WHAT S IN A BIOPSY REPORT?

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1 WHAT S IN A BIOPSY REPORT? HISTOPATHOLOGY REPORTS Peter Piper Memorial Conference Dr. William Ratterree, DACVIM (O) DACVR (RO) Sponsored by: CancerVets August 12, 2017 HISTOPATHOLOGY REPORTS OVERVIEW Important information in the report Degree of differentiation Cell and nuclear pleomorphism Mitotic index Nucleoli Amount of necrosis Tissue demarcation Completeness of excision Vascular or lymphatic invasion OVERVIEW Immunohistochemistry role of special stains Specific tumors molecular markers Tumor Grading Clinical-Pathologic Correlation and 2 nd Opinions Specific Tumor Types Mast cell tumor Soft tissue sarcoma Melanoma Hemangiosarcoma IMPORTANT INFORMATION IN THE REPORT 1

2 DEGREE OF DIFFERENTIATION DEGREE OF DIFFERENTIATION Overall differentiation Hyperplasia Normal Benign neoplasia Disorganized, well differentiated Malignant neoplasia Disorganized, well differentiated to poorly differentiated In some cases, these features are can be difficult to observe Nodular regeneration vs. well differentiated hepatocellular CA Definitive diagnosis of malignant vs. benign vs. inflammation or hyperplasia may not always be possible Repeat biopsy immediately or after a period of clinical observation Consider sample size DEGREE OF DIFFERENTIATION CELL AND NUCLEAR PLEOMORPHISM Hyperplasia None Benign neoplasia Minimal Malignant neoplasia Moderate to marked The degree of anisocytosis (cell) and anisokaryosis (nuclei) is usually reported. MITOTIC INDEX MITOTIC INDEX In a population of cells, the ratio of the number of cells undergoing mitosis (cell division) to the number of cells not undergoing mitosis Typically reported as the number of mitotic figures per 10/hpf (400x) When sample sizes are small, the number may be reported per 1/hpf (400x) Hyperplasia Variable, usually low Benign neoplasia Usually low Malignant neoplasia Often high Mitotic index alone can be a strong indicator of the biological behavior of the certain tumors Mast cell tumors Melanomas 2

3 MITOTIC INDEX NUCLEOLI Hyperplasia Normal Benign neoplasia Normal Malignant Large and/or multiple Romansik 2007 AMOUNT OF NECROSIS Hyperplasia None Benign neoplasia Usually minimal Malignant neoplasia Minimal to abundant Inflammatory mediators responsible including prostaglandins, interleukins (IL), tumor-necrosis factor alpha (TNF-alpha), microvessel density (MVD) TISSUE DEMARCATION Hyperplasia Blends with normal tissue Benign neoplasia Expansive, compressive Malignant neoplasia Invasive Individual or groups of tumor cells infiltrate extensively into surrounding tissue May invade into vascular or lymphatic spaces May invoke a schirrous or desmoplastic response characterized by an excessive fibrous reaction May destroy normal tissue or obliterate normal tissue architecture COMPLETENESS OF EXCISION Lateral and deep margins should be reported in either (mm) or (cm) If the tumor cells extend to the lateral or deep margin incompletely excised Subjective terms Narrowly, completely and widely excised margins The extent of the margin depends on the type of tumor COMPLETENESS OF EXCISION Epithelial tumors may have a capsule Example - Thyroid carcinoma Can act as a margin Capsular invasion may occur and should be reported and additional local control is recommended Mesenchymal tumors have a pseudocapsule (compressed tumor cells) Example - Fibrosarcoma Capsule does not act as a margin Cannot be shelled out 3

