Soecimen Handling

1. Specimen Handling
in Biomarker
Testing Non Small
Cell Lung Cancer

2. Relative Frequency of Lung Adenocarcinoma
Oncogene Drivers2
• The development of targeted systemic
cancer therapies has been enabled by
the identification of tumor genetic
markers that drive oncogenesis1
• At present, some marker mutations are
not actionable and do not have
approved therapies1
Current Developments in Driver Mutations
for Lung Cancer
AKT = v-akt murine thymoma viral oncogene; ALK = anaplastic lymphoma kinase; BRAF =
B-Raf proto-oncogene, serine/threonine kinase; EGFR = epidermal growth factor receptor; HER2 = human epidermal growth
factor receptor 2; HRAS = Harvey Rat Sarcoma Viral Oncogene Homolog; KRAS = Kirsten rat sarcoma viral oncogene
homolog; MAP3K1 = mitogen-activated protein triple kinase 1; MET = mesenchymal-to-epithelial transition factor; NRAS =
neuroblastoma RAS viral (v-ras) oncogene homolog; NTRK1 = non-receptor tyrosine kinase 1; PIK3CA = phosphatidylinositol-
4,5-bisphosphate 3-kinase, catalytic subunit alpha; RET = ret proto-oncogene; ROS1 = ROS proto-oncogene 1, receptor
tyrosine kinase.
1. Blakely CM, Bivona TG. Cancer Discov. 2012;2(10):872–875. 2. Bubendorf L et al.
Eur Respir Rev. 2017;26:17.0007. Image adapted from Bubendorf L et al. Eur Respir Rev. 2017;26:17.0007.
25%
10%
4%
1.9%
0.9%
1%
3%
3%
2%
1%
1%
1%
3%
1%
42%
KRAS
EGFR
ALK
ROS1
RET
NTRK1
HER2
BRAF
PI3KCA
HRAS
NRAS
AKT
MET exon 14
MAP3K1
Unknown
Unknown
KRAS
EGFR
ALK

4. ID-KEY-00090
Biomarker Status can be a Roadmap in
personalizingTreatment in Advanced NSCLC
• Biomarkers help guide the selection of effective
targeted therapies based on an individual’s
tumor biology1
• Targeted therapies selectively inhibit molecular
pathways that drive tumor growth and
proliferation1
1. Domvri K et al. J Cancer. 2013;4(9):736–754.
2. Modur V et al. Clin Chem. 2013;59(1):102–109.
Improve Prevention2 Improve Detection2
Improve Diagnosis2 Improve Treatment2

5. NSCLC: Patient pathway to diagnosis and treatment
5
IHC Testing
Molecular
ID-KEY-00090

6. TheWorkflow of Diagnosing Lung Cancer
Ipsos HealthCare, September 2015
ID-KEY-00090
Diagnostic imaging
whole organ
Biopsy Tissue sample Histological
classification Cellular
structure / staining
and protein analysis
protein analysis by
IHC and mutational
analysis DNA

7. TissueSamplingMethodsinNSCLC
Excised during surgery Bronchoscopic biopsy Biopsy Cytology
Fine Needle Aspiration
ID-KEY-00090
Brush Cytology

8. Schematic workflow of various cytology specimen
preparations and processing methods
ID-KEY-00090
Jain D et al. Arch Pathol Lab Med. 2018;142(9):1127-1133. doi:10.5858/arpa.2017-0444-RA

9. Whyisfixation
SoImportant??
• Prevent autolysis
• Ensure that the level of protein expression
measured is correct and not an artefact
• Preserve tissue as near to its original form as
possible
ID-KEY-00090

10. ID-KEY-00090
Indonesian Pathologist Guideline

11. SpecimenHandling
TISSUE SPECIMEN
• Immediately place
biopsy/surgical resection
specimens into 10% buffered
formalin within the first 20
minutes (maximum) after the
specimens are obtained
• Fixation volume is 5-10x
specimen volume. All part of
specimens should be
completely covered by
formalin
CYTOLOGY
SPECIMEN
• Smear slides are immediately place
into 96% alcohol fixation, and
allow other smear slides to air-dry
at room temperature
• Place the specimen into 10%
buffered formalin for cell block
preparation
• Fluid specimens, such as pleural
effusion, should be transported
immediately to Department of
Anatomical Pathology
• How to handle specimens on
non-working day?
• Smear slides are prepared as the
aforementioned procedures
• Fluid specimens should be stored in
refrigerator. Do not store fluid
specimens in freezer as it may
alter/damage cell morphology
ID-KEY-00090

12. Surgery
Gross
examination
Dissection
Fixation
FFPE
Cut sections
ThePathologyWorkflow
FFPE
H&E
Assesment
IHC
Result
Diagnosis
ID-KEY-00090

