05/07/2018

1

Maastricht Pathology 2018June 19-22 Maastricht

The Netherlands

Thyroid Cytology in the MDT

setting

R.Dina MD, FIAC, FRCPathDept of HistopathologyHammersmith Hospital

Thyroid CancerIntroduction

♦ The incidence of thyroid cancer appears to be increasing slowly.

♦ In the period 1971-1995 the annual UK incidence was reported at 2.3 per 100,000 women and 0.9 per 100,000 men, with approximately 900 new cases and 250 deaths recorded in England and Wales due to thyroid cancer every year.

♦ In 2006 data from Cancer Research UK reported 1,933 new cases in the UK, with an annual incidence of 4.4 per 100,000 women and 1.6 per 100,000 men. Thyroid cancer is the commonest malignant endocrine tumour, but represents only about 1% of all malignancies.

05/07/2018

2

Thyroid Cancer

♦ 5-10% of the adult population develop a palpable thyroid nodule

♦ 5% of nodules are malignant

♦ Increasing in incidence?(increased sensitivity of tests and the higher detection rate, rather than a true change in incidence)

CLINICAL PATHWAYPRIMARY CARE:URGENT REFERRAL

♦ Symptomatic patient GPGPs may wish to arrange blood tests for TFTs, Corrected Calcium - but should not wait for the results before

making a referral if an urgent referral is indicated as set out below:

♦ All patients with Thyroid Swellings and any of:

Enlarging thyroid lumpThyroid lump in patient >65 or<18 years of agePrevious neck irradiationAssociated cervical lymphadenopathyVoice change / hoarsenessFamily history thyroid cancer

Thyroid Swelling AND Stridor

Referral to the Regional Thyroid MDT is required for all

patients with

♦ • Newly diagnosed thyroid cancer (Thy5) or suspicious thyroid cancer (Thy4) on FNA cytology.

♦ • Atypical follicular/equivocal cytology (Thy3)

♦ • Histological diagnoses of thyroid cancer

♦ • Patients with inherited thyroid cancer syndromes

♦ • Recurrent Cancer♦ • All Paediatric Thyroid Lumps

♦ The Thy classification adopted by the Royal College of Physicians (http://www.british-thyroid-association.org/Guidelines/)

� Thy1: Non-diagnostic (inadequate or where technical artefact precludes interpretation; smears must contain 6 or more groups of at least 10 thyroid follicular cells to be considered adequate).Thy2: Non-neoplastic (features consistent with a nodular goitre or thyroiditis or cysts).Thy3 (i): All follicular lesions.(ii): There may be a very small number of cases where the cytological findings warrant inclusion in this category rather than Thy2 or Thy4. This will be indicated in the report.Thy4: Abnormal, suspicious of malignancy (suspicious, but not diagnostic, of papillary, medullary or anaplastic carcinoma or of lymphoma).Thy5: Diagnostic of malignancy (unequivocal features of papillary, medullary or anaplastic carcinoma, or of lymphoma or of metastatic tumour).

05/07/2018

3

The Thy classification adopted by the Royal College of Physicians

♦ Blood only♦ Only few bare nuclei

without evidence of origin from a cyst (macrophages colloid,fluid)

♦ Technical artefacts

� Thy1: Non-diagnostic (inadequate or where technical artefact precludes interpretation; smears must contain 6 or more groups of at least 10 thyroid follicular cells to be considered adequate).Action: FNAC should be repeated with or without ultrasound guidance.

Diagnostic pitfalls

♦ Adequacy of material (skills of the aspirator, cellularity of the lesion and target)

♦ Overlapping morphologic features

Thyroid cytology coming from other Institutions

♦ First pass(es): conventional smears (alcohol and air dried (1:1)

♦ Rest of material: LBC

♦ Rationale: avoid excessive No of blood stained smears

Unsatisfactory rate♦ With experience, the average

“unsatisfactory” rate is about 5% to 10%. It is incorrect to consider unsatisfactory or non diagnostic tests as negative.

♦ The lowest rate will be achieved by close cooperation between the clinician and the cytopathologist, with immediate assessment of material (Clinical Departments must allocate resources to Pathology!)

05/07/2018

4

Criteria of Adequacy♦ At least 6 groups of

follicular cells, each with approx. 10 cells

♦ Risk of malignancy is similar to diagnostic, benign samples (Thy2)

Unsatisfactory rate♦ The number of inadequates appears to be directly

related to the immediate assessment by a cytologist rather than by a clinician or a cytopathologist performing the procedure, experience being a more important determinant in the success of a thyroid FNA (3).