4 COMPLETENESS OF EXCISION VASCULAR OR LYMPHATIC INVASION Tumor emboli may be present either in the blood vessels or in the lymphatic vessels More commonly reported in epithelial tumors than mesenchymal tumors Advise staging with thoracic radiographs, abdominal ultrasound and nodal cytology Adjuvant systemic therapy (chemotherapy, targeted therapy) recommended if there is the presence of either type of invasion HISTOCHEMICAL STAINS IMMUNOHISTOCHEMISTRY ROLE OF SPECIAL STAINS Consist of chemical substances that result in a direct chemical reaction with the tissue constituents when applied to the tissue sections Used commonly to assist in the DX of certain poorly differentiated tumors or for prognosis Toluidine blue and Giemsa aid in the ID of mast cell granules in poorly differentiated MCT Silver staining of nucleolar organizer regions prognostic in LSA and MCT IMMUNOHISTOCHEMISTRY (IHC) Can aid in the classification of several tumors Widely used diagnostic technique Staining procedure that employs antibodies to ID specific cellular and extracellular molecules ex vivo (such as cell surface markers) Can be performed on frozen sections or formalinfixed paraffin blocks IHC also used for determining cellular proliferation or tumor growth fraction Ki67 and PCNA staining CD117/c-Kit TUMOR TYPES MOLECULAR MARKERS Carcinoma: cytokeratin+, vimentin Neuroendocrine: chromogranin A+, Synastophysin+, NSE+ GIST: c-kit+, SMA+/-, Desmin-, S100- Leiomyoma/sarcoma: c-kit-, SMA+, Desmin+/-, S100-4

5 TUMOR TYPES MOLECULAR MARKERS Melanoma: MelanA+/-, PNL2+/-, tyrosinase+/-, TRP1 and 2+/-, vim+, S100+/- Histiocytic SA: CD18+, CD3-, CD79a-, lysozyme+ TUMOR TYPES MOLECULAR MARKERS Lymphoma B cell: CD18-, CD79a+, CD3-, Pax5+ T cell: CD18-, CD79a-, CD3+, Pax5- Null cell: CD18-, CD79a-, CD3-, Pax5- Plasma cell tumors: MUM1+, CD18+/-, CD79a+/-, CD3- Mast cell tumors: Tryptase+, c-kit+ TUMOR GRADING TUMOR GRADING Grading is somewhat subjective Reproducibility between pathologists can be variable Tumors are heterogeneous, so patterns as well as features of malignancy may vary from area to area Limitations of small sample size Accurate grading is not possible Grades should be interpreted with caution Example - Punch biopsy of a large sarcoma FEATURES EVALUATED TO ASSESS GRADE DETERMINING GRADE Objective features Mitotic index Amount of necrosis Nucleolar features Subjective features Degree of differentiation Degree of cellular or nuclear pleomorphism Invasiveness Stromal reaction Overall cellularity Lymphoid response Individual features are scored Each score is added for a total tumor score Tumor scores are then separated into ranges and associated with a tumor grade Tumor grade may have prognostic significance and may impact treatment recommendations May correlate with survival, metastatic rate, DFI, or with frequency and/or speed of local recurrence 5

6 DETERMINING GRADE TUMOR GRADING PREDICTIVE OF BIOLOGIC BEHAVIOR Tumors whose grade or histologic features are predictive of biologic behavior in dogs Round cell tumors LSA, MCT (cutaneous, SQ) Carcinomas Lung, mammary, TCC, SCC Sarcomas STS, synovial cell, OSA (primary, metastatic, mandibular), MLO, appendicular CSA, HAS, non-lymphoid/nonangiomatous splenic SA, fibrohistiocytic nodules of spleen Melanoma dermal, oral/lip, uveal TUMOR GRADING PREDICTIVE OF BIOLOGIC BEHAVIOR Tumors whose grade or histologic features are predictive of biologic behavior in cats Lung carcinoma Mammary gland carcinoma Conflicting reports regarding mast cell tumors and fibrosarcoma CLINICAL-PATHOLOGIC CORRELATION AND 2 ND OPINIONS CLINICAL - PATHOLOGIC CORRELATION Provide an accurate history with pertinent clinical results at time of submission Photographs of the tumor site can be helpful in some cases Clinical correlations can help to make an accurate diagnosis CLINICAL - PATHOLOGIC CORRELATION Clinical correlation is especially true for some primary or secondary tumors involving bone Surface or juxtacortical OSA is based both on radiographic and histologic features Osteoma may be difficult to distinguish from reactive bone without radiographs Acanthomatous epulis vs fibrous epulis may depend if underlying bone involvement is present based on imaging if sample size is small 6

