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SALIVARY GLAND NEOPLASMS:

Classification, Molecular Insights and Biomarkers

DIANA BELL, M.D.

Associate Professor

Head and Neck Section

SALIVARY GLAND NEOPLASMSSALIVARY GLAND NEOPLASMS

~3300 new cases/year (2.5 per 100,000 U.S.)

5% of all Head & Neck neoplasms

~3300 new cases/year (2.5 per 100,000 U.S.)

5% of all Head & Neck neoplasms

Location:

80% Parotid (20% malignant)

15% Submandibular (50% malignant)

5% minor salivary glands (majority malignant)

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SALIVARY GLAND NEOPLASMS

Characteristics:

Phenotypic heterogeneity

Variable intra- and intertumoral behavior

Lack of a defined precursor

SALIVARY GLAND NEOPLASMS

Management:

Primary: Surgery ± XRT

Advanced, recurred and metastatic disease: limited options

Efforts are focused on:

understanding of their biology • genetic changes

• chromosomal changes

• epigenetic changes

identifying new targets of potential

therapeutic applications

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Acinusinter-

calated duct

striated duct excretory

duct Oral cavity

SALIVARY GLAND HISTOGENESIS

Acinic cell Ca Basaloid Tumors Warthin’s MucoepidermoidAdenoid Cystic Ca Oncocytic Salivary Duct Ca

PLGA Tumors AdenocarcinomaMyoepithelial Ca

Epi-myoepithelial CaPleomorphic adenoma

Diagnosis:

FNA

Pathologic examination

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FNA- indications:

Metastasis

Lymphoma

Infection

Reactive lesions

Planning surgery

FNA- limitations:

Degree of malignancy Basal cell tumors

Ca Ex-PA

Myoepithelial tumors

Oncocytic lesions

Overcalling

FNA- complications:

Infarction

Collapse/ obliteration of cystic lesions and tumors

In doubt:

Core biopsy

Frozen section

Categorical Classification ofSalivary Gland Lesions

Reactive/Developmental Primary Neoplasms Metastasis

Tumor-like Cysts

Benign Malignant

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Phenotypic Heterogeneity

Acinic cell carcinoma Secretory Carcinoma

(MASC) Adenoid cystic carcinoma Mucoepidermoid carcinoma Polymorphous

adenocarcinoma Epi-myoepithelial

carcinoma Clear cell carcinoma, NOS Hyalinizing clear cell

carcinoma (HCCC) Basal cell adenocarcinoma Sebaceous carcinoma Sebaceous

lymphadenocarcinoma Mucinous adenocarcinoma Cystadenocarcinoma

Oncocytic carcinoma Adenocarcinoma, NOS Myoepithelial carcinoma Carcinoma ex-PA Carcino-sarcoma Squamous carcinoma Small cell carcinoma Large cell neuroendocrine

carcinoma Lymphoepithelial carcinoma Sialoblastoma Intraductal carcinoma Salivary duct carcinoma Metastasizing pleomorphic

adenoma

WHO 2017

Most common SGCs

MEC(30%)

Adenoca NOS / SDCa(20%)

AdCC(25%)

ACC(15%)

A) Low-grade malignancies

low grade MEC

most acinic cell

most PLGA

epi-myoepithelial carcinoma

basal cell adenocarcinoma

most myoepithelial carcinoma

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B) High-grade malignancies

Solid, AdCC

High-grade MEC

PD/DD Acinic

Salivary duct carcinoma

Carcinosarcoma

Most Ca Ex-PAs (SDCa-exPA)

Incidence of Local Recurrence in Salivary Gland Carcinoma

Diagnosis Recurrence (%)

Adenoid cystic carcinoma 61

Carcinoma ex-PA 60

Adenocarcinoma, NOS 48

Acinic cell carcinoma 15

Mucoepidermoid carcinoma 5

Incidence of LN Metastasis in Salivary Gland Carcinoma

Histology Metastasis (%)

