Update on SMARCA4 Deficient Lung Tumors Natasha Rekhtman, MD, PhD Memorial Sloan Kettering Cancer Center, NY Disclosure of Relevant Financial Relationships

None to disclose SMARC and SWI/SNF Glossary

• SWI/SNF complex - Switch/Sucrose Non-fermenting

• BAF complex (alternative name for SWI/SNF) - Brahma-associated factors

• SMARC = SWI/SNF Related, Matrix Associated, Actin Dependent Regulator Of

• SMARCA4 = BRG1 - Brahma-related 1

• SMARCA2 = BRM - Brahma

• SMARCB1 = INI1 - Integrase interactor 1 SWI/SNF complex: Background

• SMARCA4 (BRG1) and SMARCB1 (INI1) are members of SWI/SNF is a multi-subunit complex.

• Inactivating mutations and loss of expression of SWI/SNF components occurs commonly in human cancers, and several tumor types are defined by the loss of individual SMARCs:

SMARCB1 (INI1, BAF47) SMARCA4 (BRG1)

Arnaud O et al Cancer Letters 2018; Hollman and Hornick Am J Surg Pathol 2011; Agaimy A Semin Diagn Pathol 2019

SWI/SNF complex: Background

• SMARCA4 (BRG1) and SMARCB1 (INI1) are members of SWI/SNF is a multi-subunit chromatin remodeling complex.

• Inactivating mutations and loss of expression of SWI/SNF components occurs commonly in human cancers, and several tumor types are defined by the loss of individual SMARCs:

SMARCB1 (INI1, BAF47) • SMARCB1 (INI1)-deficient tumors: SMARCA4 (BRG1) - Malignant rhabdoid tumors – MRT (renal, extra-renal, AT/RT) - Other: renal medullary carcinoma, , etc

• SMARCA4 (BRG1)-deficient tumors: - Minority of MRTs with intact SMARCB1 - Small cell carcinoma of the ovary, hypercalcemic type (SCCOHT)

• SMARCB1 or SMARCA4: Undifferentiated/de-differentiated carcinomas, frequently with rhabdoid features, including endometrial carcinomas (primarily SMARCA4), pancreatic, bladder, other Arnaud O et al Cancer Letters 2018; Hollman and Hornick Am J Surg Pathol 2011; Agaimy A Semin Diagn Pathol 2019 SMARCA4 in lung tumors

1) Conventional NSCLC - Prevalence and patient characteristics - Histology and IHC - Prognostic and predictive significance 2) Rhabdoid sarcoma-like tumors SMARCA4 in conventional NSCLC: Prevalence and patient characteristics

- ~5-8% of NSCLC, most commonly in adenocarcinomas, harbor mutations and loss of SMARCA4 expression.1

- Associated with h/o smoking, consistent lack of EGFR/ALK/ROS1 alterations.1,2

1. Jordan EJ et al Cancer Discov 2017 2. Matsubara D et al Cancer Science 2013 SMARCA4 in conventional NSCLC: Histology and IHC features

- Enrichment in solid pattern and complete lack of lepidic pattern1-3

- High rate of negative TTF-1 (~70%)2,3

SMARCA4 SMARCA4

1. Matsubara D et al Cancer Science 2013 2. Agaimy a et al Virchows Arch 2017 3. Montecalvo … Rekhtman. Mod Pathol 31, suppl 2; p. 742 SMARCA4 in conventional NSCLC: Emerging biomarker? Prognostic significance Predictive significance Emerging data suggest that SMARCA4 mutations • increased sensitivity to platinum-based represent a strong predictor of poor clinical outcome chemotherapy (Bell EH et al Clin Cancer Res 2016) in NSCLC (Bell EH et al Clin Cancer Res 2016, Reisman DN et al Cancer Res 2003, Matsubara D et al Cancer Science 2013) SMARCA4 mutations/loss – not druggable, but several preclinical studies show distinctive drug sensitivity of SMARCA4-deficient NSCLC:

• sensitivity to Aurora kinase A inhibition (Nature Commun 2017)

• increased sensitivity to CDK4/6 inhibitors, such as abemaciclib and palbociclib (Xue Y et al Nature Commun 2019)