7 SECOND OPINIONS Two major categories of errors Technical errors Improperly labeled specimens, tissue blocks or slides All of the critical tissue is not processed Artifacts can occur from improperly processed tissue due to equipment malfunction or tissue is poorly sectioned SECOND OPINIONS Two major categories of errors Errors in interpretation May occur in difficult cases Pathology services with physicians may be unfamiliar with tumors that do not have a human counterpart (such as TVT, perianal gland adenoma) If a pathologic diagnosis is not consistent with clinical presentation, a 2 nd opinion should be requested SECOND OPINIONS Lucy- 12 yr old, FS, GR FIRST OPINIONS Consider sending initial sample to sites of specialization CSU (Barb Powers) Oncology and radiation pathology UC Davis (Peter Moore), VDx Veterinary Diagnostics (Ted Valli), Purdue (Pepe Ramos-Vara) Hematopathology UF (Pamela Ginn) - Dermatopathology The Comparative Ocular Pathology Laboratory of Wisconsin (Richard Dubielzig) - Ocular pathology MSU (Matti Kiupel) Mast cell tumors, melanoma MAST CELL TUMORS SPECIFIC TUMOR EXAMPLES Incidence Most common cutaneous tumor (dog) 16-21% of all cutaneous tumors Histopathology Round cell neoplasia - Round to polygonal cells Round central to slightly eccentric nucleus Often surrounded by a rim of eosinophils Granules may not stain in H&E - Toluidine Blue, Astral blue or Giemsa 7

8 MAST CELL TUMORS MAST CELL TUMORS Two grading schemes for dermal MCT Patnaik: Grades I III 2-tier: low and high grade Both systems usually reported Low grade I or II, high grade II or III Subcutaneous MCT grading low, high Prognostic factors Grade Stage 0 - IV Anatomic location (aural, oral, visceral, digital, mucocutaneous) Prognostic factors Mitotic index MST for dogs with an MI < or =5 was significantly longer (70 months) than for those with an MI >5 (2 months), regardless of grade. MCT Prognostic Panel Excision with a 2-cm lateral margin and a deep margin of 1 fascial plane may result in satisfactory excision of grades I and II MCTs in dogs MAST CELL TUMOR - PROGNOSTIC PANEL MAST CELL TUMOR - PROGNOSTIC PANEL Proliferative indices AGNOR Ki67 c-kit staining pattern localization Pattern I - normal Pattern II aberrant Pattern III - aberrant Mutations c-kit exon 8 and 11 MAST CELL TUMOR - PROGNOSTIC PANEL MAST CELL TUMOR - PROGNOSTIC PANEL Molly 5 yr old, FS, Beagle Vulvar MCT Grade II Incompletely Excised 8

9 MAST CELL TUMORS THE GOOD The Really Good Low grade I MCT Oncologists rarely see Loosa 4 year old FS Catahoula Leopard Dog Completely excised low grade 2 MCT right lateral shoulder in August 2016 Scar revision; No adjuvant therapy recommended No evidence of recurrence to date MAST CELL TUMORS THE GOOD MAST CELL TUMORS THE BAD Tizzie 8 year old FS Boxer Narrowly excised (3 1.4 mm low grade subcutaneous MCT MCT Prognostic Panel kit pattern staining II 20% of dogs with pattern II were dead within 10 months MAST CELL TUMORS THE BAD TX options RT Definitive, palliative Chemotherapy VBL, CCNU, CTX, metronomic CHB Targeted therapy Palladia Tizzie SX December 2016, underwent 6 months of metronomic CHB and prednisone no evidence of recurrence to date MAST CELL TUMORS THE UGLY Nanook 15 1/2 yr FS Am Eskimo Dog Incompletely excised high grade II MCT right lateral hock with equivocal right popliteal LN mets MI > 30 mitosis per 10 hpf C-kit pattern II No mutations exon 8/11 9

10 MAST CELL TUMORS THE UGLY TX recommended definitive RT to the primary and node SX - December 1, 2016: no evidence of recurrence to date Vinblastine/lomustine - January completed 4th cycle April 27, 2017 Chlorambucil maintenance May 25, suspended June 22, 2017 due to positive nodal metastasis, vinblastine restarted June 29, 2017 Lomustine restarted July 27, responding August 9, 2017 SOFT TISSUE SARCOMAS SOFT TISSUE SARCOMAS SOFT TISSUE SARCOMAS Family of tumors that have a similar biological behavior locally aggressive with high recurrence rates and a variable potential for metastasis Examples include - fibrosarcoma, hemangiopericytoma, nerve sheath tumor. Metastatic potential predicted based on tumor grade Tumor grade based on combined score of degree of differentiation, mitotic index, amount of necrosis Grade I 10% chance for metastasis Grade II 20% chance for metastasis Grade III 50% chance for metastasis Pseudocapsule compressed tumor cells Margin evaluation and recommendations Incompletely or marginally excised tumors (any grade) Scar revision Definitive radiation protocol: daily Monday Friday x 19 treatments Coarse fraction radiation therapy: weekly x 4 weeks Metronomic chemotherapy Widely excised tumors > 1 cm Systemic chemotherapy for grade III tumors SOFT TISSUE SARCOMA THE GOOD SOFT TISSUE SARCOMA THE GOOD Woody 10 year old MN Standard Poodle Grade I hemangiopericytoma of the right craniolateral hip diagnosed via surgery Underwent definitive radiation therapy 19 x 3 Gy No significant adverse effects 10

