Nailing the diagnosisHelp your pathology help you

Small Animal Specialist HospitalDr Sophia Tzannes

Nailing the diagnosisHelp your pathology help you

• Cytology• When, how, what and where?• Advanced cytology and additional tests

• Histopathology• Pre-op, post-op- when does it matter?• When do we ask for immunohistochemistry?

Nailing the diagnosisHelp your pathology help you

• ‘Hugo’

• ‘Gywna’

• ‘Forster’

• ‘Dudley’

CYTOLOGY

• When?

• How?

• What?

• Where?

CYTOLOGY:WHEN

Pre-surgical diagnosis is encouraged

• Owners expectations: prognosis and financial considerations

• Determine whether further information is required prior definitive surgery

• Staging

• May result in non-surgical treatment as best option

• Not considered a substitute for histopathology

CYTOLOGY:WHEN

Staging and monitoring when diagnosis known

• Staging

• Monitor for recurrence of lesions cytologically

CYTOLOGYHOW

• Make sure what you are sampling is what you wish to sample

• Make sure what you have sampled is of diagnostic quality

• repeat multiple times if necessary, use different techniques

• Make sure that you submit an adequate number of slides

CYTOLOGYHOW

fat

lesion

• Make sure what you are sampling is what you wish to sample

• ‘Hugo’ is a 12 y MN Rottweiler X

• Presented August 2014 for oncological and surgical assessment of an infiltrating lipoma

• Fine needle aspirates by referring vet had consistently revealed droplets of free fat and adipocytes

• CT performed to evaluate pre-operatively

CYTOLOGYHOW: Make sure what you are sampling is what you wish to sample

• Visualisation is the key

• Superficial lesions: estimate trajectory

• Deep lesions: consider imaging as guidance

CYTOLOGY:HOW

Make sure what you have sampled is of diagnostic quality

9y FN Irish SetterPresented with mammary massesVulval mass detected during prepFine needle aspirate (FNA)

CYTOLOGY:HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY

• Equipment needed• Common problems encountered with slide preparation:

• Blood contamination

• Low cellularity

• Cell rupture or lysis

• Smear too thick

• Artefacts

CYTOLOGY:HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY

• Common problems encountered with slide preparation:

• Blood contamination

• Low cellularity

• Cell rupture or lysis

• Smear too thick

• Artefacts

-Lymph nodes-Mast cell tumours-Internal organs

Liver, spleen-Vascular tumours-Ulcerated lesions-Mucous

membranes

Spindle cell tumours

Easy to exfoliate:Round cell tumours

+/- epithelial cells

CYTOLOGY:HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY

• Common problems encountered with slide preparation:

• Blood contamination

• Low cellularity

• Cell rupture or lysis

• Smear too thick

• Artefact

CYTOLOGY:HOW : MAKE SURE WHAT YOU HAVE SAMPLED IS OF DIAGNOSTIC QUALITY

1. Is the slide of diagnostic quality?2. Are there different types of cells present? 3. Are inflammatory cells present? 4. Can you identify the cells belonging to the tissue of origin? 5. Unusual cells?

a. Shapeb. Cluster or occur individuallyc. Cytoplasmic detail and nuclear detail

6. Extra cellular material/infectious agents

CYTOLOGY:WHAT IS IT?

Is the slide of diagnostic quality?

Yes

Are there different types of cells present?

Yes

Are inflammatory cells present? Yes

Can you identify the cells belonging to the tissue of origin?

Yes, epithelial cells

Unusual cells? No

Extra cellular material/infectious agents

No

CYTOLOGY:WHAT IS IT?

Is the slide of diagnostic quality?

Yes

Are there different types of cells present?

Yes

Are inflammatory cells present? Yes

Can you identify the cells belonging to the tissue of origin?

Yes, epithelial cells

Unusual cells? No

Extra cellular material/infectious agents

No

Inflammatory

Neoplastic

Degenerative

CYTOLOGY:WHAT IS IT?

Inflammatory

Neoplastic

DegenerativeMCT: Toluidine blue

CYTOLOGY:WHAT IS IT? Neoplastic

• Cytological criteria of malignancy:

• Nuclear criteria

• e.g.: variation in nuclear size and shape- multinucleation, especially if nuclei within same cell vary in size; increased nuclear:cytoplasmic ratio or variation within same population of cells; coarse and ropey chromatin pattern; nuclear moulding; increased number of mitotic figures; variation in nucleolar size, shape and number, especially within same nucleus- bizarre forms,

• Cellular criteria

• e.g. variation in cell size and shape (greater in malignancy), cell population in abnormal location

• Cytoplasmic criteria

• e.g. cytoplasmic basophilia, vacuolation and granulation

CYTOLOGY:WHAT IS IT? Neoplastic

CYTOLOGY:WHAT IS IT?

