Ancillary techniques in breast FNAC - Ancillary techniques in the...Advantages and disadvantages direct smears •Limited number of spare slides •Few AB’s can be investigated •Unwanted

Ancillary techniques in breast FNAC:

Immunocytochemistry and in situ hybridization for determination of routine diagnostic and predictive markers

Immunocytochemistry

• General ICC issues

• Determination of prognostic and predictive markers

– ER/PgR

– HER-2

– Ki-67

• Predictive ILC marker E-cadherin

• Diagnostic markers

• QC

• Surrogate markers for genetic subtypes

Torill Sauer, Akershus University Hospital, Norway

E-cadherin

What kind of material/preparations do we use

• Cell block

• Direct smears

– air dried

– In CO2 ice

– wet fixed

• Liquid based preparations


Cell block

• Main disadvantage availability of enough material

• Main advantages

– very similar to histology in protocol for fixation, embedding, AB concentration and evaluation of immunostaining

– Possible to cut several sections an investigate multiple AB

• Cellient

• Histogel

• Thrombin, blood clot or equivalent

• Other


ER

Direct smears

• Wet fixed in ethanol

• Air dried and post fixed

– Ethanol

– Methanol

– Aceton

– Formalin

– Sequential fixation


synaptophysin

“Specific” but disturbing background

staining due to disruption of cells

resulting in cytoplasmic content lying

as a granular debris in the background

Advantages and disadvantages direct smears

• Limited number of spare slides

• Few AB’s can be investigated

• Unwanted background staining due to necrosis of tumour cells

• Insufficient blocking of endogen peroxidase in RBC

• “specific” but disturbing staining of disrupted cytoplasmic content

• Both air dried and wet fixed unstained smears can be used (but require different protocols!)

• Prestained smears may be used, but QC with controls problematic

– Ethanol fixed smears may be stained with the same protocol as histology in most cases


CK7

Liquid based preparations (LBC= ThinPrep: Cytolyt + Preservcyt)

• Commercial

– Surepath

– ThinPrep

– others

• Home made

• Type of fixative in LBC

– Ethanol

– Methanol

– Formalin

– Others

• Physiological saline with/without calf serum or equivalent medium


Advantages (and disadvantages) of LBC based ICC

Cytolytic agent in the fluid will remove the RBC

Transfer from preliminary fixative with cytolytic agent (Cytolyt) to long term fixative (Preservcyt)

Removes practically all unwanted background that might disturb the interpretation of ICC

Possible to make several preparations (cytospin or other method), but restricted when material is limited

Long time storage of preparations and residual material in liquid for weeks and months (-40C, -200C and -800C, respectively) without loss of anticenicity

– Liquid based material from fine needle aspirates from breast carcinomas offers the possibility of long-time storage without significant loss of immunoreactivity of estrogen and progesterone receptors. Torill Sauer, Kristin Ebeltoft, Mette Kristin Pedersen, Rolf Kåresen Cytojournal 2010

• Stored, residual material suitable as positive/negative controls


Specimen handling prior to ICC from LBC

• Direct smears for morphological diagnotics

• Aspirated material is rinsed in Cytolyt or equivalent. This ensures a mild but adequate fixation and hemolysis.

• Fixation for at least 2-3 hrs, overnight fixation is OK

• Centrifugation of liquid cell material; remove supernatant and immerse the rest in Preservcyt (or equivalent)

• Cells are left in Preservcyt until the day of ICC and cytospins or equivalent are prepared shortly before immunostaining


ICC of prognostic and predictive markers

• ER, PgR, Ki-67 and HER-2/neu status are strong prognostic and predictive markers and thus are routine markers in breast carcinomas.

• There is a general recommendation to do ER/PgR preoperatively, evt also HER-2

• General recommendation to repeat ER/PgR and HER-2 in distant metastases and locoregional recurrences

• FNAC may be used for investigating all of them, both as part of the primary preoperative work-up and when diagnosing recurrences and metastases.

