ΕΛΕΥΘΕΡΙΟΣ ΕΛΕΥΘΕΡΙΑΔΗΣ MD, PhD, MBA · Table 1. FFPE-Related Preanalytical...

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



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


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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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




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Parameters can influence analysis of nucleic acids, proteins, and

morphology in different ways and to different extents.





≤24 h for PCR

<12 h <12 h <6 h


available


Decalcification

EDTA Ultrasound or

EDTA





DECAL, Cal-Exc, D-






FFPE block size or

section thickness

Whole sections

preferable to cores

or isolated nuclei

Evidence not

available

2–4 μm 2–3 μm


block storage

≤5 y ≤1 y ≤ 25 y for IHC,

<10 y for platforms

requiring protein

extraction



evidence

<3 mo at

ambient



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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


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







Standardize Preanalytic Data Elements—Robb et al., Arch Pathol Lab Med—Vol 138, April 2014





≤24 h for PCR

<12 h <12 h <6 h


available


Decalcification

EDTA Ultrasound or

EDTA





DECAL, Cal-Exc, D-






FFPE block size or

section thickness

Whole sections

preferable to cores

or isolated nuclei

Evidence not

available

2–4 μm 2–3 μm


block storage

≤5 y ≤1 y ≤ 25 y for IHC,

<10 y for platforms

requiring protein

extraction



evidence

<3 mo at

ambient



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Standardize Preanalytic Data Elements—Robb et al, Arch Pathol Lab Med—Vol 138, April 2014
















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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