ΕΛΕΥΘΕΡΙΟΣ ΕΛΕΥΘΕΡΙΑΔΗΣ
MD, PhD, MBA
ΕΕΠΑ, ESP, IFCAP, ISUP
Pathologic Specimen Types
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Surgical biopsy / resection (wedge resection, lobectomy, pneumonectomy)
Core biopsy
Trans/endobronchial biopsy (+/- ultrasound guidance)
Fine needle aspiration (cores, cell blocks, smears)
Cytology (smears, washings, brushing)
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FFPE
Molecular Diagnostic Testing in Non-Small Cell Lung
Cancer: Naidoo J., Drilon A., AJHO, VOL. 10, NO. 4, September 2014
Multiplex Testing for Gene
Mutations
Multiplex hotspot mutational
testing
Multiplex sizing assays
Next-Generation or Massively
Parallel High-Throughput
Sequencing (NGS)
Single-Gene Molecular
Diagnostic Assays
Sanger sequencing
Immunohistochemistry (IHC)
Reverse transcriptase-
polymerase chain reaction (RT-
PCR)
Fluorescence in situ hybridization
(FISH)
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AdenoCa
SCC
MSKCC
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FFPE and Molecular Testing
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▪ Standardization of Diagnostic Immunohistochemistry Lin F., Shi J. in Handbook of Practical
Immunohistochemistry 2nd ed Springer 2015
▪ Guidance for laboratories performing molecular pathology for cancer patients. Cree IA, et al. J Clin Pathol 2014
▪ DNA Yield From Tissue Samples in Surgical Pathology and Minimum Tissue Requirements for Molecular
Testing. Austin et al, Arch Pathol Lab Med—Vol 140, February 2016
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Weigh the benefits versus the consequences of using archival FFPE tissue when the
handling, fixation, processing, and storage parameters for a specimen are unknown.
The use of FFPE tissue that has been handled improperly may not generate data or may yield
data that are representative of the FFPE processing conditions applied
rather than the biospecimen donor’s disease condition
When more than 1 analyte is to be investigated using the same FFPE specimen, the more
stringent conditions should be used as a guideline
Preanalytical Factors Affecting FFPE Tissue—Bass et al, Arch Pathol Lab Med—Vol 138,
November 2014
FFPE: Acceptable Thresholds for Preanalytical Factors Based on Literature Evidence for
Specific Analytes
Preanalytical Factor DNA RNA Protein Morphology
Cold ischemia<1 h for FISH,
≤24 h for PCR
<12 h <12 h <6 h
Specimen size3–10 mm3 Evidence not
available
1.2–3.5 mm3 Evidence not available
Decalcification
EDTA Ultrasound or
EDTA
Thresholds are tissue
and antigen specific
Ultrasound; EDTA, nitric,
formic or acetic acid,
DECAL, Cal-Exc, D-
calcifier, Plank-Rychlo,
Ebner’s, or Jenkin’s solution
Fixative buffer NBF NBF NBF Evidence not available
Fixation duration <72 h 8–48 h 6–24 h <1 y
Fixation temperature 4ºC or ambient 4ºC or ambient 4ºC or ambient Evidence not available
FFPE block size or
section thickness
Whole sections
preferable to cores
or isolated nuclei
Evidence not
available
2–4 μm 2–3 μm
Duration of paraffin
block storage
≤5 y ≤1 y ≤ 25 y for IHC,
<10 y for platforms
requiring protein
extraction
Evidence insufficient
Tissue section storageInsufficient
evidence
<3 mo at
ambient
<1 wk Evidence not available
CAP Pre-Analytics for Precision Medicine Project
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PPMPT Proposed Benchmarks: Tissue
1. Time to stabilization
• 60 minutes or less
2. Method of processing
• Section thickness: ≤5 mm
• Mass/volume ratio: ≥4:1, optimal ≥10:1
• Transport temperature: ambient
3. Method of stabilization
• Type of fixative: 10% neutral phosphate-buffered formalin
• Time in fixative: 6-24 hours (includes time in formalin in processor)
4. Tissue processor variables
• Maintenance schedule: Manufacturer’s recommendation or a validated deviation
• Paraffin type: low melt <60°C
• Total time in processor: 7.5-8 hours
5. Storage conditions
• Ambient (eg, 20-25°C)
6. [Metadata to be collected]
• Any deviation from the above recommendations
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Impact of the preanalytical
steps on PD-L1 staining.