• increased sensitivity investigational agents targeting oxidative phosphorylation (Deribe YL et al Nature Med 2018) Tumor Genomic Profiling Predicts Recurrence in Completely Resected Early- Stage Lung Adenocarcinoma Brandt WS et al submitted (MSKCC, surgery) SMARCA4 in lung tumors

1) Conventional NSCLC - Prevalence and patient characteristics - Histology and IHC - Prognostic and predictive significance

2) Rhabdoid sarcoma-like tumors SMARCA4-deficient thoracic sarcoma-like tumors N=19 • Defining features: Nature Genet 2015 - thoracic tumors - distinctive morphology - SMARCA4 mutations and loss of expression

N=12 • Histologic and IHC features

Mod Pathol 2017 • Distinctive patient characteristics

N=12 • Are these rhabdoid sarcomas or sarcomatoid carcinomas?

Mod Pathol 2017 SMARCA4-deficient thoracic sarcoma-like tumors: Hallmark histology = rhabdoid features.

Hallmark feature = rhabdoid cells In most cases, classic rhabdoid cells are In some cases, rhabdoid cells ✓ eosinophilic cytoplasmic inclusions present only focally. are entirely lacking ✓ macronucleoli Nevertheless, cells are ~ to rhabdoid cells ✓ monomorphic minus prominent inclusions: ✓ discohesive ✓ plasmacytoid/epithelioid Only few cases are diffusely rhabdoid! ✓ macronucleoli (but size varies) ✓monomorphic (variably) ✓ discohesive SMARCA4-deficient thoracic sarcoma-like tumors: Hallmark histology = rhabdoid or rhabdoid-like.

Pediatric MRT≈: anaplastic but Proximal-type≈ epithelioid monotonous discohesive cells sarcoma without overt inclusions

Image from Agaimy A Semin Diagn Pathol 2019 Image from Hollmann and Hornick AJSP 2011

Yoshida et al: Rhabdoid cells in 6/12 cases. SMARCA4-deficient thoracic sarcoma-like tumors: Morphologic spectrum.

Monomorphism can vary Some areas – ↑ pleomorphism Immunohistochemical features: SMARCA4 loss

From Yoshida et al Mod Pathol 2017 Either complete loss (most cases) OR “severe global reduction” (truncated may be partially expressed and detected by the N-terminal epitope) Immunohistochemical features: other key features Stem cell markers Low/absent kertains frequently positive: (EMA usually +) SALL4, CD34, SOX2, CD99

Monomorphic undifferentiated high grade tumor + • CD34+  ? epithelioid vascular tumor, epithelioid sarcoma, NUT SALL4 • CK-/CD99+  ? Ewing • SALL4+  ? germ cell tumor Immunohistochemical features: other key features Stem cell markers Low/absent kertains frequently positive: Claudin-4 (EMA usually +) SALL4, CD34, SOX2, CD99 consistently negative

SALL4 - Tight junction protein -Consistently neg. in MRTs , SCCOHTs, Epith.Sarc. -vs- pos. in CAs1 -Consistently neg. in SMARCA4 sarcoma-like thoracic tumors -vs- pos. in NSCC2,3

1. Schaefer IM et al Mod Pathol. 2017 2. Yoshida A et al Mod Pathol 2017 3. Montecalvo … Rekhtman et al Mod Pathol 31, suppl 2 Immunohistochemical features: other key features Stem cell markers Low/absent kertains frequently positive: Claudin-4 SMARCA2 (BRM) (EMA usually +) SALL4, CD34, SOX2, CD99 consistently negative co-deficient (most)1-3

SALL4 vs NSCC usually SMARCA4-intact1-3 IHC profile (variable CK, stem cell marker expression, Dual loss of SMARCA4/A2 predicts negative Claudin-4, SMARCA2 co-deficiency) – sensitivity to a novel class of drugs similar to pediatric MRTs, SCCOHTs, undif/dedif endometrial CA… - EZH2 inhibitors and distinct from NSCC Januario et al PNAS 2017, Wang Y et al J Pathol 2017 SMARCA4-deficient thoracic sarcoma-like tumors: Distinctive clinicoradiologic features.

