1. Anna Long and Judith Hogarth Cellular Pathology Newcastle upon Tyne Hospitals NHS Foundation Trust North of England Cancer Network HER2 testing Audit

2. We will cover… HER2 testing – an introduction Audit parameters Aims Results Summary Limitations

3. HER2 HER2 is a trans-membrane tyrosine kinase that mediates cell growth and survival HER2 signalling induces cell proliferation Herceptin® binds to HER2 receptors and inhibits proliferation by disrupting HER2 mediated downstream signalling Only those patients that exhibit elevated expression of the HER2 antigen will benefit from treatment Getting the test right is critical!

4. HER2 testing HER2 status is routinely used in breast cancer testing and is recognised as a prognostic and predictive marker. Eligibility for treatment assessed by immunocytochemistry and/or in situ hybridisation IHC ISH If ≥ 2.0 = Amplified If < 2.0 = Non-amplified If < 2.0 = Non-amplified Negative PositiveEquivocal 1+ 0 0 2+ 3+

5. NECN HER2 testing At time of audit, undertaken by three centres in the NECN Gateshead Health NHS Foundation Trust (QEH) Newcastle upon Tyne Hospitals NHS Foundation Trust (RVI) North Tees and Hartlepool NHS Foundation Trust (UHNT) CentreTests referred from IHC method ISH performed? ISH method QEH SRH UHND Dako HercepTest No, referred to RVIN/A RVI CRI NTGH Ventana 4B5YesVentana SISH UHNT JCUH Ventana 4B5No, referred to Source BioScience N/A (Vysis FISH at Source BioScience)

6. UKNEQAS All centres performing HER2 testing must participate in the relevant NEQAS HER2 IHC and ISH modules UKNEQAS cite two recommended HER2 testing guidelines: ASCO/CAP (Wolff et al. 2006) UK updated guidelines (Walker et al. 2008)

7. Walker et al. (J Clin Path) Laboratories undertaking tests should be defined by each Cancer Network. UK minimum laboratory workload: 250 IHC cases per annum 100 ISH cases per annum Total HER2 positive cases: 15-17% Total IHC 2+ HER2 cases: average range 18-19% Number of IHC 2+ cases that exhibit HER2 gene amplification: 17-24%

8. Data collected from January 2011-December 2012 inclusive Aims of the audit Are centres within the cancer network meeting these standards? Therefore we have established within the NECN: (1) How many tests are performed by each centre (2) The HER2 positivity rate (3) The number of 2+ cases generated by IHC (4) The number of 2+ cases that exhibit HER2 gene amplification

9. (1) How many tests are performed by each centre

10. All centres meet minimum benchmark for IHC tests performed Number of IHC tests performed annually

11. Number of ISH tests performed annually *Equivocal QE cases stained using SISH at RVI, UHNT cases performed by Source BioScience All centres meet minimum benchmark for ISH tests performed

12. In summary (1) – Minimum number of tests performed All centres satisfy the minimum requirement for number of IHC and ISH tests performed.

13. (2) HER2 positivity rate

14. NECN HER2 profile – positivity 2011 - Positive vs. negative rates fits within the standard set out by Walker et al (15-17%). 2012 sees this rate drop 0.7% outside this range. 461/2816 426/2974

15. Positivity profile for each centre Quoted range 2006-2007 = 15-17% RVI and UHNT fell within the expected positivity range in 2011. QEH rate slightly above expected range in 2011 = 17.7% Both QE and RVI have seen a drop in the positivity rate in 2012 (below 15%) UHNT has shown an increase in positivity rate in 2012 (above 17%). UKNEQAS data 2009-2012= 14.5% 135/764 135/976 181/1185 148/1212 145/867 145/794

16. Positivity rates - QEH and RVI Positivity rate of cases has dropped across all hospitals (range = 2.4 - 5.0%) Positivity rate of cases has dropped across all hospitals (range = 0.5 - 10.1%) NTGH positivity rate has more than halved (19.9 – 9.8%) 56/362 60/508 19/105 27/199 60/297 48/269 53/406 52/412 77/523 66/495 51/256 30/305 15.5 18.1 11.817.820.213.1

17. Positivity rates in referring hospitals UHNT Positivity rate of cases has increased across all hospitals (range = 0.5 - 3.1%) 82/491 82/477 63/376 63/317

18. In summary (2) – Positivity rates The NECN as a whole falls below the expected frequency of positive HER2 cases. The audit period spans 2011-2012, however the published literature details HER2 cases from 2006-2007. UKNEQAS data spanning 2009-2012, quotes a positive rate of 14.5%, which means the NECN in fact meets the expected frequency of positive cases. 2/3 referral centres in 2012 fell below 14.5% (QEH and RVI) QEH – all referring hospitals showed a similar decline in positivity rate This would suggest some common factor ?the test itself. RVI - one referral hospital (NTGH) showed a marked decline in positivity rate (19.9-9.8%) This would suggest an upstream factor is responsible for the observed shift.

