1. Hege G. Russnes, et al: Breast Cancer Molecular Stratification From Intrinsic Subtypes to Integrative Clusters. The American Journal of Pathology. Vol. 187, October 2017.

2. Maurice Chan et al: Development of a Next-Generation Sequencing Method for BRCA Mutation Screening. The Journal of Molecular Diagnostic. Vol. 14, November 2012.

4. Metodologie di Anatomia Patologica per lo studio di Biomarcatori Predittivi di risposta terapeutica

5. BRCA2 Gene Variants from Formalin Fixed Paraffin Embedded Samples
A Multi-Center Assessment of a next-Generation Sequencing Assay fpr Detection of Germline and Somatic BRCA1 and
BRCA2 Gene Variants from Formalin Fixed Paraffin Embedded Samples
Jon Sherlock, Charles Scafe, Gary Bee, Goutam Nistala, Adam Broomer, & John Bushop – Thermo Fisher Scientific Oyster Point Blvd South San Francisco CA 94080
Workflow Analysis
Participating Laboratories
Results

6. Maurice Chan et al: Development of a Next-Generation Sequencing Method for BRCA Mutation Screening. The Journal of Molecular Diagnostic. Vol. 14, November 2012.

8. Prognostic (Recurrence Risk) Predictive (Treatment Benefit)
Prognostic & Predictive Markers Used in Breast Cancer Management
Prognostic (Recurrence Risk)
Predictive (Treatment Benefit)
•Axillary node status
•Histologic type/grade
•Tumour size
•Patient age
•Lymphatic/Vascular invasion
•ER/PR status
•HER2 neustatus
•Endopredict
•Prosigna
•uPAPAI1
•OncotypeDX® test
•ER/PR status
•HER2 neustatus
•OncotypeDX® test
Sunali Mehta et al: Predictive and prognostic molecular markers for cancer medicine.

9. The Oncotype DX® Breast Cancer Test
16 Cancers and 5 Reference Genes from 3 studies
It is a 21 gene genomic test (16 tumourgenes and 5 reference genes).
• Uses RT-PCR technology on formalin fixed tissue.
• Quantitatively predicts the likelihood of breast cancer recurrence in women with newly diagnosed, invasive EBC.
• Is the only assay that has been demonstrated to be predictive of likelihood of benefit from chemotherapy.
• Is included clinical practice guidelines (St Gallen, ESMO, ASCO®, NCCN®) and NICE4-7
Proliferation
Ki-67
STK 15 Survivin
Cyclin B
MYBL2
OESTROGEN ER
PR Bcl-2
SCUBE 2
GSTM1
BAG1
Invasion Stromelysin 3
Cathepsin L2
CD86
Reference
b-actin
GAPDH RPLPO GUS TFRC
HER2
GRB7
Harris L, et al. J ClinOncol. 2007;33(25): Paik S, et al. JClinOncol

10. Elements of analytic validation
OncotypeDX (Genomic Health, USA)
Elements of analytic validation
• Analytical sensitivity (limits of detection and quantitation)
• Assay precision and linear dynamic range
• Analytical reproducibility
• PCR amplification efficiency
• Sample and reagent stability
• Reagent calibration
• Instrument validation and calibration

11. Roberta Sgariglia, Pasquale Pisapia, Mariantonia Nacchio, Caterina De Luca, Francesco Pepe, Maria Russo, Claudio Bellevicine, GiancarloTroncone, Umberto Malapelle. Multiplex digital color-coded barcode technology on RNA extracted from routine cytological samples of Non Small Cell Lung Cancer patients: pilot study. Journal of Clinical Patholgoy In press

12. neoplastic cell percentage
Staining
[RNA]
RIN 1 40
Papanicolau
4.14
3.8 2 60
4.80
4.0 3 35
5.04 4 55
Diff-Quik
4.32
4.2 5 75
4.02
4.6 6 80
3.90 7 30
4.50 8 50
12.96 9
3.96 10 70
9.54 11
4.26
3.4 12
3.24
3.6 N
FOV Counted
FOV Count
FOV ratio (%) BD 1
180
194 93
0,47 2
192 99
0,37 3
193
0,34 4
0,42 5 6
190 98
0,29 7
0,4 8
0,36 9
0,43 10 31 16 11
188 97 12
191
0,16
Roberta Sgariglia, Pasquale Pisapia, Mariantonia Nacchio, Caterina De Luca, Francesco Pepe, Maria Russo, Claudio Bellevicine, GiancarloTroncone, Umberto Malapelle. Multiplex digital color-coded barcode technology on RNA extracted from routine cytological samples of Non Small Cell Lung Cancer patients: pilot study. Journal of Clinical Patholgoy In press