COLONIC ADENOCARCINOMAS HARBORING NTRK FUSION GENES: A CLINICOPATHOLOGIC AND MOLECULAR GENETIC STUDY OF 16 CASES AND REVIEW OF THE LITERATURE

Abstract

This study was undertaken to determine the frequency, as well as the clinicopathologic and genetic features, of colon cancers driven by neurotrophic receptor tyrosine kinase (NTRK) gene fusions. Of the 7008 tumors screened for NTRK expression using a pan-Trk antibody, 16 (0.23%) had Trk immunoreactivity. ArcherDx assay detected TPM3-NTRK1 (n=9), LMNA-NTRK1 (n=3), TPR-NTRK1 (n=2) and EML4-NTRK3 (n=1) fusion transcripts in 15 cases with sufficient RNA quality. Patients were predominantly women (median age 63 years). The tumors involved the right (n=12) and left colon unequally and were either stage T3 (n=12) or T4. Local lymph node and distant metastases were seen at presentation in 6 and 1 patients, respectively. Lymphovascular invasion was present in all cases. Histologically, tumors showed moderate to poor (n=11) differentiation with a partly or entirely solid pattern (n=5) and mucinous component, (n=10) including 1 case with sheets of signet ring cells. DNA mismatch repair deficient phenotype was seen in 13 cases. Tumor-infiltrating CD4/CD8 lymphocytes were prominent in 9 cases. PD-L1(+) tumor-infiltrating immune cells and focal tumor cell positivity were seen in the majority of cases. CDX2 expression and loss of CK20 and MUC2 expression were frequent. CK7 was expressed in 5 cases. No mutations in BRAF, RAS and PIK3CA were identified. However, other genes of PI3K-AKT/MTOR pathway were mutated. In several cases, components of Wnt/β-catenin (APC, AMER1, CTNNB1), p53 and TGFβ (ACVR2A, TGFBR2) -pathway were mutated. However, no SMAD4 mutations were found. Two tumors harbored FBXW7 tumor suppressor gene mutations. NTRK fusion tumors constitute a distinct but rare subgroup of colorectal carcinomas.

INTRODUCTION

Colonic adenocarcinoma (from here on “colon cancer”) is genetically heterogenous and characterized by a range of genomic and epigenomic alterations, most of which are mutations in oncogenes or tumor suppressor genes.¹ Through recent advances in sequencing technology, oncogenic fusion genes, earlier known from sarcomas and lymphomas, have been also identified in epithelial cancers, including colorectal carcinoma (CRC).^2–4 Fusions frequently involve genes encoding receptor tyrosine kinases (RTKs) and result in the expression of chimeric proteins. These proteins typically contain activated kinases, inducing MAPK and AKT downstream and signaling pathways that promote tumorigenesis.⁵

Neurotrophic receptor tyrosine kinase (NTRK) genes, NTRK1 (chromosome 1q21-q22), NTRK2 (chromosome 9q22) and NTRK3 (chromosome 15q25) encode a family of transmembrane receptor tyrosine kinase proteins, Trk-A, -B and -C. Trk proteins, activated by neurotrophins, are expressed in neuronal tissue and play a central role in the development and function of the human nervous system.⁶ Molecular alterations (predominantly gene fusions) and aberrant activation of NTRK genes have been reported in different types of epithelial, hematopoietic, and mesenchymal malignancies. Typically, the 3’ region of NTRK gene fuses to the 5’ region of a gene partner, forming a chimeric gene/oncogene that expresses constitutively activated tyrosine kinase.⁷ Detection of an oncogenic NTRK fusion has immediate clinical implications. Recently developed Trk inhibitors have shown significant efficacy in the treatment of advanced and metastatic tumors, including colorectal carcinomas harboring oncogenic NTRK fusion genes.^7–9

In colon cancer, the first fusion between TPM3 (Tropomyosin 3) and NTRK1 was identified more than 35 years ago.¹⁰ Subsequently, a small number of CRCs driven by NTRK1- or NTRK3- fusions have been described. However, only limited clinicopathologic data of these tumors are available.^{4,8,9,11–15} A recent study summarized clinicopathologic and molecular genetic features of metastatic CRCs driven by different tyrosine kinase fusions, including NTRK fusions. ¹⁶ The clinicopathologic profile of primary tumors harboring oncogenic NTRK fusions remains to be elucidated. A summary of clinicopathologic data of previously published CRCs harboring NTRK fusions is available in supplemental data Table S1A and Table S1B.