– Kocjan G. The role of breast FNAC in diagnosis and clinical management: a survey of current practice. Cytopathology 2008;19:271-8

– Marinsek et al. Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates. Cytopathology 2013; 24(1):7-20

– ASCO/CAP guidelines for ER/PgR and HER-2 testing


ASCO/CAP guidelines for ER/PgR and HER-2 testing

• “If laboratories choose to use alternative fixatives other than buffered formalin, the laboratory is obligated to validate that fixative’s performance against the results of testing of the same samples fixed also in buffered formalin and tested with the identical ER/PgR/HER2 assay, and concordance in this situation must also be 95%.”


CAP FAQ website

• “Do the guidelines exclude HER2/ER/PgR testing of cytology specimens (fluids and aspirates) that have been fixed in 95% ethanol rather than formalin?”

• “Since cytology specimens are not ordinarily fixed in formalin, that sentence is not directly applicable, but labs performing ER/PgR/HER2 testing on such specimens must document that they validated their methods and achieved acceptable concordance, in this case by comparing staining of alcohol fixed cytology specimens with routinely processed (formalin-fixed) tissue sections”.


ICC QC of ER/PgR, Ki-67 and HER-2/neu

• In histology, all of these are subject to rigorous quality control and standardizing.

• Is it possible to apply QC to ER/PgR, Ki-67 and HER-2/neu ICC?

• How do we obtain material for positive and negative controls?

• Some argue that immunocytochemistry (ICC) is more unreliable and unpredictable than immunhistochemistry (IHC)

– due to lack of QC and standardizing

– In a routine setting, some have found that a number ICC ER and/or PgR negative cases are positive on IHC

– Some ICC HER-2 3+ cases turn out negative on IHC


Ki-67


As to FN results of ER/PgR

• Do not let the

slides dry out

at any time (during pre-staining and

staining procedure) =

• From the ER/PgR

datasheet/information from

Abbott


What happens when we air dry the smears?

• Ruptures in the cytoplasmic membrane

• Ruptures in the nuclear ”membrane”

• Do we also cause ruptures and changes in the

tertiary structure of large molecules as f.ex.

ER/PgR?

– Random sites

– Predilection sites?

– Irreversible


We do not know for sure what causes the ICC/IHC discrepancy, but

• Probably the old Abbott recommendation is still good

advice:

• So, whatever procedure you use for ER/PgR ICC:

– ” Do not let the slides dry out at any

time ”

Use LBC to minimize FN ER/PgR

The cytological literature favour ER/PgR ICC based on cell material in liquid suspensions

Methanol based fixative seems more effective than ethanol for investigating steroid receptors

Concordance between cytology and histology generally good

ER/PgR to have a very robust cut off as to positivity/negativity

Evaluate percentage of positive tumour cell nuclei

Staining intensity irrelevant


HER-2 ICC

• must be validated against the histological protocol and have a concordance of at least 95 % with the histological results

• May at least be relevant in a metastatic setting

• Cut off in IHC subtle and difficult to reproduce

• The cytological literature shows that it is possible using cell blocks


HER-2/neu ICC

• Standardised kits and protocols are for IHC

• In-house optimization of all steps in handling and ICC staining is crucial

– Direct smear/liquid based preparation

– Air drying vs wet fixation

– Fixation/postfixation

– Length of fixation

– Demasking

– Concentration of primary antibody

– Incubation time

– Type of buffer

– Counterstain


HER-2/neu in situ hybridisation

• Cytological material is ideal! No capping of nuclei

– FISH

– CISH

– SISH

• As in ICC: all handling and protocols must be optimized

– Direct smears: air dried or wet fixed

– (Liquid based preparations)

• Standardised, marketed FISH HER-2 kits and protocols identical for cytology and histology

• CISH and SISH need optimation



Dual FISH Dual CISH


Fixation for ISH

• Primary fixation

• Methanol/acetic acid

• Methanol

– Formalin

• Postfixation

– Formalin!!!

Crucial steps in CISH/SISH protocol

• Pretreatment/demasking +/-

– Is an advantage when using formalin fixation

• Length of pretreatment, concentration of pretreatment enzyme

• Posthybridisation washing

• Type of buffer


CISH


How do we report our results?