Immunohistochemical analysis
of a lung adenocarcinoma after:
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Ilie M. et al., Virchows
Arch (2016) 468:511–525
3 h 8 h
24 h 48 h
72 h 96 h
Tumor Tissue Management
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Inadequate Tissue for Testing - Size Matters
Complexities in Personalized Medicine: A Pathologist’s Perspective Leonard D. G.B, M.D., Ph.D., FDA-
AACR-ASCO Public Workshop: Complexities in Personalized Medicine 24 March 2015
Diagnostic Ancillary Techniques in NSCLC
IMMUNOHISTOCHEMISTRY
Primary lung vs. extrapulmonary origin
CK7, CK20, TTF-1, CDX-2, PAX-8, Thyroglobulin, RCC, Heppar1, DPC4, calretinin,
WT-1, D2-40, GCDFP, PSA etc
Lung adenocarcinoma markers
TTF-1, Napsin-A, Surfactant (PE-10)
Squamous cell carcinoma markers
P63, p40, CK5, CK5/6(CK20), CK903
Markers of special NSCLC subtype
B-catenin (fetal adenoCa)
EBER (lymphoepithelioma like carcinoma)
ALK positive adenoCa
Neuroendocrine markers
Synaptophysin, chromogranin, CD56,NSE
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For small biopsies
• Minimum number of stains
should be used to exclude
squamous differentiation
• Material should be preserved
for molecular studies
(mutation analysis, in situ
hybridization
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Tissue Salvage Management
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Metastatic / Primary
Tumor Type
ADC / SQC ...
ALK, PDL-1 ..
Malignancy / Morphology
RT-PCR, FISH
Multiplex, NGSEach Step Wastes
Valuable Tissue
Pre-Defined Tissue Handling Protocol
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Selection of Targeted Therapy on FFPE
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Reporting
Tissue Acquisition
Pre-Analytical Stage
Pathology
Molecular / Biomarkers
FFPE
Pneumonologist
Thoracic Surgeon
Oncologist
PATIENT
FFPE and Molecular Testing
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Improvements in technology during the past decade allow most molecular tests to be
performed on formalin-fixed, paraffin-embedded (FFPE) samples
ever-growing number of tumor markers that can be tested at the molecular level, and no
single laboratory can aspire to offer them all.
Many requests for molecular testing will be sent out to off-site laboratories
Pre analytic variables and storage time affect molecular testing results
The molecular testing laboratory has no control over these pre analytic events
Tissue type and volume requirements differ for each molecular testing methodology Το
είδος και η ποσότητα του ιστού που απαιτείται για κάθε μέθοδο διαφέρει
ethical and legal expectations for proper informed consent and counseling for patients have
grown.
• Biobanking in Genomic Medicine, Zhou et al., Arch Pathol Lab Med. 2015;139:812–818
• Stuck Between a Scalpel and a Rock, or Molecular Pathology and Legal-Ethical Issues in Use of Tissues
for Clinical Care and Research, Dry S., Grody W.W, Papagni P., Am J Clin Pathol 2012;137:346-355
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Table 1. FFPE-Related Preanalytical Factors Categorized by the Extent Each Has Been
Investigated in the Literature for Potential Effects on DNA, RNA, Protein, and Morphology
Analytes (limited to evidence available for FFPE fixation and processing factors)
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Preanalytical Factors Affecting FFPE Tissue—Bass et al, Arch Pathol Lab Med—Vol 138, November 2014
Parameters can influence analysis of nucleic acids,
proteins, and morphology in different ways and to
different extents.
Standardization of pre-analytical data elements
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Standardize Preanalytic Data Elements—Robb et al, Arch Pathol Lab Med—Vol 138, April 2014
Weigh the benefits versus the consequences of using archival FFPE tissue when the
handling, fixation, processing, and storage parameters for a specimen are unknown.