Mucoepidermoid carcinoma 44

Adenocarcinoma, NOS 36

Carcinoma Ex-PA 21

Acinic cell carcinoma 13

Adenoid cystic carcinoma 5

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Incidence of Distant Metastasis in Salivary Gland Carcinoma

Histology Metastasis (%)

Adenoid cystic carcinoma 44

Adenocarcinoma, NOS 27

Carcinoma Ex-PA 21

Squamous cell carcinoma 15

Acinic cell carcinoma 14

Mucoepidermoid carcinoma 9

Prognostic Factors in Salivary Gland Carcinoma

Favorable features:

Low-grade histology

Low stage

Parotid location

Unfavorable features:

High-grade histology

High stage

Submandibular location

Cervical LN metastasis

Facial nerve paralysis

Skin involvement

Recurrence

Molecular Testing and Targeted Therapy in Salivary Gland Carcinomas

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MEC

MUCOEPIDERMOID CARCINOMA

1. Considerable cellular heterogeneity

- epidermoid, intermediate and mucin producing cells

2. Intra- and interobserver variability

- reproducibility of grading schemes

Treatment:

Influenced by conventional clinico-pathologic parameters (age, disease stage, grade).

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MUCOEPIDERMOID CARCINOMA

3. Uniquely characterized by a specific translocation

t(11; 19) (q12; p13)

Fusion transcript t(11;19)(q21;p13)

mucoepidermoid carcinoma translocated 1

MECT1/ CRTC1 gene at 19p13

AND

mastermind-like gene family MAML2 at 11q21

Fusion transcript t(11;19)(q21;p13) (cont.)

chimeric gene MECT1-MAML2: fuses exon 1 of MECT1 with exons 2–5 of MAML2.

MECT1-MAML2 fusion product disrupts the Notch signaling pathway.

identification of the MECT1 gene product as a potent co-activator for genes that are regulated by cAMP responsive elements (CRE).

MECT1-MAML2 may be disrupting both Notch and CREB signaling pathways to induce tumorigenesis.

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Copyright ©2006 American Association for Cancer Research

Kaye, F. J. Clin Cancer Res 2006;12:3878-3881

Fusion in MEC

It is generally accepted that more than 50% of this entity manifest the MECT1-MAML2.

Up to 2016

Fusion (+)ve cases showed significantly better survival than

(-)ve cases (?)

MECT1-MAML2 represents a specific prognostic molecular marker in MEC (?)

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28 patients

90 patients

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Genomic profiles and MECT1-MAML2 fusion distinguish different subtypes of MEC

fusion (+) MEC significantly lower CNV

fusion (-) MEC multiple genomic imbalances

Fusion in MEC

Represents a distinct mechanism in the development of this entity.

Fusion (+) MEC, regardless of grade, manifest a more stable genome.

Fusion (-) MEC represent a distinctly different pathway characterized by marked genomic instability.

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Clinical scenarios with potential benefit from CRTC1/MAML2 translocation testing

Diagnostic!

Preoperative

(+)ve CRTC1/MAML2 fusion transcript

Dgn of malignancy on preoperative FNA and bx of a low grade salivary gland neoplasm, in the absence of classic morphology.

Diagnostic

Confirmation of diagnosis of HG MEC and exclusion of MEC mimics (adenosquamous carcinoma, acantholytic SCC, adenoca NOS, SDca).

Confirmation of diagnosis of histologic variants of MEC (e.g., oncocytic), and exclusion of benign MEC mimics (e.g., Warthintumor with mucinous metaplasia).

Confirmation of diagnosis in MEC recurrence or metastasis.

Objective evidence for a separate neoplasm in a patient with multiple synchronous or metachronous tumors withmorphologic overlap (salivary MEC with adenosquamous cell carcinoma of lung).

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AdCC

AdCC- what is new in biological markers?

Cytogenetics and CGH

MYB Oncogene

Kit and EGFR in AdCC

DNA Methylation in AdCC

Xenograft Models

Cancer Stem-like Cells

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c-kit 70-90%

EGFR 20-60%

Cell dependent expression:

c-kit (ductal epithelial cells), and

EGFR (myoepithelial cells)

Studies of Chromosome 6q Alterations

Reference Total no. of ADCC

Deletion No. Translocation No.