In this study, a large, unselected cohort of colon cancers was screened using immunohistochemistry and targeted RNA sequencing to find tumors with NTRK gene fusions. Sixteen colon cancers harboring NTRK1 or NTRK3 fusions were identified and their clinicopathologic, immunohistochemical, and molecular genetic features were studied in detail.

MATERIAL and METHODS

This study evaluated 7008 anonymized colon carcinomas from Europe (Czech Republic, Finland, Germany, Italy and Poland), Japan, and the United States. Staging of tumors were provided by contributors following The American Joint Committee on Cancer (AJCC) tumor/node/metastasis (TNM) classification and staging recommendation (http://cancerstaging.org) or Dukes staging system.¹⁷ Histological classification of tumors was based on WHO classification of tumors of the digestive system.¹⁸ The density of tumor infiltrating lymphocytes (TILs) was scored following previously reported methods.¹⁹ A score of at least two lymphocytes by high power field was requiered for high level TIL.

All patients underwent a partial colectomy. Clinical information regarding adjuvant chemotherapy was available in 8 cases; it was administered in five patients. None of the patients were known to receive tyrosine kinase-inhibitor therapy.

Tumor samples were analyzed employing tissue microarrays (TMAs) or multitumor blocks. TMAs were constructed using core biopsies and MicaArray kit (MicaArray, New York, NY) or Manual Tissue Arrayer MTA-1 (Beecher Instruments Inc./Estigen, Tartu, Estonia). Multitumor blocks were built manually using rectangular tissue samples, as previously reported.²⁰

Immunohistochemistry

In all 7008 tumors, NTRK1, −2 and −3 expression was evaluated using a pan-Trk antibody clone A7H6R (# 92991, Cell Signaling Technology, Inc., Danvers, MA) and Leica Bond-Max automated immunohistochemistry, Leica Biosystems, Inc. (Buffalo Grove, IL) with 25-minute heat induced epitope retrieval in ER2 buffer.

All pan-Trk-positive tumors were evaluated for the expression of several antigens including DNA-mismatch repair (MMR) proteins [MutL Homolog 1 (MLH1), PMS1 Homolog 2 (PMS2), MutS Homolog 2 (MSH2) and MutS Homolog 6 (MSH6)], Caudal Type Homeobox 2 (CDX2), Catenin Beta 1 (CTNNB1), Cytokeratin 7 and 20 (CK7, CK20), Ki-67, Mucin 2 (MUC2), Tumor Protein P53 (P53) and Programmed death-ligand 1 (PD-L1). In addition, tumor-infiltrating lymphocytes (TIL) and macrophages were characterized with antibodies against cluster of differentiation 4 (CD4), CD8, and CD68. A detailed description of antibodies and immunohistochemical protocols are provided in supplemental data Table S2.

DNA and RNA extraction

DNA and RNA were recovered from formalin fixed paraffin-embedded colon carcinoma specimens using a Maxwell® RSC instrument, DNA or RNA FFPE Kit (Promega, Madison, WI), according to the manufacturer’s protocols.

Archer Dx assay

Target-specific libraries for next-generation sequencing (NGS) were constructed using Archer Universal® RNA Reagent Kit v2 (ArcherDx, Boulder, CO). Library sequencing was accomplished using a MiSeqDx instrument (Illumina, San Diego, CA). NGS data were analyzed using the Archer Analysis Pipeline Virtual Machine (https://archerdx.com). In one case, the result of ArcherDx assay was confirmed in a secondary laboratory that performed a blinded experiment (the prior finding was withheld until after the experiment was completed).

Ion Torrent next-generation sequencing

Next-generation sequencing was performed by Macrogen USA (Rockville, MD) using the Ion Torrent™ (Life Technologies/Thermo Fisher Scientific, Waltham, MA) next-generation sequencing platform. Depending on the DNA quality, either Ion AmpliSeq™ Comprehensive Cancer Panel (409 gene targets) or Ion AmpliSeq™ Cancer Hotspot Panel v2 Kit (50 gene targets) was employed. All fifty genes targeted by the Cancer Hotspot Panel were included in the Comprehensive Cancer Panel.