Is the slide of diagnostic quality?

Yes

Are there different types of cells present?

Yes

Are inflammatory cells present? Yes

Can you identify the cells belonging to the tissue of origin?

Yes, epithelial cells

Unusual cells? No

Extra cellular material/infectious agents

No

Inflammatory

CYTOLOGY:WHAT IS IT?

• Limitations

• Mammary > histopathology

• Cannot grade

• May not be definitive

CYTOLOGY: WHERE?WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?

• External tissues

• Internal tissues and fluid

• Contra-indications include: Vascular lesionCoagulopathy

Impaired accessHollow organ

Cancer seeding (TCC)

CYTOLOGY: WHERE?WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?

• Staging

• Performed on patients with malignant lesions

• Knowing pattern of metastatic spread is helpful

• Less differentiated MCT recommend lymph node, liver and spleen aspirates

• Soft tissue sarcoma: haematogenous route to lungs (other soft tissue, liver) common

• Subtype dependent: histiocytic/synovial cell sarcoma to regional LN (plus lung, etc)

• Carcinoma: lymphatic route

CYTOLOGY: WHERE CAN WE SAMPLE, WHERE SHOULD WE SAMPLE?

CANINE LYMPHOMA- SPECIAL CONSIDERATIONS

• Multi-centric lymphoma• Which lymph nodes?

• How many samples?

• What should I be requesting to maximise my diagnosis?

• Immunocytochemistry and immunohistochemistry refer to the process of detecting antigens in tissues or cells by exploiting the antigen-antibody binding process found in biological tissues.

CYTOLOGY:LYMPHOMA- SPECIAL CONSIDERATIONSIMMUNOPHENOTYPING AND FLOW CYTOMETRY

IMMUNOPHENOTYPING

• The diversity of immunophenotyping markers used in diagnostic pathology is substantial and more markers are becoming available in the veterinary field. Some examples of commonly used markers include:– CD3: T cell lymphoma– CD79a: B cell lymphoma– CD18, 11: Histiocytic disease– CD117: KIT used to identify MCT’s and GIST’s– Vimentin: a marker common to sarcomas

PARR AND FLOW

What is the difference between flow cytometry and PARR?

• The PARR assay is a PCR assay in which we are amplifying DNA to evaluate lymphocyte (LCT) receptor gene length. In a heterogenous LCT population the LCT’s are all genetically distinct so the LCT receptor genes vary in length.

• Homogenous or clonal LCT population, is often malignant and have identical gene length throughout the population.

• The results tell us if the majority of cells in the sample are derived from the same original clone (most

consistent with neoplasia), or from multiple clones (most consistent with a reactive process- inflammation,

immune mediated disease or infection)

PARR AND FLOWWhat is the difference between flow cytometry and PARR?

• Flow cytometry (FC) allows identification and quantitation of cell surface markers

• The FC study involves staining live cells (blood, BM, LCT) with labelled antibodies that bind to proteins expressed on the cell surface

• The cells are analyzed on a flow cytometer, which tells us how many cells of each type are present. This information allows us to determine the lineage of the cells present, and whether they are homogeneous (more consistent with neoplasia) or heterogeneous (more consistent with a reactive process

• Limitation – sample collection- appropriate cytofixative for FNA samples (48 hours), or blood/bone marrow into EDTA (24 hours)

CYTOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONSFLOW CYTOMETRY

• Immunophenotyping lymphoma• Prognosis

• Treatment guidance

CD3 (T cell marker)CD4 (helper T cells)CD5 (T cell marker)CD8 (cytotoxic T cells)CD21 (B cells, but not their precursors)CD34 (stem cell marker, expressed in acute leukaemia)CD3-e (pan T cell marker) CD79a (pan B cell marker)Myeloperoxidase (expressed by myeloblasts and granulocyte precursors)MAC387 (expressed by monoblasts, and some granulocyte precursors)CD14 (expressed by monoblasts and monocytes)

CYTOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONSFLOW CYTOMETRY

• Differentiation of hyperplasia and neoplastic populations• Characterisation of leukaemias CD3 (T cell marker)