♦ Nasuti JF, Gupta PK, Baloch ZW. Diagnostic value and cost-effectiveness of on-site evaluation of fine-needle aspiration specimens: review of 5,688 cases.Diagn Cytopathol. 2002 Jul;27(1):1-4

Crucial factors in the interpretation of scanty cellular FNAs

♦ The availability of complete clinical information as more often clinically benign lesions are the ones which yield fewer cells.

♦ Identification of background colloid ♦ Bare thyroid nuclei (not lymphocytes!)♦ Pigmented macrophages

How to improve♦ The nonaspiration technique and use of smaller

needles (25 g) has been advocated to reduce blood contamination

♦ It probably decreases the incidence of WHAFFTs♦ Cell blocks prepared from FNA biopsies have also

been advocated to increase diagnostic accuracy .

♦ Kamal MM, Arjune DG, Kulkami HR. Comparative study of fine needle aspiration and fine needle capillary sampling of thyroid lesions.Acta Cytol. 2002 Jan-Feb;46(1):30-4.

05/07/2018

5

Baskin HJ. Ultrasound-guided fine-needle aspiration biopsy of thyroid nodules and multinodular goiters. Endocr Pract. 2004 May-Jun;10(3):242-245.

♦ Analysis of inconclusive fine-needle aspiration of thyroid follicular lesions suggests a malignancy rate of 2.2%, which is no worse than patients with a benign preoperative diagnosis.

♦ A balanced approach with careful follow-up for nondiagnostic cytology is prudent

The Thy classification adopted by the Royal College of Physicians

� Thy2: Non-neoplastic (features consistent with a nodular goitre or thyroiditis).Action: Two diagnostic benign results 3-6 months apart required to exclude neoplasia. Decision to intervene surgically dependent on clinical factors (e.g. clinical suspicion and the presence of manifestations causing physical or psychological distress i.e. breathing difficulties or unfavourable cosmetic appearance).

The Thy classification adopted by the Royal College of Physicians

� Thy3 (i): All follicular lesions.Action: Lobectomy. Complete thyroidectomy will be necessary if histology proves malignant.(ii): There may be a very small number of cases where the cytological findings warrant inclusion in this category rather than Thy2 or Thy4. This will be indicated in the report.Action: Discuss with cytopathologist to determine course of action.

Thy 3f (follicular neoplasm)♦Hypercellular♦Microfollicular arrangement♦Scanty colloid♦Regular nuclear outlines♦Fine chromatin♦Fragile cytoplasm♦No nuclear pseudoinclusions

05/07/2018

6

♦Thy3a: cytological/nuclear or architectural atypia, or other features that raise the possibility of neoplasia, but which are insufficient to enable confident placement into any other category.

Thy3 a and Thy3f subcategories

The Thy classification adopted by the Royal College of Physicians

♦ Thy4: Abnormal, suspicious of malignancy (suspicious, but not diagnostic, of papillary, medullary or anaplastic carcinoma or of lymphoma).Action: Surgical intervention indicated for differentiated tumour. Further treatment dependent upon pathology report. For lymphoma, metastatic tumour or undifferentiated i.e. anaplastic thyroid carcinoma, further investigation indicated.

H.R. 19-05-08

05/07/2018

7

♦ Highly cellular aspirate containing numerous clusters and sheets of follicular cells showing nuclear crowding;

♦ Nuclear grooving and pseudoinclusions are occasionally identified

♦ The epithelial fragments are seen within a background of moderate fluid colloid and mixed inflammatory cells.Therefore this is a Thy 4

The Thy classification adopted by the Royal College of Physicians

♦ Thy5: Diagnostic of malignancy (unequivocal features of papillary, medullary or anaplastic carcinoma, or of lymphoma or of metastatic tumour).Action: Surgical intervention indicated for differentiated thyroid cancer, depending on tumour size, clinical stage and other risk factors such as gender and extremes of age. Appropriate further investigation indicated alongside radiotherapy/chemotherapy for anaplastic carcinoma, lymphoma or metastatic tumor.

What are the proportions of patients in each Thy classification?

05/07/2018

8

The distribution of Thy categories in 2014

Histology 118

Which are the factors that may influence cyto-histo correlations?• The operator• The cytopathologist• The MDT

Reproducibility of Thy3 category in FNA of the Thyroid and the effect of MDT discussion on the management of Thy3 cases in a Multilaboratory setting

Thy1, 9%

Thy2, 75%

Thy3, 9%

Thy4, 3% Thy5, 4%

Total=2157

All Thy3-5 cases were discussed at the Central MDTMeeting, although the two labs (HH and SMH) were still independently reporting.The guidelines followed were the same.