Bioinformatics analysis of NGS-data was processed by Torrent Server Suite 4.2 and sequences aligned to human genome reference sequence HG-19 (The Genome Reference Consortium). Variant calling was performed using Variant Caller v4.2, which is compatible with the Integrative Genomics Viewer (Broad Institute, Cambridge, MA), a high-performance visualization tool for interactive exploration of large, integrated data sets. Mutation nomenclature was based on recommendations from Human Genome Mutation Society (www.hgvs.org). The FATHMM (Functional Analysis Through Hidden Markov Models), SIFT (Sorting Intolerant from Tolerant) and PolyPhen (Polymorphism Phenotyping) scores predicting functional consequences of coding variants were either obtained from the COSMIC (Catalogue of Somatic Mutations in Cancer) at https://cancer.sanger.ac.uk or assessed during bioinformatic analysis.

MLH1 promotor hypermethylation analysis

MLH1 promotor hypermethylation was evaluated in two tumors with loss of MLH1 expression detected by immunohistochemistry. Sodium bisulfite conversion of genomic DNA was executed using EZ DNA Methylation Gold-kit (Zymo Research, Burlington, ON, Canada) and provided by Zym Research procedure. The methylation-specific PCR amplification of the MLH1 promotor and evaluation of PCR amplification products were done as previously reported.²¹

RESULTS

Trk immunohistochemistry and NTRK fusion gene transcripts

Pan-Trk immunostaining was seen in 16 of 7008 analyzed colon cancers (0.23%). Fifteen of the 16 Trk-positive colon cancers contained RNA sufficient for molecular studies. The ArcherDx assay detected NTRK1 (n = 14) and NTRK3 (n = 1) fusion gene transcripts. Nine of these tumors harbored TPM3-NTRK1 chimeric transcripts with either exon (e) 8 to e10 (n=7) or e8 to e13 (n=2) fusion breakpoints. In three cases, LMNA-NTRK1 fusions with three distinctive fusion breakpoints – e4 to e10, e10 to e11, and e11 to e8 – were detected. TPR-NTRK1 chimeric transcripts with identical e21 to e10 fusion breakpoints were identified in two tumors. One colon cancer harbored EML4 (EMAP Like 4)-NTRK3 transcripts with an e2 to e14 fusion breakpoint.

Immunohistochemical patterns for pan-Trk of different types of NTRK fusion colon cancers are listed in Table 1. Tumors harboring TPM3-NTRK1 fusions displayed strong cytoplasmic and membrane pan-Trk positivity and lacked nuclear staining (Figure 1A). Colon cancers with LMNA-NTRK1 fusions showed strong cytoplasmic but weak membrane immunoreactivity and focal nuclear staining (Figure 1B). The latter was not seen in a tumor harboring a LMNA(e4)-NTRK1(e10) fusion.

Table 1.

Type of NTRK fusion and pan-Trk expression pattern identified in 15 colon carcinomas.

Fusion gene	No. of tumors	Membrane pan-Trk staining	Cytoplasmic pan-Trk staining	Nuclear pan-Trk staining
TPM3-NTRK1	9	+++	+++	-
LMNA-NTRK1	3	+ or +/−	+++	Focal or scattered cells
TPR-NTRK1	2	+	++	-
EML4-NTRK3	1	-	+	-

Figure 1.

Trk-positive immunohistochemistry in colon cancers. TPM3(e8)-NTRK1(e10) fusion tumor (Case 8), displayed strong, diffuse cytoplasmic and membrane staining (A); LMNA(e10)-NTRK1(e11) fusion tumor (Case 14) with an area of strong nuclear staining and weaker cytoplasmic staining (B); TPR(e21)-NTRK1(e10) fusion tumor (Case 5) showed weaker cytoplasmic and membrane staining than TPM3- and LMNA-NTRK1 fusion tumors and lacked nuclear immunoreactivity (C); EML4(e2)-NTRK3(e14) fusion tumor (Case 12) displayed weak but distinct cytoplasmic staining and no membrane or nuclear immunoreactivity (D).