CD4 (helper T cells)CD5 (T cell marker)CD8 (cytotoxic T cells)CD21 (B cells, but not their precursors)CD34 (stem cell marker, expressed in acute leukaemia)CD3-e (pan T cell marker) CD79a (pan B cell marker)Myeloperoxidase (expressed by myeloblasts and granulocyte precursors)MAC387 (expressed by monoblasts, and some granulocyte precursors)CD14 (expressed by monoblasts and monocytes)

CYTOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONSPARR

• Sample factors:• cell lysis/poor preservation

• low diagnostic yield

• location of sample- e.g. intestinal tract

• Disease factors:• equivocal diagnosis- ‘reactive node’

• early diagnosis

• certain lymphoma sub-types versus

• persistent lymphocytosis

HISTOPATHOLOGY

• Pre-op, post-op- when does it matter?• When do we ask for immunohistochemistry?

HISTOPATHOLOGY

• Definitive diagnosisPunchTru-cut

Grab/pinchIncisional biopsy

HISTOPATHOLOGYPre-op, post-op- when does it matter?

• Pre-operative histopathology especially important when grading may influence surgical plan

• Surgical margins

Grade 1 and 2:Minimum 2 cm and one deep

fascial plane

Grade 3:Minimum 3 cm and one deep

fascial plane Mast cell tumour, H+E

HISTOPATHOLOGYPre-op, post-op- when does it matter?

• Pre-operative histopathology especially important when grading may influence surgical plan

• Surgical approach when surgery may be challenging:

• large

• localisation

HISTOPATHOLOGYPre-op, post-op- when does it matter?

• Pre-operative histopathology is not indicated where there is no influence on surgical plan

• Location

• renal

• spleen

• mammary

Always obtain post-operative histopathology

HISTOPATHOLOGY:When do we ask for immunohistochemistry?

• Diagnosis is inconclusive

• Diagnosis does not fit clinical picture

• Where a more precise diagnosis will influence prognosis or treatment•Round cell neoplasia•Fibrohistiocytic splenic neoplasia•Intestinal neoplasia (sarcoma, other)

HISTOPATHOLOGY:When do we ask for immunohistochemistry?

• ‘Forster’, 12y MN Border Collie

• Presented as a referral for a recurrent oral melanoma

Forster’s biopsy:Round cell population

• Further diagnosis with histopathology and immunohistochemistry

Epitheliotropic T cell lymphoma

Amelanotic melanoma

Round cellsHistiocytic cells

Mast cellsLymphomaPlasma cells

TVT(Melanoma)

HISTOPATHOLOGY:

When do we ask for immunohistochemistry?

Lip mass. Extensively infiltrating through the submucosa are myriadround cells with scant amounts of lightly eosinophilic to clearcytoplasm and no obvious pigment. The cells have atypical lymphoidappearance with round nuclei containing granular to branched chromatinand 1 nucleolus. Some of the nuclei are indented while occasional cellshave a slightly larger nucleus. The cells show aggregation aroundvessels as well as infiltration into bundles of nerve and skeletalmuscle. The mitotic index is 5. There are scattered interstitialeosinophils, small dark lymphocytes, occasional plasma cells andmacrophages. Within the overlying haired skin is a similar population ofround cells showing epitheliotropism, and also affecting hair follicles,sebaceous glands and sweat glands. These cells often have clearcytoplasm and infiltrate epithelium as individual cells and smallclusters. Affected regions of the epithelium are subtended by plasmacells, small dark lymphocytes and a few of the atypical lymphocytes.Distribution of this change within the affected haired skin is notuniform. The mucosal surface is affected but to a lesser extent.Neoplastic cells are not seen at mucosal and haired skin margins.

IMMUNOHISTOCHEMISTRY REPORTThe neoplastic lymphocytes within epithelium and deeper tissue arestrongly and uniformly labelled with CD3

Lip mass. Extensively infiltrating through the submucosa are myriadround cells with scant amounts of lightly eosinophilic to clearcytoplasm and no obvious pigment. The cells have atypical lymphoidappearance with round nuclei containing granular to branched chromatinand 1 nucleolus. Some of the nuclei are indented while occasional cellshave a slightly larger nucleus. The cells show aggregation aroundvessels as well as infiltration into bundles of nerve and skeletalmuscle. The mitotic index is 5. There are scattered interstitialeosinophils, small dark lymphocytes, occasional plasma cells andmacrophages. Within the overlying haired skin is a similar population ofround cells showing epitheliotropism, and also affecting hair follicles,sebaceous glands and sweat glands. These cells often have clearcytoplasm and infiltrate epithelium as individual cells and smallclusters. Affected regions of the epithelium are subtended by plasmacells, small dark lymphocytes and a few of the atypical lymphocytes.Distribution of this change within the affected haired skin is notuniform. The mucosal surface is affected but to a lesser extent.Neoplastic cells are not seen at mucosal and haired skin margins.