Thy1,14%

Thy2,65%

Thy313%

Thy4,3%

Thy5,5%

St Mary's

Thy1,6%

Thy2,80%

Thy3,6%

Thy4,4%

Thy5,4%

HH

ResultsHH Malignanc

yBenign PPV = M/(M+B)

Thy3 11 25 30.6%Thy4 32 8 80%Thy5 23 1 95.8%SMHThy3 9 33 21.4%Thy4 2 2 50%Thy5 16 1 94.1%HH+SM

HThy3 20 58 25.6%Thy4 34 10 77%Thy5 39 2 95.1%

05/07/2018

9

Conclusions• In 2008 the proportion of Thy 3 cases, after almost 2 years of

joint MDTs,changed (SM 13% vs 14%, HH 8%vs 5%) while PPV is more divergent.

• This is associated with a significant difference in the proportion of Thy3 cases receiving surgery in the two groups (HH=59%,SM=36%).

• MDT discussion of Thy3 cases influences significantly the management and outcome of such patients.

What is the positive predictive value of Thy3-5 categories?

How many patients have cyto-histo correlation?

36 3752

89

4747

125

84

99

2015 2016 2017

Total 308 cases

SMH HH TOTAL

2015-2017 cyto-histological correlations(340 cases)

69

90

107

20

54

THY1 THY2 THY3 THY4 THY5

Chart Title

05/07/2018

10

Distribution of cases 2015-17

20%

27%31%

6%

16%

CHART TITLE

THY1 THY2 THY3 THY4 THY5

Correlated cases 2015-17

Proportion of malignancy

♦THY5 100%♦THY4 100%♦THY3 25.3 %♦THY2 13.3%

Review of false negatives (12 cases-11 patients)

05/07/2018

11

Conclusions

♦ 50% of false negatives are associated with incidental microcarcinoma

♦ 25% are “true” false negatives (no cytological criteria of suspicion or malignancy)

♦ 17.5% are sampling errors (not representative of the nodule)

♦ 7.5% is an interpretation error

Progress in Identifying Driver Mutations in Thyroid Cancer

Ref Nikiforov et al 2015

The Cancer Genome Atlas Project

• Proposes “a reclassification of thyroid cancers into molecular subtypes that better reflect their underlying signalling and differentiation properties, which has the potential to improve their pathological classification andbetter inform the management of the disease”.

Our study• Large number of point mutations seen in BRAF gene but the most

common one is the T → A transversion at base 1799 of exon 15 (valine to glutamic acid at position 600)

• This transversion is an important oncogenic mutation in PTC and occurs roughly in 45% of cases

• A molecular diagnostic test for the BRAF Val600Glu mutation could help improve accuracy in difficult to determine thyroid nodules

• Aim: compare Sanger sequencing, pyrosequencing and next generation sequencing for the detection of the BRAF Val600Glu mutation

05/07/2018

12

Discussion (1)• Molecular testing is nowadays becoming a routine approach for

the diagnosis of several disorders and the identification of biomarkers an integral tool in patients’ management

• Quality and quantity of material isolated from diagnostic samples is of vital importance.

• In this study, the frequency and the amount of DNA extracted from LBC-FNAs was consistently higher than that of smear-FNAs. However the quality of the material was equally comparable as shown by the complete concordance in results irrespective of the origin of DNA

• From our study we would recommend that LBC-FNAs, if available, are preferably used for molecular testing because DNA

Advantages, disadvantages and cost analysis of Sanger sequencing, pyrosequencing and Next Generation Sequencing in our lab

Sequencing method Advantages Disadvantages Cost analysis

Sanger -Relatively low cost/sample

-Very stable technology

-Relatively low sensitivity

-Non quantitative

-EC: £50,0001-£160,0002

-PT: 10-12hrs

-CPS: £253

Pyro -Relatively low cost/sample

-Quantitative

-Sensitive

-Quick

(single working day)

-Detects single type of mutation

-Sensitive to background noise

-Foreknowledge of mutation

-EC: £85,000

-PT: 8-10hrs

-CPS: £15-203

NGS -Small amount of DNA required

-Quantitative

-Very sensitive

-Simultaneous analysis of multiple cancer

hotspots

-High cost/sample

-Takes up to three days

-Requires sufficient knowledge of bioinformatics

-Requires a lot of hands-on time

-Requires specialist training

-EC: £80,000

-PT: 3 days

-CPS: £200

EC: equipment cost; PT: personnel time; CPS: cost per sample; 1: Applied Biosystem’s 3130 sequence analyser; 2: Applied Biosystem’s 3500xL sequence analyser; 3: includes cost of PCR reaction