TPR-NTRK1 fusion tumors displayed weaker cytoplasmic and membrane pan-Trk staining than TPM3- and LMNA-NTRK1 fusion tumors and lacked nuclear immunoreactivity (Fig. 1C). The sole colon cancer with an EML4-NTRK3 fusion revealed weak but distinct cytoplasmic pan-Trk staining and no membrane or nuclear immunoreactivity (Fig. 1D). In this case, the results of the ArcherDx assay were confirmed by a blinded experiment in a secondary laboratory.

Demographic and clinicopathologic features of NTRK fusion colon cancers

Clinical characteristics of NTRK fusion colon cancers are summarized in Table 2. Most of these cancers (13/16, 81%) were diagnosed in women. Median ages for women and men were 63 and 71 years. 38.5 % (5/13) of female patients were in the fifth and sixth decades (age range: 46–56 years). Tumors harboring NTRK fusions involved different portions of the large intestine including cecum (n=2), ascending colon (n=1), hepatic flexure (n=4), transverse colon (n=2), splenic flexure (n=3), descending colon (n=3), and rectosigmoid (n=1). Based on the TNM criteria, NTRK fusion colon cancers were either T3 (n=12) or T4 (n = 4). Five patients had local lymph node metastases. In one case, both local and distant metastases (liver and lung) were diagnosed at presentation. Follow-up data were available in 12 cases. One patient (Case 1) with local and distal metastases died of the disease in 1 month. Two elderly (84- and 86-years old) patients (Case 7 and 9) died of unknown causes after 7 days and 24 months, respectively. Nine patients were alive without disease with follow-up ranging from 11 months to 17 years (median follow-up, 28 months).

Table 2.

Clinical characteristics of 16 colon cancers with positive pan-Trk immunohistochemistry.

Case	Age	Sex	Tumor site in colon	Staging system (pTNM, Dukes)	Follow-up	NTRK fusion gene
1	54	F	Cecum	pT4aN2bM1b	‡DOD (1m)	TPM3(e8)-NTRK1(e10)
2	68	F	Cecum	pT3N0M0	§ ANED (1y 5m)	LMNA(e4)-NTRK1(e10)
3	46	F	Ascending	*Dukes C	⸸ANED (17y)	⁑Unknown
4	50	F	Hepatic flexure	pT3N0M0	⸸ANED (1y 10m)	TPM3(e8)-NTRK1(e13)
5	53	F	Hepatic flexure	pT3N1aM0	⸸ANED (2y 4m)	TPR(e21)-NTRK1(e10)
6	63	M	Hepatic flexure	pT3N1M0	‡ANED (7y)	TPM3(e8)-NTRK1(e13)
7	86	F	Hepatic flexure	pT3N0M0	§ DOC (7d)	LMNA(e11)-NTRK1(e8)
8	77	F	Transverse colon	pT3N0M0	NA	TPM3(e8)-NTRK1(e10)
9	84	M	Transverse colon	pT3N0M0	§ DOC (2y)	TPM3(e8)-NTRK1(e10)
10	63	F	Splenic flexure	pT3N0M0	⸸ANED (3y 9m)	TPM3(e8)-NTRK1(e10)
11	68	F	Splenic flexure	pT3N0M0	‡ANED (11m)	TPR(e21)-NTRK1(e10)
12	71	F	Splenic flexure	pT3N0M0	‡ANED (12y 4m)	EML4(e2)-NTRK3(e14)
13	56	F	Descending	pT4aN2bM0	NA	TPM3(e8)-NTRK1(e10)
14	62	F	Descending	pT4aN2bM0	⸸ANED (1y 1m)	LMNA(e10)-NTRK1(e11)
15	71	M	Descending	pT3N0M0	NA	TPM3(e8)-NTRK1(e10)
16	70	F	Rectosigmoid junction	pT4aN0M0	NA	TPM3(e8)-NTRK1(e10)

Abbreviations and symbols: ANED alive, no avidance of disease; DOC died of unknown causes; DOD died of disease; NA not available; y-years, m-months, d-days;

^⸸Adjuvant chemotherapy;

^§No adjuvant chemotherapy;

^‡adjuvant chemotherapy status unknown.

^*6 cm tumor, metastases in local lymph nodes.

^⁑RNA failed quality control test.