Sandros et al 11 6q22-q24 5 t(6;14)(q22;q11) 1

Mark et al. 2 - 0 - 0

Highashi et al. 2 - 0 t(6;9)(q21-22;p13-21),t(X;6)(p22;q23)

2

Lopez -Gines et al. 1 6q23 1 - 0

Jin et al. 2 6q21 1 t(6;9)(q21-22;p13-21) 1

Hrynchak et al. 2 - 0 t(6;9)(q23;q22) 1

Martins et al. 3 - 0 t(6;9)(q23-25;p22-24) 1

Nordkvist et al. 10 6q21 1 t(6;9)(q23;p21) 2

El-Naggar et al. 1 - 0 t(6;15)(q25-15) 1

Martins et al. 3 6q23add 9p22

0 t(6;12)(p21;q13) 2

Bell et al. 1 - 0 t(6;14)(q25;q13) 1

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G-Banding

SKY Analysis

MYB Oncogene

chr6 q23

NFIB

p23‐22.3chr9

MYB exon1‐ 8         NFIB exon12

1 2 3 4 5 6 7 8 9

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11 12 13 14 15 16

MYB

main

break point

12345678

91011

12 main

break point

15 variants

t(6; 9) (q22-23; p23-24) with fusion transcript involving MYB and NFIB

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Identification of the

MYB-NFIB Fusion and

Therapeutic Applications

RT-PCR, FISH, IHC

Few therapeutic options available

that directly target MYB.

1. Reports of DNA vaccines and antisense

MYB oligonucleotides - potential role in

the treatment of AdCC.

2. Therapies directed against MYB-NFIB

transcriptional downstream targets may

prove more feasible.

3. Various antibodies and inhibitors BCL2,

FGF2, MYC, and COX-2, as potential

chemotherapeutic agents; their efficacy

in AdCC requires further investigation.

18 NOTCH1 mutations identified in 15 patients

Ferrarotto R et al, J Clin Oncol, 2016

IHC for NICD1 to evaluate pathway activation: 40/72 (56%) were +

NOTCH1 mutations in AdCC

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Adenoid Cystic Carcinoma (AdCC)

Morris LGT et al, JAMA Oncology, 2016

NOTCH1 mutations define a distinct disease phenotype

Stage I-III Stage IVA-C

NOTCH1 mut vs. wt:

86% vs. 46% stage IV (P=0.02)

79% vs. 38% solid (P<0.001)

Ferrarotto R et al, J Clin Oncol, 2016

Tubular Cribriform Solid

(HYALINIZING) CLEAR CELL CARCINOMA(HCCC)

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t(12;22)(q21;q26) EWSR1-ATF180-90 %

SECRETORY CARCINOMA OF SALIVARY GLANDS

(MASC)

t(12;15(p13;q25)ETV6-NTRK3

Secretory Carcinoma of Salivary Gland

Morphology and genetics similar to secretory carcinoma of

the breast

43-93% harbors the fusion of the transcriptional regulator

(ETV6) with membrane receptor kinase (NTRK3)

ETV6-NTRK3 encodes a chimeric TK with potent transforming

activity in fibroblasts

PKC Ras/MAPK PI3K

Differentiation & Survival

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LOXO-101 (Larotectinib), a TRKA/B/C inhibitor, in patients with NTRK fusions

Hyman DM et al, ASCO Annual Meeting, 2017

ORR: 76%

LOXO-101 (Larotectinib), a TRKA/B/C inhibitor, in patients with NTRK fusions

Hyman DM et al, ASCO Annual Meeting, 2017

POLYMORPHOUS ADENOCARCINOMA (PLGA)

Site: Minor salivary glands, palate, buccal mucosa

Growth:“eye of the storm”, tubular, trabecular, papillary, solid, cribriform

Nuclear features: vesicular and ovoid nuclei

Clinical: cervical LN mets very rare

Tissue invasion: PNI

Molecular alterations: Hotspot activating PRKD1 somatic point mutation (E710D)

