BEYOND LINEAGE:

DIAGNOSTIC AND PREDICTIVE

MOLECULAR IMMUNOHISTOCHEMISTRY
Jason L Hornick, MD, PhD
Director of Surgical Pathology
and Immunohistochemistry
Brigham and Women’s Hospital
Professor of Pathology
Harvard Medical School
Boston, MA, USA
 Evolution of Immunohistochemistry

20th Century 21st Century

DIFFERENTIATION MOLECULAR
(LINEAGE) GENETICS
 Conventional Immunohistochemistry
Line of differentiation IHC markers
Epithelial Keratins
Neural crest
S100 protein, SOX10
(melanocytic/Schwann cell)
Hematopoietic CD45 (LCA)
Smooth muscle SMA, desmin
Skeletal muscle Desmin, MyoD1, myogenin
Vascular (endothelial) CD31, ERG
 “Next-Generation” Immunohistochemistry

• Protein loss due to tumor suppressor gene inactivation

• Epigenetic alterations of proteins (e.g., methylation)

• Protein products of gene rearrangements for diagnosis

and therapy predictive testing

• Mutant oncoprotein and fusion-specific antibodies

 Protein Loss Due to Tumor Suppressor
Gene Inactivation – Examples

Gene Tumor types

BAP1 Mesothelioma, melanoma, renal cell carcinoma

CDC73 (parafibromin) Parathyroid carcinoma

SMARCB1 (INI1) Malignant rhabdoid tumor, epithelioid sarcoma

SMAD4 Pancreatic ductal adenocarcinoma

SMARCA4 (BRG1) Small cell “carcinoma” of the ovary, hypercalcemic type

 BAP1 Tumor Predisposition Syndrome
• Germline mutations in BAP1
• Autosomal dominant inheritance
• Predisposition to development of benign melanocytic
neoplasms, uveal melanoma, cutaneous melanoma,
pleural malignant mesothelioma, renal cell carcinoma
• Sporadic counterparts of these tumors may harbor
somatic BAP1 mutations
• Loss of BAP1 by IHC helpful to diagnose mesothelioma
Early Malignant Mesothelioma
Early Malignant Mesothelioma
Early Malignant Mesothelioma

BAP1
Early Malignant Mesothelioma

BAP1
 Small Cell Carcinoma of the Ovary,
Hypercalcemic Type
• Adolescents and young women (peak, 18 – 30 years)

• Some cases familial

• Large abdominal or pelvic mass

• Hypercalcemia in two-thirds of cases

• Very poor prognosis (33% survival when diagnosed

at early stage)
 Small Cell Carcinoma of the Ovary,
Hypercalcemic Type
• Sheet-like growth pattern most common
• Follicle-like structures with eosinophilic fluid in 80%
• Relatively uniform cytomorphology
• Round cells with hyperchromatic nuclei and minimal
cytoplasm
• Large cell component in 50% of cases: epithelioid cells
with abundant eosinophilic cytoplasm, often rhabdoid
• Proposal to re-name “malignant rhabdoid tumor of the
ovary”
Small Cell Carcinoma of the Ovary, Hypercalcemic Type
Small Cell Carcinoma of the Ovary, Hypercalcemic Type
Small Cell Carcinoma of the Ovary, Hypercalcemic Type
 Small Cell Carcinoma of the Ovary,
Hypercalcemic Type and SMARCA4
• Consistent mutations in SMARCA4 (most often
nonsense or frameshift)

• Loss of heterozygosity (tumor suppressor)

• Up to 50% of patients: germline mutation

(variable penetrance)

• Consistent loss of SMARCA4 (BRG1) by IHC

Small Cell Carcinoma of the Ovary, Hypercalcemic Type

SMARCA4
 Epigenetic Alterations:
Malignant Peripheral Nerve Sheath Tumor
• Arise in patients with NF1, sporadically, or following
radiation therapy
• Challenging diagnosis
• Diagnostic criteria:
1. Origin from a nerve or a neurofibroma
2. Spindle cell sarcoma in a patient with NF1
3. Evidence of Schwann cell differentiation by IHC or EM
» S100 protein and SOX10 only 30-50% sensitivity
• Diagnosis in sporadic setting relies on distinctive
histology and exclusion of mimics
Malignant Peripheral Nerve Sheath Tumor
 Polycomb Repressive Complex