Discussion (3)• The British Thyroid Association has produced draft guidance on

differentiated thyroid cancer that includes discussion of BRAF p.Val600Glu testing in thyroid FNA cytology, stating that “molecular analysis (e.g. BRAF p.Val600Glu mutation for PTC, alone or as part of a panel) is an emerging field and may refine the prediction of both benignity and malignancy in thyroid cytology samples”

• Overall NGS can detect other mutations that a single gene analysis does not provide.

• NGS may in the future provide a plethora of additional information and a more comprehensive set of data to guide treatment choice in the context of personalised medicine.

From Darkness into Light

No molecular marker Many molecular markersOne molecular marker

05/07/2018

13

The Thyroid Nodule

♦ Congenital anomalies– Thyroglossal duct cyst

♦ Inflammatory Lesions– Inflammatory foci– Compensatory regenerative nodules (?TSH, ?TGI)

♦ Hyperplasia♦ Neoplasia

– Benign– Malignant

What to look for♦ Type of cells

(thyrocytes,macrophages, lymphocytes etc)

♦ Amount and type of colloid vs cellularity

♦ Bare nuclei♦ Architecture

Review of discordant casesCriteria♦ Architecture (monolayers, crowded clusters)♦ Cellularity (scanty,moderate,marked)♦ Colloid (scanty-abundant,fluid-dense)♦ Pseudoinclusions (absent,rare,abundant)♦ Nuclear groovings (absent,rare,abundant)♦ Chromatin pattern♦ Nuclear membrane (smooth,irregular)♦ Cytoplasm (amount, staining pattern)♦ Naked nuclei (present, absent)♦ Lymphocytes

Follicular lesions♦ The morphological distinction of

hyperplastic adenomatous nodules, well-differentiated follicular carcinomas, and follicular variants of papillary carcinoma is difficult, even for cytologists with extensive experience of thyroid fine needle aspiration.

♦ Attempts to improve the preoperative diagnosis of thyroid nodules by use of strict instructions for obtaining adequate specimens and inclusion of' clinical characteristics (such as sex, dimcnsion of' thc nodule, character of the gland by palpation) have been reported.

05/07/2018

14

Granulomatous Thyroiditis (De Quervain-Subacute)♦ Follicular cells with

degenerative features (paravacuolar granules)

♦ Lymphocytes and polymorphs(subacute)

♦ Epithelioid and giant cells (granulomatous)

Lymphocytic Thyroiditis (Hashimoto)♦ Polymorphic

lymphocytes (isolated and intraepithelial)

♦ Plasmacells♦ Hurtle cell metaplasia♦ Degenerative changes

in follicular cells

05/07/2018

15

Lymphocytic Thyroiditis (Hashimoto)

Goitre

♦ Abundant fluid colloid♦ Bland thyrocytes in

follicles or sheets♦ macrophages

05/07/2018

16

♦ Epithelioid aggregates can look very worrying!

♦ Put in context♦ Previous FNA?

?Background of goitre

Classification of Thyroid Neoplasms♦ Primary follicular epithelial neoplasms

– Benign Follicular adenoma– Malignant Differentiated: Papillary,

FollicularPoorly Differentiated (Insular)Anaplastic

♦ Primary parafollicular epithelial neoplasms– Medullary carcinoma– ?Mixed/composite follicular cell-C cell

♦ Other primary epithelial neoplasms♦ Primary non-epithelial neoplasms♦ Secondary tumors

Follicular lesion,cytologically benign♦ Less fluid colloid♦ Predominance of

bland thyrocytes, usually arranged in microfollicles

♦ Few macrophages♦ Naked nuclei♦ Signs of hyper or

hypofunctionality

05/07/2018

17

Cellular Thy 2

♦ Abundant fluid colloid♦ Bare nuclei♦ Groups of follicular

cells♦ Stromal fragments♦ 3D rounded follicles

05/07/2018

18

Adenomas

Hot

Hurtle

Follicular lesion,cytologically suspicious♦ Scanty or absent colloid♦ Abundance of