IMMUNOHISTOCHEMISTRY REPORTThe neoplastic lymphocytes within epithelium and deeper tissue arestrongly and uniformly labelled with CD3

• IMHC: CD3 +(CD8+)

HISTOPATHOLOGY:

When do we ask for immunohistochemistry?

Epitheliotropic lymphoma

• Histopathology

• 50% (7/14) of oral cases were conclusively diagnosed with histopathology alone (Nemec et al, 2012)

• early stages can have marked mixed inflammatory infiltrate, late stages > B lymphocytes

• >50% there is a mix of lymphocyte cell size reported

• IMHC

• humans: 5-10% lymphoid neoplasia cannot be diagnosed with use of histopathology and IMHC

• PARR

• T cell receptor (TCR) gamma rearrangement

• Sensitivity 80-95%, Specificity 96-100%

Clinical features

Histopathology

Immunohistochemistry

Clonality testing

Clinical follow up and repeat biopsy

HISTOPATHOLOGY:

When do we ask for immunohistochemistry?

Epitheliotropic lymphoma: diagnosis

HISTOPATHOLOGY:

When do we ask for immunohistochemistry? Diagnosis

Change in diagnosisChange in management

Surgical > Medical

HISTOPATHOLOGY:

When do we ask for immunohistochemistry?

• ‘Dudley’, 12y MN Staffordshire Bull Terrier

• Presented as a referral for haemabdomen

• Diagnosed with splenic mass

• Splenectomy after staging

• Diagnosis: splenic sarcoma

The splenic masses reflect a malignancy which consists of variably pleomorphic spindloid to histiocytic cells admixed with lymphoid nodules and extramedullary haematopoiesis, consisting with a fibrohistiocytic nodule.The regions of necrosis and areas with a relatively high mitotic rate warrants a diagnosis of malignancy. Diagnosis: splenic sarcoma

HISTOPATHOLOGY:

When do we ask for immunohistochemistry?

• Immunohistochemistry requested

• Diagnosis: fibrohistiocytic sarcoma

VimentinDesmin

Smooth muscle actinFactor VIII

CD3CD79aCD18

• Histologic and immunohistochemical review of splenic fibrohistiocytic nodules (SFHN) in dogs (Moore

2012)

• Splenic FHN cases were re-evaluated in 32 dogs

• Histopathology Grade I(2) Grade II (9) Grade III(21) dogs

• CD3, CD20, CD79a, CD18, CD11d, K1-67 used to reclassify

• Grade I- MZL and lymphoid nodular hyperplasia

• Grade II-MZ hyperplasia(1), MZL (1), complex hyperplasia (2), LNH(1), stromal sarcoma (3)

• Grade III- MZH(1), diffuse large B cell LSA(1), MZL(1)CNH (6),LNH (1), SS (5), HS (6)

• Prognosis:

HISTOPATHOLOGY:

When do we ask for immunohistochemistry? Prognosis

HS- poor 74 daysStromal sarcoma 488 days, 56% alive 1 year

CNH MST 387 days 70% alive 1 yearLNH MST 570 days 60% alive 2 years

HISTOPATHOLOGY:When do we ask for immunohistochemistry? Therapy

• Intestinal tumours

• Reclassification of small intestinal and cecal smooth muscle tumours in 72 dogs (Maas et al, 2007)

• Retrospective study of 47 dogs with a prior diagnosis of leiomyoma or leiomyosarcoma

• 85% reclassified as gastrointestinal stromal tumour (GIST) or GIST-like

• Therapeutic relevance

• GIST + c-kit, target for tyrosine kinase inhibitors (TKIs)

• Diagnosis is inconclusive

• Diagnosis does not fit clinical picture

• Where a more precise diagnosis will influence prognosis or treatment

•Round cell neoplasia

•Fibrohistiocytic splenic neoplasia

•Intestinal neoplasia (sarcoma, other)