Epigenetic modification of chromatin:

• PRC2 recruits to chromatin and trimethylates histone H3 at lysine 27
Physiologic regulation of cell fate and proper stem cell differentiation
Deregulation → cancer development
Modified from Sauvageau et al. Cell Stem Cell 2010
Oct 2014

Nov 2014

Nov 2014
 PRC2 and MPNST
• PRC2 alterations (SUZ12 or EED mutations) in 85-90%
of MPNST

• Homozygous mutations result in loss of H3K27me3

(histone H3 lysine 27 trimethylation) in ~65% of MPNST

• Rate of H3K27me3 loss depends on grade

• IHC for H3K27me3 highly specific diagnostic marker

Schaefer et al. Mod Pathol 2016

Prieto-Granada et al. Am J Surg Pathol 2016

 Immunohistochemistry for H3K27me3 in
Malignant Peripheral Nerve Sheath Tumors

Grade H3K27me3 loss

Low grade 35%

Intermediate grade 60%

High grade 80%

Schaefer et al. Mod Pathol 2016

Prieto-Granada et al. Am J Surg Pathol 2016

Immunohistochemistry for H3K27me3 in
Other Spindle Cell Neoplasms
Tumor type H3K27me3 loss
Cellular schwannoma 0%
Atypical neurofibroma 0%
Monophasic synovial sarcoma 0%
Leiomyosarcoma 0%
Myxofibrosarcoma 0%
Malignant solitary fibrous tumor 0%
Low-grade fibromyxoid sarcoma 0%
Spindle cell rhabdomyosarcoma 0%
Gastrointestinal stromal tumor 0%
Fibrosarcomatous DFSP 0%
Dedifferentiated liposarcoma 6%
Spindle cell melanoma 7%
Malignant Peripheral Nerve Sheath Tumor

H3K27me3
Malignant Peripheral Nerve Sheath Tumor

H3K27me3
Monophasic Synovial Sarcoma

H3K27me3
Protein Correlates of Gene Rearrangements for
Therapy Predictive Testing
Gene Tumor types Frequency

Lung adenocarcinoma 4%
ALK
Inflammatory
60%
myofibroblastic tumor

Lung adenocarcinoma 1%
ROS1
Inflammatory
5%
myofibroblastic tumor
Anaplastic Lymphoma Kinase

CD30
Dramatic Responses to Crizotinib
Dramatic Responses to Crizotinib

Kwak et al. N Engl J Med 2010

Tumor Types with ALK Rearrangements
Hematolymphoid
ALK+ anaplastic large-cell lymphoma
ALK+ diffuse large B-cell lymphoma
Systemic histiocytosis
Epithelial
Lung adenocarcinoma
Renal cell carcinoma
Other carcinomas (very rare)
Melanocytic
Spitz tumor
Mesenchymal
Inflammatory myofibroblastic tumor
Epithelioid fibrous histiocytoma
 ALK Fusion Partner Sometimes Results in
Distinctive Patterns of Staining by IHC
Fusion partner Tumor types ALK staining pattern

NPM ALCL, DLBCL Cytoplasmic/nuclear/nucleolar

TPM3 ALCL, IMT Diffuse cytoplasmic

EML4 Lung AdCA, IMT Diffuse cytoplasmic

CLTC ALCL, DLBCL, IMT Punctate cytoplasmic

RANBP2 EIMS Nuclear membrane

AdCA, adenocarcinoma; ALCL, anaplastic large-cell lymphoma; DLBCL, diffuse large B-cell lymphoma;
EIMS, epithelioid inflammatory myofibroblastic sarcoma; IMT, inflammatory myofibroblastic tumor
Anaplastic Large Cell Lymphoma
NPM::ALK

ALK
Lung Adenocarcinoma
EML4::ALK

ALK
Epithelioid Inflammatory Myofibroblastic Sarcoma
RANBP2::ALK

ALK
Marked Differences in ALK Protein Expression
Levels: Implications for Antibody Selection
Tumor Level of fusion Monoclonal
IHC result
type protein expression antibody
ALK1 Positive
ALCL High
5A4, D5F3 Positive
ALK1 Usually positive
IMT Intermediate
5A4, D5F3 Positive

Lung ALK1 Usually negative

Low
AdCA 5A4, D5F3 Positive
AdCA, adenocarcinoma; ALCL, anaplastic large-cell lymphoma; IMT, inflammatory myofibroblastic tumor
Multifocal Recurrent EIMS Treated
with ALK Inhibitor Crizotinib

3 months

Butrynski et al. N Engl J Med 2010

Lung Adenocarcinoma
CD74::ROS1

ALK ROS1
 IHC as a Surrogate for FISH or NGS:
Stand-Alone Predictive Testing for NSCLC?