• As in histology

• Use same cut-off as on histological specimens

• Single probe vs dual probe

• 2/2 normal

• Non-countable clusters of the HER-2 gene = amplified

(small and large clusters)

• Borderline counts

• Polysomy

• Give ratio in dual probes

• Very good correlation between cytological and histological

results (practically 100 % as to amplified or not amplified)

ICC of Ki-67

• An easy marker!?

• Can be determined on

– Direct smears

– Prestained smears

– LBC preparations

• No specific technical problems

• IHC interpretation is problematic, no universal agreement

• ICC validation and optimation a problem due to non-agreement of how/where to count and cut offs


Preoperative diagnosis of ILC

• Might trigger a rapid IHC on frozen section to detect metastasis in a sentinel node

• A definite cytomorphological distinction of ductal and lobular carcinoma is difficult

• E-cadherin can be used on preoperative breast FNAC specimens (direct smears, pre-stained smears and from LBC)

• Immunophenotype of ILC

– ER/PgR positive

– E-cadherin negative


E-cadherin

• a cell adhesion molecule that forms complexes with β -, Υ -, α -catenins and p120 catenin

• expression and mutations in the E-cadherin gene, CDH1 , have shown a relationship between LCIS and invasive lobular carcinoma

• gene located at 16q21.1

• The CDH1 gene is inactivated in both in situ and invasive lobular carcinomas by genetic (inactivating mutations of deletions) or epigenetic (promoter methylation) mechanisms.

• Loss of E-cadherin-mediated cell adhesion is believed to account for the dyshesive nature of lobular neoplasia and invasive lobular carcinoma


Diagnostic immunocytochemistry

• Presence/lack of myoepithelial markers in papillary tumours

– p63, CK5/6

• Markers for metaplastic carcinomas/carcinosarcomas and sarcomas

– vimentin

– myoepithelial markers (p63, CK5/6)

– HMW markers (squamous differentiation)

– endothelial markers

• Neuroendocrine differentiation in breast carcinomas

– chromogranin

– synaptophysin

• Metastases

– MM

– Carcinomas from lung, GI tract, genitalia interna etc


panMel

Is it possible to apply QC to ICC?


Material for positive and negative controls

• Breast cancer cell lines (MCF-7, SKBR and others) with known ER/PgR, Ki-67 and HER-2 status and other markers

• Spare direct slides, air dried or wet fixed (stored at -700C) with carcinoma cells with known Ki-67 and HER-2 status as well as other markers

• Slides made from liquid suspensions (stored at -700C) with carcinoma cells with known ER/PgR, Ki-67 (and HER-2 status)

• Residual, stored liquid suspensions with carcinoma cells with known ER/PgR, Ki-67 (and HER-2 status) as well as other markers

• Tumour cell preparations and ICC procedure that omit the primary antibody as negative control


Common causes of FN and FP findings in ICC

• Misinterpretation of neoplastic cell population

• Insufficient peroxidase blocking

• Necrotic cells

• Dried preparations during staining procedure

• AB cross reactivity, AB not specific

• Nonspecific AB binding

• Inappropriate fixation (too short- prolonged formaline fixation)

• Antigen diffusion

• AB concentration to low/high


ER false negative

Internal/intralaboratory QC of immunocytochemical (ICC) determination of markers in breast FNAC

– Adequate cell material with known positive controls

• Cell lines

• Direct smears

• Cell suspensions

– Xtra slide for negative control, omitting primary AB

– If using cell block, QC as in IHC: parafin embedded histological sections of tumour tissue

– Use of prestaining smears for ICC should be an exception, an emergency procedure

– Repeat all ER/PgR ICC negative cases on histology

– keep a record of discrepant cases

– Standard for maximum discrepant cases?


External/interlaboratory QC in immunocytochemistry (ICC)

– Circulation of smears/slides from liquid suspensions containing cells with known immunophenotype

• Breast cancer cell lines

• Cancer cells from actual patients (FNAC material)

– Marinsek et al. Multinational study of oestrogen and progesterone receptor immunocytochemistry on breast carcinoma fine needle aspirates. Cytopathology 2013; 24(1):7-20

• Scrape material from the cut surface of fresh, unfixed breast carcinoma specimens?