19. (3) 2+ cases generated by IHC

20. 2+ rate for NECN Av. Quoted range 2006-2007 = 18-19% UKNEQAS data 2009-2012 = 21.7% The NECN falls below the quoted range for the whole audit period, and significantly below the UKNEQAS data.

21. 2+ rate for centres Av. Quoted range 2006-2007 = 18-19% UKNEQAS data 2009-2012 = 21.7% RVI and UHNT have shown <2% variation in 2+ rate. QEH sees a 7.2% decrease 112/764 87/986 152/889 123/810180/1193 209/1230 16.015.117.015.217.18.8

22. % 2+ rates for referring hospitals QEH Fall in 2+ rate observed in all hospitals (range = 4.9 – 8.8%) Most significant decrease seen in SRH cases – more than halved. 58/363 52/518 18/107 16/199 36/300 19/269 16.010.016.88.012.07.1

23. % 2+ rates for referring hospitals - RVI and UHNT Small increase seen in CRI and RVI cases (1.2% and 0.2 % respectively). Significant increase seen in NTGH cases (5.4%) Small decrease seen in JCUH and UHNT cases (1.4% and 2.3% respectively). 57/409 63/417 43/257 69/312 80/527 77/501 64/386 49/322 88/503 74/488

24. In summary (3) – 2+ rate 2+ rate within NECN falls below the frequency cited in published and UKNEQAS data. RVI and UHNT saw slight (<2%) fluctuations in 2+ rate between 2011 and 2012. QEH showed a 7.2% decrease in 2+ reporting All referring hospitals showed a decrease in 2+ rate (4.9-8.8% fall – SRH rate more than halved).

25. (4) 2+ cases that exhibit HER2 gene amplification

26. Number of 2+ cases that exhibit HER2 gene amplification - NECN There has been a decline in the number of 2+ cases that exhibit HER2 gene amplification (3.8%). Av. Quoted range 2006-2007 = 17-24% UKNEQAS data 2009-2012 = 14.7% 70/441 49/406 15.912.1

27. 2+ cases that exhibit HER2 gene amplification by centre UHNT HER2 gene amplified cases increased 2.2%. RVI saw a decrease of 6.7% in 2+ cases that demonstrated gene amplification….Why is this? 53/292 33/28817/149 16/118 18.213.611.411.5

28. Amplified ISH performed at RVI Quoted range 2006-2007 = 17-24% UKNEQAS data 2009-2012 = 14.7% There is significant variation in the reporting of HER2 gene amplified cases (range 0-36.8%). Most significant outliers = QEH (decrease of 10.3% to 0% amplified), UHND (increase of 22.1%), NTGH (decrease of 18.6%) 6/58 0/52 3/18 3/16 5/36 7/19 9/57 6/63 17/80 11/77 13/43 8/69 10.30.016.713.918.836.89.515.811.630.214.321.3

29. Amplified ISH UHNT cases (Source BioScience) There is variation in the reporting of HER2 gene amplified cases (range 0.7-5.3%). 9/85 11/69 7/64 5/49

30. In summary (4) – 2+ amplified cases There is marked variation in the positivity rate by HER2 gene amplification. Significant outliers QEH (0% amplified 2012) UHND (increase of 22.1% from 2011 to 2012) NTGH (decrease of 18.6% from 2011 to 2012) This may be a symptom of variable 2+ rate by IHC

31. In summary

32. Change in rates 2011 – 2012 by hospital HospitalChange in positivity rate (%) Change in 2+ rate (%) Change in amplified 2+ rate (%) QEH  2.2  6.0  10.3 SRH  5.0  8.8  2.1 UHND  2.4  4.9  22.9 NTGH  10.1  5.4  18.6 RVI  1.4  0.2  7.0 CRI  0.5  1.2  6.3 UHNT  0.5  2.3  5.3 JCUH  3.1  1.4  0.7

33. Limitations

34. Limiting factors Limitations Pre-analyticalAnalyticalPost-analytical Cold ischaemia timeSubjective interpretation Core vs excisionChosen HER2 testStaffing FixationMethodology of testReporting confidence ProcessingStaining platforms Decalcification Drying time We need standardisation!

35. Future considerations Look at 2013 data?

36. Thank you Trudy Johnson - QEH Sharron Williams, Jim France – UHNT Merdol Ibrahim - UKNEQAS Immunocytochemistry colleagues, RVI

37. Thank you for listening… Questions? References: Walker et al. HER2 testing in the UK: further update to recommendations. J Clin Path. 2008:61; 818-824. Wolff et al. American Society of Clinical Oncology/college of American Pathologists guideline recommendations for HER2 testing in breast cancer. J Clin Oncol. 2007: 25; 1-28. Ibrahim M. UKNEQAS HER2 audit data. UKNEQAS. 2009-2012. Personal communication.