Histologic features

The majority of NTRK colon cancers showed moderate to poor (n=11) or poor (n=4) differentiation. (Figure 2A) Eight tumors displayed focal (n=6) or extensive (n=3) solid growth areas, while one revealed solid ribbon-like growth pattern consistent with medullary morphology (Figure 2B). Focal to extensive mucinous component was seen in eight tumors, including one with sheets of signet ring cells (Figure 2C). A vague nested pattern was present in 1 case (Figure 2D). The lymphovascular invasion was present in all tumors. Nine colon cancers had a high level (≥2 TIL/HPF) of tumor infiltrating lymphocytes. The histologic features of NTRK fusion tumors analyzed in this study are summarized in Table 3.

Figure 2.

Histologic features of Trk-positive colon cancers. TPM3(e8)-NTRK1(e10) fusion tumor (Case 13) with moderate to poor differentiation (A); LMNA(e11)-NTRK1(e8) fusion tumor (Case 7) with solid areas displaying ribbon-like growth pattern (B); LMNA(e10)-NTRK1(e11) fusion tumor (Case 14) with prominent sheets of signet ring cells (C); EML4(e2)-NTRK3(e14) fusion tumor (Case 12) showed a vague nested growth pattern (D).

Table 3.

Histopathological characteristics of 16 colon cancers with positive pan-Trk immunohistochemistry.

Case	Degree of glandular differentiation	Solid growth area	Mucinous component	Tumor infiltrating lymphocytes (TILs)
1	Moderate to poor	Focal	No	αLow
2	Poor	Extensive	Focal	βHigh
3	Moderate to poor	No	Focal	Low
4	Moderate to poor	Focal	§Extensive	Low
5	Moderate to poor	No	Focal	High
6	Moderate	No	No	Low
7	Poor (medullary subtype)	Extensive	No	Low
8	Moderate to poor	Focal	Focal	High
9	Moderate to poor	No	No	High
10	Moderate to poor	No	No	High
11	Moderate to poor	*Focal	§Extensive	Low
12	None	All	No	High
13	Moderate to poor	Focal	Focal	High
14	Poor	⸸Focal	§Extensive	Low
15	Moderate to poor	No	Yes	High
16	Poor	Extensive	Focal	High

^*Minimal;

^⸸Sheets of signet ring cells;

^§Luminal;

^α< 2 TIL/HPF;

^β≥ 2 TIL/HPF

Immunohistochemical profile

Thirteen of 16 (81%) NTRK fusion colon cancers revealed loss of MLH1 and PMS2 expression, indicating mismatch repair protein deficiency. Loss of MSH6 expression was seen in one MLH1/PMS2-deficient tumor. All colon cancers expressed MSH2. CK 20 expression was variable and presented in 9/16 tumors (56%), including 6 with focal immunostaining (Figure 3A). CK7 was expressed in 5/16 tumors (31%); in 4 cases showed extensive expression. While most tumors were CDX2-positive, complete or focal loss of expression was seen in 4 cases (Figure 3B). MUC2 expression was absent (8/16) or focally present (6/16) in 87.5% of tumors (Figure 3C). The majority (13/16) of colon cancers revealed a high proportion (80–100%) of Ki67 positive tumor cells (Fig 3D). Confluent nuclear p53 expression was identified in four colon cancers, including three with MMR-proficiency (Figure 4A, B). One tumor was entirely negative, while the remaining eleven displayed a variable number of tumor cells with p53 positive nuclei. Membrane and cytoplasmic β-catenin immunostaining were seen in all cases. However, in one MMR-deficient tumor, prominent nuclear β-catenin accumulation was noted (Figure 4C, D). Detailed tumor immunoprofiles are presented in Table 4.

Figure 3.

Immunohistochemistry of Trk-positive colon cancers. Focal CK20 expression (A) in TPM3(e8)-NTRK1(e10) fusion tumor (Case 15); Focal CDX2 expression (B) in LMNA(e11)-NTRK1(e8) fusion tumor (Case 7); Focal MUC2 expression (C) in LMNA(e4)-NTRK1(e10) fusion tumor (Case 2); Diffuse (near 100%) Ki67 expression (D) in TPR(e21)-NTRK1(e10) fusion tumor (Case 5).

Figure 4.

