephone 08 93461862.

PATHWEST DoCTorS ForUMS

The 2013 PathWest series of Doctors Forums are well under way with sessions in Kalgoorlie, Karratha and Port Hedland already held.

The proposed schedule for the 2013 is below.

Date locationTuesday 14th May Broome

Wednesday 15th May DerbyTuesday 25th June Collie

Thursday 27th June MandurahWednesday 14th August AlbanyTuesday 10th September Busselton

Wednesday 23rd October Northam

Wednesday 13th November Armadale

*This schedule is subject to change.

To register your interest in attending a forum or if you would like a particular topic to be presented, please contact PathWest Marketing via email marketing@pathwest.com.au or telephone (08) 9346 7418.