homogeneous or mildly atypical thyrocytes, usually arranged in microfollicles

♦ Absent macrophages♦ Few or absent naked

nuclei

Follicular neoplasm

Follicularcarcinoma

Insular carcinoma

Criteria for Diagnosis of Follicular Carcinoma

♦ As per criteria of follicular adenoma

and

♦ Capsular or vascular invasion

05/07/2018

19

Solitary nodule in left lobe of a 48 y.o woman

Thy 3 (follicular neoplasm)♦ Hypercellular♦ Microfollicular

arrangement♦ Scanty colloid♦ Regular nuclear

outlines♦ Fine chromatin♦ Fragile cytoplasm♦ No nuclear

pseudoinclusions

Solitary nodule in left lobewoman 48 y.o.♦ Minimally invasive

follicular carcinoma

Incidental micropapillary microcarcinoma

05/07/2018

20

Cytology

Follicular carcinomaIncidental papillary

microcarcinoma

♦ Solitary cold nodule♦ Cellular smear♦ Devoid of colloid♦ Clusters and follicular

groups

♦ Nuclear crowding♦ Abundant dense

cytoplasm♦ Discohesive cells with

preserved cytoplasm

♦ Nucleoli♦ Fine cytoplasmic

granulations, occasionally with a polar pattern

05/07/2018

21

♦ Suspicious♦ Thy 5 or Thy 4?♦ DD medullary♦ Oncocytic variant of

papillary♦ Hurthle cell tumour♦ BUT calcitonin

negative

Hurtle cell tumours♦ Cytologically

suspicious by definition

♦ Abundant orangiophilic cytoplasm (granular)

♦ Vesicular nucleus, prominent nucleolus

Hurtle cells

♦ R lobe solid nodule in MNG

♦ FNA of the L lobe showed Thy 2

♦ Images are from the R lobe nodule

05/07/2018

22

♦ Nuclear crowding♦ Irregular nuclear

outlines♦ Occasional

pseudoinclusions

Hyalinising trabecular adenoma (Thy 4)♦ Palisading, elongated

cells arranged perpendicularly to metachromatic material

Papillary Carcinoma:A Cytologic Diagnosis

♦ Architecture irrelevant– Papillary, Follicular, Mixed, Solid , Cystic– Diffuse sclerosis variant is hard to recognize

♦ Psammoma bodies are rare♦ Invasion not a criterion

– Encapsulated variant♦ Nuclear features predict behavior

Papillary Carcinoma

05/07/2018

23

Cytologic Features ofPapillary Carcinoma

♦ Crowded overlapping nuclei

♦ Irregular nuclear membrane

♦ Nuclear grooves♦ Pale vacuolated

nucleoplasm♦ Peripheral margination

of chromatin♦ Nuclear

pseudoinclusions

Thy 5, papillary carcinoma

05/07/2018

24

Pitfalls♦ Pseudoinclusions can

be mimicked by red blood cells overlying nuclei or by air bubbles ( which however normally involve both cytoplasm and nucleus)

Pitfalls in thyroid tumour pathologyJ Rosai, E Kuhn, ML CarcangiuHistopathology 2006 (Aug)49:107-120

Medullary Carcinoma♦ Plasmacytoid and /or

spindle cells♦ Cytoplasm granularity♦ Usually dispersed but

also trabecular or follicular pattern

♦ positive with calcitonin

05/07/2018

25

05/07/2018

26

Colloid in LBC and conventional smears

Nuclei in LBC and conventional smears

Nuclear Pseudoinclusons and membrane irregularity

05/07/2018

27

Papillary carcinoma in LBC

♦ Nuclear pseudoinclusions and membrane irregularity are equally identifiable in LBC albeit hyperchromatism is usually more prominent

www.eurocytology.eu/ecc2018

05/07/2018

28

RCPath Part 2 examination♦ 8 non-gynae cytology cases in pairs in 20’ slots; the cases

are marked centrally according to a predetermined scoring system

♦ 20 Histology cases in 10 pairs of H&E slides in 20’ slots over 3 hours and 20’ on the second morning

♦ 4 macro cases are provided in the form of macro photographs of surgical specimens with clinical details.Two 20’ slots(2 cases each) + 20’ discussion with 2 examiners

♦ OSPEs:2x20’ examinations, one face to face with 2 examiners, the other written

♦ Long cases: 4 x 20’ (include histochemistry, immunohistochemistry, immunofluorescence and electron microscopy)

♦ Frozen sections: 6 cases in 2 x 20’ stations (3 each station)

OSPEs

♦Objective structured practical examination (OSPE)

♦Round table on how to restructure cytopathology training and exam at a Cytopathology Day which will take place on September 26, 2018 at Imperial College London

The End!

Any Questions?