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CRIBRIFORM ADENOCARCINOMA OF MINOR SALIVARY GLAND (CAT)

Site: Minor salivary glands, BOT predominant

Growth:“glomeruloid”, cribriform, tubular, solid

Nuclear features: optically clear (PTC-like)

Clinical: early cervical LN metsbilateral, no DM

Tissue invasion: LVI

Molecular alterations: PRKD1-3translocations, ARID1 A and DDX3Xpartner genes

Molecular tumor markers investigated in salivary gland carcinomas

Target therapies and clinical trials

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Chromosome

Chromosome

Chromosome

Factors Complicating Clinical Trials Outcome:

Small size

Combined histologies

Variable prior therapies

Variable inclusion criteria

A need for biologically - based target therapy!

Adenoca/ SDCa

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Mammary

DC

Salivary

DC

SDCa

HER2AR

Salivary Adenocarcinomas/ SDCa

Share in common:

Growth factors and hormonal expressions with breast / prostate Ca

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Salivary duct carcinoma SDCa is an aggressive adenoca similar to apocrine breast cancer

M:F ratio 4:1, most patients SIII/IV disease (60% N+)

Androgen receptor is overexpressed in ~ 75%

- splice variants in ~ 40%

- FOXA1, FASN mutations

ERBB2 amplifications occur in 20-30%

Genomic alterations in the PI3K/AKT/mTOR

pathway: 54%

Mitani Y et al, Clin Cancer Res, 2014Dalin MG et al, Clin Cancer Res, 2016

Wang K et al, Clin Cancer Res, 2016

Salivary Duct Carcinoma

Mutation burden: 1.7 mut/ Mb

Dalin MG et al, Clin Cancer Res, 2016

Hormonal Expression in SDCa

Positive Negative(2-3) (0+1)

66%70%

25%

0%

20%

40%

60%

80%

100%

AR ERbeta HER-2

Per

cen

t P

osi

tive

35%

EGFR

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A B

Similar to mammary and prostatic adenocarcinoma:

Hormonal and growth factors expression targeted for therapy

ERb Anti-estrogens (PD98059)

AR Anti-Androgens

Her2neu Trastuzumab (Herceptin)

EGFR Cetuximab, bevacizumab, gefitinib, erlotinib

Markers Agent

New trial concepts are needed

Locati L et al, Ann Oncol, 2003; Piha-Paul S et al, J Clin Oncol, 2011

LHRH agonist +

Bicalutamide

Docetaxel+

Trastuzumab

Sirolimus+

Bevacizumab

AR+

HER2+

PTEN-

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Molecular Alterations in Salivary Gland Carcinomas

Tumor Type Chromosomal AlterationGene Fusion/

Rearrangement%

MEC t(11:19)(q21; p13)t(11; 15) (q21; q26)

CRTC1-MAML2CRTC3-MAML2

40-80%5

AdCC t(6;9) (q22-23; p23-24)t(8; 9)

MYB-NFIBMYBL1-NFIB

25-8010-20

HCCC t(12;22)(q21;q26) EWSR1-ATF1 80-90

MASC t(12;15)(p13;q25)t(12;X)

ETV6-NTRK3ETV6-RET

95-982-5

PLGA 14q12 Hotspot activating PRKD1 somatic point mutation (E710D)

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CAT (CASG) t(1;14)(p36.11;q12)t(X;14)(p11.4;q12)

ARID1A-PRKD1DDX3X-PRKD1PRKD2 and PRKD3 rearrangements

241316

SDC 17q21.13q26.32Inv(10)(q11.21q11.22)

HER2 amplificationPIK3 CA mutationNCOA4-RET

20-4020<5

Clinical trials of salivary gland carcinomas based on:

Histologic classification

Molecular and cellular characterization

Biomarker profiling

Needs:

Tissues and cell lines infrastructure

Transgenic animal models

In vitro 3D models

Perspectives