– Large volume laboratories must contribute appropriate cell material

– Optimal storage conditions for control material, both suspensions and slides:

• How long before the reactivity is significantly reduced?

• Optimal storage temperature (RT? -20oC? -80oC?)


Surrogate markers for genetic subtypes

•Luminal A – ER/PgR positive

– HER-2 negative

– Ki-67 low proliferation index

•Luminal B – ER/PgR positive

– HER-2 +/-

– Ki-67 intermediate proliferation index

•HER-2 type – ER/PgR negative

– HER-2 positive

– Ki-67 high proliferation index

•Basal cell type – Triple negative (ER, PgR, HER-2)

– Ki-67 high proliferation index

– Myoepithelial markers positive (some cases) • EGFR positive

• CK 5/6

• CK14

• p63


Conclusions

1. ER/PgR, Ki-67 and HER-2 status on cytological material must be optimized and validated against histology and the results will have treatment consequences

2. Postfixation in formaline is recommended

3. Use liquid based material!!!!! QC (except for HER-2)

4. Direct, air dried smear for in situ hybridisation recommended for HER-2 status and with formaline as primary fixation

5. ASCO/CAP guidelines for ER/PgR and HER-2 recommends to repeat ER/PgR and HER-2 in distant metastases (and then we have a good reason to have an optimal procedure and protocol for cytological preparations and be able to do it in metastatic settings and maybe also preoperatively)

6. E-cadherin for preoperative diagnosis of ILC useful

7. Diagnostic markers (f.ex. myoepithelial and neuroendocrine markers) useful in specific cases, procedures most always be optimized

8. QC as in IHC; control material is the challenge



Thank you for your attention!

Protocols for ICC and SISH on air dried and Giemsa stained smears

• Elsa Beraki, Thale Kristin Olsen, Torill Sauer (2012) Establishing a protocol for immunocytochemical staining and chromogenic in situ hybridization of Giemsa and Diff-Quick prestained cytological smears Cytojournal 9: 8.

– Protocols for immunocytochemical staining (ICC) and in situ hybridization (ISH) of air-dried Diff-Quick or May-Grünwald

Giemsa (MGG)-stained smears have been difficult to establish. An increasing need to be able to use prestained slides for ICC and ISH in specific cases led to this study, aiming at finding a robust protocol for both methods. Materials and Methods: The material consisted of MGG- and Diff-Quick-stained smears. After diagnosis, one to two diagnostic smears were stored in the department. Any additional smear(s) containing diagnostic material were used for this study. The majority were fine needle aspirates (FNAC) from the breast, comprising materials from fibroadenomas, fibrocystic disease, and carcinomas. A few were metastatic lesions (carcinomas and malignant melanomas). There were 64 prestained smears. Ten smears were Diff-Quick stained, and 54 were MGG stained. The antibodies used for testing ICC were Ki-67, ER, and PgR, CK MNF116 (pancytokeratin) and E-cadherin. HER-2 Dual SISH was used to test ISH. Citrate, TRS, and TE buffers at pH6 and pH9 were tested, as well as, different heating times, microwave powers, and antibody concentrations. The ICC was done on the Dako Autostainer (Dako , Glostrup, Denmark), and HER-2 Dual SISH was done on the Ventana XT-machine (Ventana / Roche®, Strasbourg, France). Results: Optimal results were obtained with the TE buffer at pH 9, for both ICC and ISH. Antibody concentrations generally had to be higher than in the immunohistochemistry (IHC). The optimal microwave heat treatment included an initial high power boiling followed by low power boiling. No post fixation was necessary for ICC, whereas, 20 minutes post fixation in formalin (4%) was necessary for ISH. Conclusions: Microwave heat treatment, with initial boiling at high power followed by boiling at low power and TE buffer at pH 9 were the key steps in the procedure. Antibody concentrations has to be adapted for each ICC marker. Post fixation in formalin is necessary for ISH.


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Ancillary techniques in breast FNAC - Ancillary techniques in the...Advantages and disadvantages direct smears •Limited number of spare slides •Few AB’s can be investigated •Unwanted