The use of FFPE tissue that has been handled improperly may not generate data or
may yield data that are representative of the FFPE processing conditions applied
rather than the biospecimen donor’s disease condition
Parameters must be recorded (από μένα)
By priority of their importance, 170 preanalytic variables were ranked in 5 categories:
a) variables common to all biospecimens = 102
b) tissue-specific variables = 36
c) blood, serum and plasma specific variables = 20
d) body fluid variables = 6
e) nucleic acid variables = 16
Additional sources of preanalytical variability, including extraction methods, antigen
retrieval techniques, and uninvestigated variables, may serve as additional
confounding variables
Table 2. Acceptable Thresholds for Preanalytical Factors Based on Literature
Evidence for Specific Analytes
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Preanalytical Factors Affecting FFPE Tissue—Bass et al, Arch Pathol Lab Med—Vol 138, November 2014
Weigh the benefits versus the consequences of using archival FFPE tissue when the
handling, fixation, processing, and storage parameters for a specimen are unknown.
The use of FFPE tissue that has been handled improperly may not generate data or
may yield data that are representative of the FFPE processing conditions applied
rather than the biospecimen donor’s disease condition
Parameters must be recorded (από μένα)
When more than 1 analyte is to be investigated using the same FFPE specimen, the
more stringent conditions (Table 2) should be used as a guideline.
Human FFPE tissue specimens of 3 mm3 in size, subjected to a cold ischemia time of
less than 1 hour and were preserved in NBF for 8 to 24 hours at room temperature or
4ºC should yield acceptable DNA, RNA, protein, and morphology data if blocks are
stored for less than 1 year and if slide-mounted tissue sections are stored for less than
1 week
Additional sources of preanalytical variability, including extraction methods, antigen
retrieval techniques, and uninvestigated variables, may serve as additional
confounding variables
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Preanalytical Factors Affecting FFPE Tissue—Bass et al, Arch Pathol Lab Med—Vol 138, November 2014
Parameters can influence analysis of nucleic acids, proteins, and
morphology in different ways and to different extents.
Table 2. Acceptable Thresholds for Preanalytical Factors Based on Literature
Evidence for Specific Analytes
Preanalytical Factor DNA RNA Protein Morphology
Cold ischemia<1 h for FISH,
≤24 h for PCR
<12 h <12 h <6 h
Specimen size3–10 mm3 Evidence not
available
1.2–3.5 mm3 Evidence not available
Decalcification
EDTA Ultrasound or
EDTA
Thresholds are tissue
and antigen specific
Ultrasound; EDTA, nitric,
formic or acetic acid,
DECAL, Cal-Exc, D-
calcifier, Plank-Rychlo,
Ebner’s, or Jenkin’s solution
Fixative buffer NBF NBF NBF Evidence not available
Fixation duration <72 h 8–48 h 6–24 h <1 y
Fixation temperature 4ºC or ambient 4ºC or ambient 4ºC or ambient Evidence not available
FFPE block size or
section thickness
Whole sections
preferable to cores
or isolated nuclei
Evidence not
available
2–4 μm 2–3 μm
Duration of paraffin
block storage
≤5 y ≤1 y ≤ 25 y for IHC,
<10 y for platforms
requiring protein
extraction
Evidence insufficient
Tissue section storageInsufficient
evidence
<3 mo at
ambient
<1 wk Evidence not available
Standardization of pre-analytical data elements
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Weigh the benefits versus the consequences of using archival FFPE tissue when the
handling, fixation, processing, and storage parameters for a specimen are unknown.
The use of FFPE tissue that has been handled improperly may not generate data or may yield
data that are representative of the FFPE processing conditions applied
rather than the biospecimen donor’s disease condition
When more than 1 analyte is to be investigated using the same FFPE specimen, the more
stringent conditions should be used as a guideline
Preanalytical Factors Affecting FFPE Tissue—Bass et al, Arch Pathol Lab Med—Vol 138,
November 2014
By priority of their importance, 170 preanalytic variables were ranked in 5 categories:
a) variables common to all biospecimens = 102
b) tissue-specific variables = 36
c) blood, serum and plasma specific variables = 20
d) body fluid variables = 6
e) nucleic acid variables = 16
Standardize Preanalytic Data Elements—Robb et al., Arch Pathol Lab Med—Vol 138, April 2014
Table 2. Acceptable Thresholds for Preanalytical Factors Based on Literature
Evidence for Specific Analytes
Preanalytical Factor DNA RNA Protein Morphology
Cold ischemia<1 h for FISH,
≤24 h for PCR
<12 h <12 h <6 h
Specimen size3–10 mm3 Evidence not
available
1.2–3.5 mm3 Evidence not available
Decalcification
EDTA Ultrasound or
EDTA
Thresholds are tissue
and antigen specific
Ultrasound; EDTA, nitric,
formic or acetic acid,
DECAL, Cal-Exc, D-
calcifier, Plank-Rychlo,
Ebner’s, or Jenkin’s solution
Fixative buffer NBF NBF NBF Evidence not available
Fixation duration <72 h 8–48 h 6–24 h <1 y
Fixation temperature 4ºC or ambient 4ºC or ambient 4ºC or ambient Evidence not available
FFPE block size or
section thickness
Whole sections
preferable to cores
or isolated nuclei
Evidence not
available
2–4 μm 2–3 μm
Duration of paraffin
block storage
≤5 y ≤1 y ≤ 25 y for IHC,
<10 y for platforms
requiring protein
extraction
Evidence insufficient
Tissue section storageInsufficient
evidence
<3 mo at
ambient
<1 wk Evidence not available
Standardization of pre-analytical data elements
22/4/2017 30
Standardize Preanalytic Data Elements—Robb et al, Arch Pathol Lab Med—Vol 138, April 2014
Weigh the benefits versus the consequences of using archival FFPE tissue when the
handling, fixation, processing, and storage parameters for a specimen are unknown.
