Molecular Pathology USCAP-WADIAP

30 August 2016, Ivory Coast Behnoush Abedi-Ardekani, MD_AP/CP

Overview

•  Integral part of diagnostic pathology: the concept of personalized medicine

•  The challenge to the pathologist – Diagnosis and classification of a tumor – Production of necessary information to guide

treatment accurately and rapidly – Save the tissue for molecular analysis while

not hampering the diagnosis

Molecular pathology

•  Integration of molecular knowledge into diagnostic pathology treatment

•  Molecular techniques being performed on tissues obtained during the routine work

•  Interdisciplinary tumor boards with the presence of molecular pathologist and the availability of molecular information

•  Molecular classification of malignant tumors •  Conventional histopathology to be refined by molecular

data for selecting the optimal treatment

“Genomics” in the history

•  In 1975, the Sanger introduced the concept of DNA sequencing

•  In 2000, Jonathan Rothberg founded 454 Life Sciences, further developed the first commercially available NGS platform, the GS 20 in 2005

•  Second generation HT-NGS platforms introduced by Roche, Illumina, and SOLiD –  Map of human genome variation from population-

scale sequencing •  1000 genome project consortium, Nature, 2003

Era of third generation HT-NGS •  Sequencing of single DNA molecule without PCR

amplification –  Error reduction –  Increased read length of bases

•  Higher throughput –  Decreased time to result (from days to hours or minutes)

•  Faster turnaround time –  Higher consensus accuracy to detect rare variants –  Low cost

•  Sequencing of human genome at high fold coverage for less than 1000

–  Direct RNA sequencing instead of cDNA sequencing

Workflow for laboratories undertaking molecular pathology

Cree IA, et al. J Clin Pathol 2014

SUMMARY OF TISSUE HANDING CONDITIONS AFFECTING THE YIELD AND THE QUALITY OF NUCLEIC ACIDS FOR MOLECULAR ANALYSES

NGS in clinics •  Identification of genomic alterations at the nucleotide level •  Interest of using FFPE tissues

–  Storable at room temperature –  Suitable for large collections –  The most routine method used in pathology

•  FFPE now used for different types of molecular platforms –  Copy number variation analysis –  Detection of single nucleotide variants

•  More DNA damage in FFPE samples but still comparable with data from frozen samples

•  High frequency of base alterations arising from formalin cross linkage of cytosines leading to artificial C>T or G>A mutations

1) Prefixation

•  Prefixation – Sample size

•  Circled tumor area > 10 mm2

– Cold ischemia •  Preferably less than 1 hour

– Decalcification methods •  EDTA-based reagents lead to better DAN yield

compared to acid-based reagents

Sample size

Goswami R.S. et al., AJCP, 2016

2) Fixation

•  Use of neutral buffered formalin (NBF) – Preventing the formic acid formation and low

pH due to reaction of formaldehyde with oxygen

•  Fixation time – Less than 72 hours

•  Fixation temperature – Some reports supporting better results with

fixation at 4°C

Source of errors in FFPE samples

Do H., Dobrovic A., Clinical Chemistry, 2015

3) Processing and storage

•  Little data concerning the dehydration, clearing and paraffin quality

•  Storage duration of paraffin blocks – Controversy data

•  Storage duration of unstained sections – Preferably less than one week

Microscopic analysis •  Microscopic examination by pathologist is

mandatory for ultimate accurate and successful molecular analyses – Diagnosis – Tissue qualification – % of viable tumor cells – Presence of any other important element

(composition of tissue) – Annotation of the area of interest

Microdissection

•  To obtain the area of interest –  Manual microdissection

using scalpel or needle to scrape the selected areas

–  Thick sections unstained slides

–  Laser microdissection •  Dissection of a pure cell

population –  DepArray

CLINICAL APPLICATIONS OF MOLECULAR PATHOLOGY

Breast cancer •  Determination of molecular subtypes

subtypes – Different therapeutic approaches – Different survival rate and prognosis

•  Based on gene expression profile and IHC

Intrinsic breast cancer subtypes based on gene expression patterns

•  Low or absent ER gene expression

–  Basal-like •  High expression of EGFR, CK5 &17,

laminin •  Poor prognosis, short survival rate •  Responsive to platinum salts •  Mostly high grade ductal, medullary or

metaplastic •  Most are triple negative •  75% of TNBC are basal-like •  High frequency of TP53 mutation (80%)

+ loss of RB1 and BRCA1

–  HER2 positive •  Short survival rate •  Targeted therapy: Trastuzumab

–  Normal-like •  High expression of genes expressed by

adipose tissue

•  ER gene positive –  Luminal A

•  Responsive to HT, better prognostic than luminal B

–  Luminal B •  Variable response to HT, better

response to CT

–  Heterogenous gene expression & mutation

–  High mutation rate of PIK3CA –  Low TP53 mutation –  TP53 pathways largely intact

in luminal A, often inactivated in more aggressive luminal B

Prat et al. 2012 World breast cancer report, 2012

Ovarian cancer •  First approach in high grade serous

ovarian tumors: – Analysis of breast and ovarian cancer

susceptibility genes: BRCA1 & BRCA2 – PARP inhibitors for recurrent platinum-

sensitive high grade serous ovarian tumors • 

Algorithm for recurrent HG serous ovarian tumors

Dieter M. et al., Cancer gene therapy, 2015

Colorectal cancer •  EGFR-targeting antibodies

– Exclusively in patients with no KRAS mutations

•  KRAS and NRAS hotspot mutations in exons 2-4 – Microsatellite instability due to DNA mismatch

repair deficiency •  HNPCC/Lynch Syndrome •  Sporadic CRC

–  IHC analysis of the mismatch repair proteins •  MLH1, MSH2, MSH6, PMS2

NSCLC •  EGFR and ALK alterations targeted therapies

–  Erlotinib, gefitinib, afatinib

•  Exclusion of KRAS mutation for efficiency of EGFR-targeted therapies –  Exclusivity of mutations in KRAS, EGFR, ROS1 and

ALK •  KRAS and EGFR mutation analysis by PCR •  Carefully validated ALK-IHC

–  Crizotinib as targeted therapy against ALK-TKI –  Re-biopsy and re-analysis of metastatic resistant

tumors

Overview of molecular tests for therapeutic implications in different tumor types

Success of molecular analyses •  Carefully controlled pre-analytical variables •  Standard Operating procedures (SOPs) •  Precise microscopic evaluation •  Tumor cell content threshold •  Choosing the best and the most adapted

analysis method based on the available specimen –  Targeted sequencing, WES, WGS, etc.

Conclusion “Since currently the vast majority of the assays are tissue-based the responsibility of accurate performance lies in the hand of pathologists. The scientific societies have to be alert to cover this chance. Education of doctors and technicians, quality control of technical procedures and the intellectual interpretation of the results are crucial to provide reliable results. Clearly this will play an increasing role in the future structure of tissue-based diagnostics.”

Dieter M. et al., Cancer gene therapy, 2015