11 SOFT TISSUE SARCOMA THE BAD Porter 11 year old MN mixed breed dog Grade II (intermediate) hemangiopericytoma diagnosed via incisional biopsy in December 2016 Underwent surgery and followed up with 6 months of metronomic chemotherapy SOFT TISSUE SARCOMA THE BAD Suspected recurrence one month after chemotherapy was discontinued CT scan and FNA cytology confirmed recurrence Coarse fraction radiation therapy 4 x 8 Gy Median tumor-free progression time - 9 months SOFT TISSUE SARCOMA THE UGLY SOFT TISSUE SARCOMA THE UGLY Kara Mudge 10 ½ year old FS Golden Retriever Incompletely excised grade 3 soft tissue sarcoma of the left perineum surgery Staging thoracic radiographs and abdominal CT scan negative for metastatic disease Had recurrence of the tumor at the scar at the time of suture removal Completed coarse fraction RT - 6 x 6 total of 36 Gy Adjuvant chemotherapy was advised SOFT TISSUE SARCOMA THE UGLY MELANOMA 11

12 Incidence 5 7% of all skin tumors 30 40% oral tumors Most common primary intraocular tumor CANINE MELANOMA MELANOMA THE GOOD Cutaneous 85% are benign Predictors of benign behavior MSU Melanoma Prognostic Panel Mitotic index Ki67 Surgery is often curative, multifocal tumors may occur Oral Small subset may have a more favorable prognosis To more accurately predict the outcome panel evaluating nuclear atypia score, the mitotic index, Ki67, and pigmentation MELANOMA THE BAD AND THE UGLY Bubba 11 yr MN English Bulldog Acute onset dysphagia and cough Skull CT scan done for staging and RT planning Large, heterogeneous mass in both the oral and nasal cavity MELANOMA THE BAD AND THE UGLY Coarse fraction RT 4 x 8 Gy = 32 Gy Regression after the first treatment No significant AE Response rate: 80 90% for MELANOMA THE BAD AND THE UGLY HEMANGIOSARCOMA 12

13 HEMANGIOSARCOMA THE GOOD HEMANGIOSARCOMA THE BAD Stage I Dermal (does not extend into the deep dermis) Surface of skin, dark red to purple, may bleed Secondary to chronic sun exposure, field cancerization effect Glabrous skin (ventral abdomen, medial thigh) Predisposed breeds - Whippet, Greyhound, Dalmatian, Pit Bull, Boxer Treatment options SX, laser therapy, cryotherapy MST 3 yrs, high chance for recurrence, less likely to metastasize unless non-predisposed breed Maximus 2 yr M Doberman had a mass on the medial aspect of the thigh SX - narrowly excised (lateral margin mm and deep margin 5.5 mm) dermal hemangiosarcoma It expanded the dermis and subcutis, making it a Stage II tumor CT scan for staging negative Adjuvant RT and chemo advised MST 6 12 months HEMANGIOSARCOMA THE UGLY HEMANGIOSARCOMA THE UGLY Stage II and III SQ or Intramuscular 71 dogs MST 6 mos, 25% survival at 1 year TX - SX, RT, chemo, supplements Stage III visceral Standard = Sx, followed by adjuvant chemo, supplements Splenectomy, liver lobectomy Goal: remove all macroscopic disease, prevent hemorrhage CONCLUSION QUESTIONS Histopathology is not back and white-shades of grey Mitotic index, Grade, Margins, Vascular/ Lymphatic Invasion Oncology Network Second options (cytology, histopathology) Oncologists Always exceptions MCT location Dr. William Ratterree- drratterree@cancervetsfl.com Dr. Carrie Kosarek- drkosarek@cancervetsfl.com 13