- Predominantly (~90%) smokers, but few never smokers - Extremely large, locally-infiltrative tumors, frequently involving both lung and mediastinum - Uniquely broad age range, including typical age for NSCC and younger (30-50 yo) patients, predominantly smokers

Combination of young (<50) + smoker + large thoracic mass  think of this tumor! SMARCA4-deficient thoracic sarcoma-like tumors: Distinctive clinicoradiologic features.

- Predominantly (~90%) smokers, but few OS by two groups of stage IV n = 59 never smokers 1.0 BRG-def undif n = 19 BRG def carc n = 40 - Extremely large, locally-infiltrative tumors, Log-rank p=0.004 frequently involving both lung and 0.8

mediastinum 0.6

Log-rank p = 0.004 - Uniquely broad age range, including typical NSCC (n=40) 0.4 age for NSCC and younger (30-50 yo) Survivalprobability median survival: 20.7 mo

patients, predominantly smokers 0.2 sarcoma-like (n=19)

- Dismal survival – significantly worse than 0.0 median survival: 5.2 mo 1 for SMARCA4-def. NSCC 0 10 20 30 40 50 60 Months since diagnosis

1. Montecalvo … Rekhtman et al Mod Pathol 31, suppl 2 Are these sarcomas or sarcomatoid (undifferentiated/dedifferentiated) carcinomas?

• Le Loarer F et al: In expression profiling studies, these tumors clustered with MRTs & SCCOHTs, but were distinct from SMARCA4-def. lung carcinomas, leading to the conclusion that these represent primary thoracic sarcomas. Plus, no carcinomatous components documented.

• Indeed, by morphology and marker expression these tumors have the phenotype analogous to MRT and SCCOHT (low/variable epithelial markers, + stem cell markers)

• Unusual features for MRT-like sarcomas included:

✓ Most patients – even younger – were smokers, many with emphysematous lung disease

✓ Sequencing (Le Loarer et al and Yoshida et al): • complex genomes, with TP53 mutations in almost all cases • Yoshida et al: 2/5 tumors had NSCLC-type driver mutations (1 KRAS, 1 KEAP1)

✓ Focal TTF-1 and p40 expression in some cases in each study Sarcoma vs sarcomatoid carcinoma? Documentation of NSCC components (i.e. SMARCA4-deficient composite rhabdoid tumors)

pan-CK claudin-4

Rhabdoid NSCC Rhabdoid NSCC

Rhabdoid NSCC SMARCA4 SMARCA2

Rhabdoid NSCC Rhabdoid NSCC Sarcoma vs sarcomatoid carcinoma? Documentation of NSCC components (i.e. SMARCA4-deficient composite rhabdoid tumors)

pan-CK claudin-4

p40 SMARCA4 Sarcoma vs sarcomatoid carcinoma? Next-generation sequencing and genomic smoking signature analysis

Genomic smoking signature analysis

NSCC-type - STK11, KEAP1, KRAS - in 8/18 cases (44%), including both combined and pure cases.1 SMARCA4-def. Conventional Together with documentation of SMARCA4-def. composite tumors, molecular data undif/rhabdoid soft tissue + support that these tumors represents undif/dedif carcinomas with rhabdoid features tumors thoracic sarcomas (n=35) rather than primary sarcomas. (n=16) This may have implications not only for taxonomy purposes, but mutational background and cell lineage may impact whether these tumors respond to EZH2 inhibitors ~ to MRTs.2 Overall, therapeutic approach to these aggressive tumors remains TBD.

1. Montecalvo … Rekhtman. Mod Pathol 31, suppl 2; p. 742. 2 Chan-Penebre et al Mol Cancer Ther 2017 Summary and conclusions • SMARCA4-deficient conventional adenocarcioma and other NSCCs • Associated with poor yet non-rhabdoid morphology, lack of TTF-1 • Strong predictor of poor prognosis, and several recent studies suggest potential druggability

• SMARCA4-deficient undif. rhabdoid sarcoma-like tumors • A distinct category for previously unclassified or misclassified tumors… BUT we are getting SMARC-ER! • Unexpected IHC profile (e.g. CD34+, SALL4+) can present diagnostic pitfall • Predominantly older smokers, but distinctive subgroup of younger (<50) yo smokers and rare never smokers • Several lines of evidence support that these tumors are nosologically related to carcinoma/epithelial precursors