Stand-
IHC Clone Sensitivity Specificity
alone test
ALK1 Low High No
ALK
5A4, D5F3 High High Yes

ROS1 D4D6 High Moderate No

Protein Products of Gene Rearrangements for
Therapy Predictive Testing: NTRK

genes proteins IHC

NTRK1 TRKA

NTRK2 TRKB Pan-TRK

NTRK3 TRKC
Drilon et al. N Engl J Med 2018
 Tumor type Pan-TRK+ (%)
Colorectal adenocarcinoma 3/223 (1.3%)
Non-small cell lung carcinoma 0/108 (0%)
Gastroesophageal carcinoma 1/71 (1.4%)
Others 0/45 (0%)
TOTAL 4/227 (0.9%)
Only 1 case with confirmed NTRK fusion!
Hechtman JF. Mod Pathol 2022
Rosen et al. Clin Cancer Res 2020
Mutant Oncoprotein-Specific Antibodies

Gene Mutation Tumor Type

EGFR L858R, E746-A750del Lung adenocarcinoma

BRAF V600E Melanoma, many others

IDH1 R132H Gliomas

H3F3A K27M Diffuse intrinsic pontine glioma

H3F3A G34W Giant cell tumor of bone

H3F3B K36M Chondroblastoma

High Grade Glioma

IDH1R132H
Melanoma

NRASQ61R
Metastatic Melanoma

BRAFV600E
Giant Cell Tumor of Bone

H3G34W
 Fusion-Specific Antibody: Synovial Sarcoma
• Relatively common soft tissue sarcoma: 7% overall
• Peak in young adults; predilection for extremities
• Aggressive: 5-yr and 10-yr survival 60% and 50%
• Harbors pathognomonic t(X;18)(p11;q11)
• Results in SS18::SSX1 >> SS18::SSX2 (rarely SS18::SSX4)
• Monophasic, biphasic, and poorly differentiated variants
• Considerable overlap with other tumor types
• IHC using conventional markers lacks specificity
Antibody Sensitivity Specificity

SS18::SSX 95% 100%

SSX 100% 96%

 IHC with SS18::SSX fusion-specific antibody
Tumor type Total cases SS18::SSX +
Synovial sarcoma 233 94%
Malignant peripheral nerve sheath tumor 128 0%
Solitary fibrous tumor 52 0%
Dedifferentiated liposarcoma 87 0%
Leiomyosarcoma 64 0%
Ewing sarcoma 35 0%
Mesothelioma (sarcomatoid) 24 0%
Sarcomatoid carcinoma 19 0%
Baranov et al. Am J Surg Pathol 2020
Zaborowksi et al. Histopathology 2020
Perret et al. Am J Surg Pathol 2021
Biphasic Synovial Sarcoma

SS18::SSX SSX
Monophasic Synovial Sarcoma

SS18::SSX SSX
Monophasic Synovial Sarcoma

SS18::SSX
Poorly Differentiated Synovial Sarcoma

SS18::SSX SSX
Myxoid Liposarcoma
High Grade Myxoid (“Round Cell”) Liposarcoma
 Genetics of Myxoid Liposarcoma