The use of FFPE tissue that has been handled improperly may not generate data or
may yield data that are representative of the FFPE processing conditions applied
rather than the biospecimen donor’s disease condition
Parameters must be recorded (από μένα)
By priority of their importance, 170 preanalytic variables were ranked in 5 categories:
a) variables common to all biospecimens = 102
b) tissue-specific variables = 36
c) blood, serum and plasma specific variables = 20
d) body fluid variables = 6
e) nucleic acid variables = 16
Additional sources of preanalytical variability, including extraction methods, antigen
retrieval techniques, and uninvestigated variables, may serve as additional
confounding variables
Ιστός για ALK Testing (χειρισμός – περιορισμοί)
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I. Βιοπτικό ιστολογικό υλικό και κυτταρολογικό δείγμα εξίσου κατάλληλα για έλεγχο
κατάστασης ALK
II. Απαιτείται κατάλληλη Επεξεργασία και Χειρισμός υλικού
III. Το δείγμα πρέπει να περιέχει επαρκή αριθμό νεοπλασματικών κυττάρων
I. Αριθμός νεοπλασματικών κυττάρων για ανοσοϊστοχημική (IHC) εκτίμηση της ALK
πρωτεΐνης παραμένει αδιευκρίνιστος
II. Για FISH ανάλυση κατ’ ελάχιστο 50 εκτιμήσιμα νεοπλασματικά κύτταρα
IV. Καταλληλότερη προσέγγιση για κυτταρολογικά δείγματα η παρασκευή cell block και στη
συνέχεια λήψη τομών οι οποίες θα τύχουν διαχείρισης ιστολογικών τομών
V. ΟΛΟ το ιστολογικό και κυτταρολογικό υλικό θα πρέπει να υποβληθεί σε επεξεργασία
I. Όγκοι διαμέτρου ≤ 3cm in toto
II. Μεγάλες πλευριτικές συλλογές = μερική επεξεργασία
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Size Matters
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Ιεράρχηση της εκτέλεσης διαγνωστικών και
προβλεπτικών δοκιμασιών
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Επαναληπτική βιοψία αδύνατη στο 20%, ανεπαρκές υλικό στο 26% των
περιπτώσεων.
Επωφελής για την καθοδήγηση της θεραπείας στο 30% των περιπτώσεων
Park S, Holmes-Tisch AJ, Cho EY et al. Discordance of molecular biomarkers associated with epidermal
growth factor receptor pathway between primary tumors and lymph node metastasis in non-small cell lung
cancer. J. Thorac. Oncol. 2009; 4; 809–815
Cree I.A. et al. PD-L1 testing for lung cancer in the UK: recognizing the
challenges for implementation. (2016) Histopathology 69, 177–186
Διασφάλιση Ποιότητας στο Παθολογοανατομικό Εργαστήριο
The histopathological diagnosis is not a laboratory test, but individual
medical art
Glen Christiansen: Molecular pathology of prostate cancer: Where are we now?,
ECP 2013, Lisbon
Virchows Arch (2016)
“In God we trust, all others bring data”
William Edwards Deming (1900 –1993)
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How do you standardize Artists ??
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