13. Chest clinician, assessment
and chest X ray, onto CT scan
and biopsy (usually EBUS –
can be sample size
challenge). Sometimes
surgeon will take sample.
Sample sent to hospital path
lab, is booked in, prepared
and then is “cut” for
diagnosis – cancer type,
histology.
Biomarker testing is
requested by the pathologist
– usually automatic/reflex
and parallel/ (EGFR/ALK and
PD-L1 at same time). Sample
ready for sending
Tissue block sent to testing
centre (usually daily van
collection or post)
Sample is booked in,
prepared and examined.
Result(s) are sent via e-mail
or loaded onto “portal”.
Remaining tissue block
returned to requesting lab
Results received by
Pathologist/Oncologist/Onc
Nurse – results and
treatment options
discussed/agreed at next
weekly MDT
Patient appointment –
Oncologists and patient agree
treatment option
2-5 days
1-3 days
5-10 days
0-2 days
1-6 days
1-2 days
Laboratory TAT : 5-8 days
Total TAT: 8-18 days
Total pathway TAT: 10-28
Collected during interviews with testing labs across the UK
Sample
Collection
Sample
Preparation
Test
Ordering
Result
Reporting
Lab
Testing
Sample
Transport
Treatment
Decision
Biomarker Testing Pathway and How it Varies
ID-KEY-00090

14. PotentialIssuesinLungCancerBiomarkerTesting
• Lung cancer  90 % cytology sample
• Tissue heterogeneity
• A tumor biopsy is a mixture of normal and tumor tissue
• Protein expression is dynamic
• IVD tests require verification prior to clinical use
• Validation for each IHC marker for each cancer type
• Obtaining validation materials and controls can be a
challenge
• Interpretative proficiency
• Pathology will have to be attentive to which scoring
algorithm is needed
Standardization and
harmonization of
assays & Improve
MDT collaboration
ID-KEY-00090

15. Time to slicing
and fixation
Method of tissue
processing
Type of
fixation
Equipment
calibration
Laboratory
procedures
Time of
fixation
Assay
validation
Staff competence
Type of antigen
retrieval
Test reagents
Controls
Assay
conditions
Use of
image
analysis
Interpretation
criteria
Reporting
elements
Scoring
system
PD-L 1 testing
variation
Post-analytic Pre-analytic
Analytic
The Important process of IHCTesting to
minimize potential issues
ID-KEY-00090

16. Pulmonologist conducts
bronchoscopy (or EBUS in
minority of cases) if tumor
accessible via bronchial tube
Radiologist conducts CT guided
biopsy (if tumor accessible
transthoracically)
Surgeon conducts surgical biopsy for
minority of late stage patients
suitable for surgery
Pathologist may attend CT scan
guided biopsy procedures to
ensure tissue sample is good
quality
Tissue extraction
Pathologist typically sends results
with some degree of interpretation
(i.e., whether patient is positive or
negative, and suitability for specific
therapies)
In some markets pathologists will
present results to MDT
Will discuss results with oncologist
if necessary
Pulmonologist/Oncologist will make
treatment decision, often in
conjunction with an MDT, consisting
of:
Pulms, Surgeons, Paths, palliative
care, radiotherapy
Treatment initiation
Pulmonologist/Oncologist typically
requests – need to ensure patient
will be suitable candidate for
treatment (i.e., good PS)
Pathologist may conduct test
reflexively if routine in hospital (esp.
PD-L 1 as a more established test)
Tissue extractor may request upfront if
test routine (minority)
Pathologist conducts or outsources
depending on specialism of lab
EGFR and Pd-L1 often outsourced as
molecular testing is required
Pathologist may discuss need for re-
biopsy with Tissue Extractor if needed
Biomarker testing
Source: MK-3475 NSCLC Biomarker Landscape, Mature Markets, Q2-2014 (Research Partnership)
MDT: Multi-disciplinary teams
Tissue Extractor Pathologist Oncologist
Stakeholders in Biomarker testing
ID-KEY-00090

17. Patient
Technician
Pulmonologist
Pathologist Pulmonologist
/Surgeon
Wolff AC, Hammond ME, Hicks DG, Dowsett M, McShane LM Allison KH, et. al. American Society of Clinical Oncology/College of American
Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007;
Successful andTimely Pathology and BiomarkerTesting
Require a Multidisciplinary Approach
ID-KEY-00090

18. Summary
Good process in pre-analytic,
analytic & post-analytic phase can
help achieve improvements in
result and quality of biomarker
testing
ID-KEY-00090

19. THANKYOU
ID-KEY-00090