Translocation Gene fusion Frequency

t(12;16) FUS::DDIT3 90%

t(12;22) EWSR1::DDIT3 10%

Baranov et al. Mod Pathol 2021
Myxoid Liposarcoma

DDIT3
Screening for Familial Predisposition Syndromes
Markers Tumor Types Syndrome
Colonic adenocarcinoma,
MMR proteins Lynch Syndrome
endometrial adenocarcinoma, others
Familial Paraganglioma/
SDHB Paraganglioma, GIST, RCC
Pheochromocytoma
Hereditary
RCC, cutaneous and uterine
FH Leiomyomatosis and
leiomyomas
Renal Cell Cancer
Atrial myxoma, malignant melanotic
PRKAR1A Carney Complex
nerve sheath tumor, others
Parathyroid adenoma and Hyperparathyroidism-
Parafibromin
carcinomas Jaw Tumor Syndrome
 Succinate Dehydrogenase
Mutations and Tumorigenesis
• Familial paraganglioma syndrome
– Most common inherited paraganglioma syndrome
– Germline mutations in SDH subunit genes (complex II of
electron transport chain/Kreb cycle)
• Carney-Stratakis syndrome (paraganglioma + gastric
GIST)
– Also caused by germline mutations in SDH subunit genes
• Carney triad (paraganglioma + gastric GIST +
pulmonary chondroma)
– Usually caused by SDHC promoter hypermethylation
 Succinate Dehydrogenase Deficiency
• Mutations in SDH subunit genes (or promoter
methylation) lead to loss of protein expression
• IHC for SDHB: loss of staining (irrespective of which
gene is mutated)
• IHC for SDHB can identify SDH-mutant GISTs
• Similar findings observed in Carney triad-associated,
pediatric, and similar adult “wild-type” gastric GISTs
(without KIT/PDGFRA mutations)
• IHC for SDHB is a good tool for identifying this clinically
distinctive class of gastric GISTs: “SDH-deficient GISTs”
 SDH-deficient GISTs
• Only arise in the stomach
• Multinodular/plexiform growth pattern
• Epithelioid >> mixed morphology
• Not that rare (~8% of gastric GISTs)
• Lymph node metastases common
• Distant metastases common – clinically indolent
• Current risk assessment criteria do not predict
behavior for this class
• Limited (if any) response to imatinib
SDH-deficient GIST
SDH-deficient GIST

Stomach
KIT-mutant GIST
SDH-deficient GIST
SDH-deficient GIST
SDH-deficient GIST
Metastatic SDH-deficient GIST
Metastases According to Risk Category
Gastric GIST
Risk category SDH-deficient GIST
(Miettinen)
None 0% 33%
Very low 2% 67%
Low 3.5% 60%
Moderate 12-16% 71%
High 55-86% 82%

Mason and Hornick Am J Surg Pathol 2016

Imatinib is Not Effective for
SDH-deficient GIST

Baseline Imatinib 6 months

 SDH-deficient GISTs: IHC

• Similar phenotype as conventional GIST (KIT, DOG1,

CD34+)

• IHC for SDHB: loss of normal granular cytoplasmic

(mitochondrial) staining

• IHC for SDHB excellent tool for confirming diagnosis

of this clinically distinctive class of gastric GISTs
KIT
 KIT DOG1

CD34 desmin
IHC for SDHB and Genotype

Doyle et al. Histopathology 2012

KIT exon 11-mutant GIST

SDHB
SDH-deficient GIST

SDHB
Multinodular architecture in GIST is highly
associated with SDH deficiency

Sensitivity 99%

Specificity 99%

Doyle et al. Histopathology 2012, with updated data

 SDHx Mutations in GIST

• SDHA mutations most common (35-40%); although

germline, usually appear sporadic

• Lead to loss of expression of BOTH SDHB and SDHA

• SDHB, SDHC, and SDHD mutations (25%) often

associated with familial clustering, sometimes with
paraganglioma (Carney-Stratakis)
 Wagner et al. Mod Pathol 2013

SDHA-
mutant
GIST

SDHB SDHA

SDHB-
mutant
GIST

SDHB SDHA
 What about SDH-Deficient GISTs that
lack SDHx Mutations?
• Nearly all show SDHC promoter-specific CpG island
hypermethylation (“epimutation”) and gene silencing

• Most GISTs in Carney triad have SDHC epimutation

• Leads to loss of expression of SDHB by IHC (similar

to tumors with mutations)

Killian et al. Sci Transl Med. 2014

 Summary

• Rapid evolution of new generation of IHC markers

• Replacement for molecular genetic testing in

many cases

• Rapid and inexpensive therapy predictive testing

• Broad screening for familial cancer predisposition

syndromes
jhornick@bwh.harvard.edu
@JLHornick

THANK YOU!