HISTOPATHOLOGY:When do we ask for immunohistochemistry? Therapy

• Mast cell tumours

• Ki-67 prognostic, may elect adjunctive treatments if high

• KIT (CD 117) if use of TKIs considered

C-Kit staining high Ki-67 low Ki-67

HISTOPATHOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONS

• Summary of Canine Malignant Lymphoma Revised From the Revised European-American Classification of Lymphoid Neoplasms/ World Health Organization Classification of Lymphoid NeoplasmsB Cell NeoplasmsPrecursor B cell neoplasmsPrecursor B lymphoblastic leukemia/lymphomaMature (peripheral) B cell neoplasmsB cell chronic lymphocytic leukemia/prolymphocyticLeukemia/small lymphocytic lymphomaB cell prolymphocytic leukemiaLymphoplasmacytic lymphomaSplenic marginal zone B cell lymphomaPlasma cell myeloma/plasmacytomaExtranodal marginal zone B cell lymphoma of mucosa-associated lymphoid tissue typeNodal marginal zone lymphomaFollicular lymphomaMantle cell lymphomaDiffuse large B cell lymphomaaMediastinal large B cell lymphomaBurkitt’s lymphoma/Burkitt’s cell leukemiaProvisional entity: high-grade B cell lymphomaBurkitt’s-likeaPrimary effusion lymphomaT Cell and Putative Natural Killer Cell NeoplasmsPrecursor T cell neoplasmPrecursor T lymphoblasticLymphoma/leukemiaMature (peripheral) T cell and natural killer cell neoplasmsT cell prolymphocytic leukemiaLarge granular lymphocyte leukemia (LGL)Aggressive natural killer (NK) cell leukemiaPeripheral T cell lymphomas, unspecifiedaAdult T cell lymphoma/leukemiaIntestinal T cell lymphoma (+enteropathy associated)Hepatosplenic gdT cell lymphomaSubcutaneous panniculitis-like T cell lymphomaMycosis fungoides/Sezary syndromeAnaplastic large cell lymphoma, T and null cell primary cutaneoustypePeripheral T cell lymphoma not otherwise specifiedAngioimmunoblastic T cell lymphomaAngiocentric T cell lymphomaa Peripheral T cell lymphomas are those that are not otherwise specified(NOS) to a specific subtype by further definition

Personalised lymphoma treatment

• Lymphomas divided into 3 major groups

• high, intermediate, low grade• Most common lymphoma was centroblastic large B cell

CANINE LYMPHOMA- SPECIAL CONSIDERATIONS(Valli et al, 2010)

HISTOPATHOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONS(Valli et al, 2010)

Personalised lymphoma treatment

• Does the specific diagnosis of lymphoma subtype have a similar impact on survival time in dogs as it did in humans?

Low grade T cell (T zone) lymphoma

Longest survival time(622 days)

T cell high grade (peripheral T cell)

lymphomaShortest survival time

(162 days)

Clinical signsLow grade often noneHigh grade frequently

unwell

HISTOPATHOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONS(Valli et al, 2010)

Personalised lymphoma treatment

• Does a specific diagnosis in a dog influence the chemotherapy required?

High Grade B and T cell lymphoma

Effective chemotherapy increased survival time

T zone lymphomaChemotherapy decreased

survival time(13 dogs with no

treatment had longest survival of 687 days)

• Indolent lymphoma comprises 5- 29% of all canine lymphomas• Limited information regarding the subtypes and biological behavior. • Canine indolent lymphoma consists of a group of diseases that are histopathologically

similar to subtypes of non-Hodgkin’s lymphoma identified in people. • Histopathological subtypes:

B cell- marginal zone (MZL), follicular lymphoma (FL) and mantle cell lymphoma (MCL ), T-zone lymphoma (TZL).

HISTOPATHOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONS(Valli et al, 2010)

• B cell predominant• Both B and T cell indolent lymphomas share a low mitotic rate,

slow rate of progression• TZL can be difficult to recognize as malignancy given the small

mature –appearing cell type and low mitotic activity.• Detection of clonality is useful adjunct to histologic examination

HISTOPATHOLOGY: CANINE LYMPHOMA- SPECIAL CONSIDERATIONS

INDOLENT LYMPHOMA

Clinical features

Histopathology

Immunohistochemistry

Clonality testing

Clinical follow up and repeat biopsy

NAILING THE DIAGNOSIS: MAKING THE MOST OF OUR PATHOLOGY

Cytology

• Make sure what you are sampling is what you wish to sample

• Make sure what you have sampled is of diagnostic quality

• Make sure that you submit an adequate number of slides

• Diagnosis is inconclusive

• Diagnosis does not fit clinical picture

• Where a more precise diagnosis will influence prognosis or treatment

•Round cell neoplasia

•Immunophenotyping

•Clonality testing

•Fibrohistiocytic splenic neoplasia

•Intestinal neoplasia (sarcoma, other)