Involvement of p53 and β-catenin pathways in Trk-positive colon cancers. TPM3(e8)-NTRK1(e10) fusion tumor (Case 1) with retained MLH1 expression (A) harbored TP53 mutation [p.Met246Arg] and displayed diffuse p53 immunostaining (B); LMNA(e11)-NTRK1(e8) fusion tumor (case 7) with loss of MLH1 expression (C) harbored APC mutation [p.Cys1502Ter] and displayed nuclear β-catenin accumulation (D).

Table 4.

Immunophenotype of 16 colon cancers with positive Trk immunohistochemistry.

Case	MLH1	PMS2	MSH2	MSH6	CK7	CK20	CDX2	MUC2
1	Retained	Retained	Retained	Retained	100	100	100	(−)
2	Loss	Loss	Retained	Retained	100	(−)	(−)	Focal
3	Loss	Loss	Retained	Retained	(−)	(−)	100	(−)
4	Loss	Loss	Retained	Retained	(−)	Focal	100	Focal
5	Loss	Loss	Retained	Retained	100	Focal	100	(−)
6	Retained	Retained	Retained	Retained	(−)	100	100	(−)
7	Loss	Loss	Retained	Retained	(−)	(−)	Focal	(−)
8	Loss	Loss	Retained	Retained	(−)	Focal	100	(−)
9	Loss	Loss	Retained	Retained	(−)	Focal	100	Focal
10	Loss	Loss	Retained	Retained	(−)	(−)	(−)	Sc
11	Loss	Loss	Retained	Retained	(−)	Focal	100	Sc
12	Loss	Loss	Retained	Retained	100	Sc	100	Sc
13	Loss	Loss	Retained	Loss	(−)	Sc	100	(−)
14	Retained	Retained	Retained	Retained	Focal	100	100	100
15	Loss	Loss	Retained	Retained	(−)	(−)	100	100
16	Loss	Loss	Retained	Retained	(−)	(−)	Focal	(−)

Sc scattered cells

In nine cases, CD4 and CD8 immunostaining showed high numbers of tumor-infiltrating T cell lymphocytes; CD4+ cells were less prominent. Cases with CD4+/CD8+ TIL immunoreactivity displayed stroma rich in CD68-positive tumor-infiltrating macrophages. Thirteen cases showed a variable PD-L1+ population of tumor-infiltrating immune cells, while tumor cell positivity was absent to minimal.

DNA methylation studies

DNA methylation study was performed on two colon cancers (Case 5 and Case 10) and revealed hypermethylation of MLH1 promotor.

Mutation profiles of NTRK fusion tumors

Four hundred-nine genes were sequenced in nine NTRK fusion tumors (Case 1, 2, 5, 7, 8, 10, 13, 15, and 16) with well-preserved DNA. Because of insufficient DNA quality, Case 11 was evaluated with a panel of 50 gene targets. The genes mutated in these NTRK fusion colon cancers are listed in Table 5 and in supplemental data Table S3. No mutations in MLH1, PMS2, MSH2, and MSH6 were identified in eight MMR-deficient NTRK fusion colon cancers with documented loss of MLH1 and PMS2 by immunohistochemistry. However, one tumor contained a subclone with a pathogenic PMS1 mutation [p.Arg93Cys].

Table 5.

Mutated oncogenes and tumor suppressor genes in NTRK-fusion colon cancers reported in this study.

Gene ID	Mutation	Predicted functional consequences*	Case
ACVR2A	p.Asp386fs	N/A	2
AKT1	p.Ser240Pro	Pathogenic	10
AMER1	p.Arg531Ter	Neutral	8
AMER1	p.Gly140Asp	N/A	16
AMER1	p.Val305fs	N/A	2
APC	p.Cys1502Ter	Pathogenic	7
APC	p.Pro750Ser	Pathogenic	16
APC	p.Cys1289Tyr	Pathogenic	11
CTNNB1	p.Arg225Cys	Pathogenic	8
CTNNB1	p.Lys354Asn	Pathogenic	2
FBXW7	p.Arg479Gln	Pathogenic	13
FBXW7	p.Trp446Ter	Pathogenic	11
MTOR	p.Ala1792Val	Pathogenic	2
PIK3C2B	p.Arg727Trp	Pathogenic	15
PIK3CD	p.Val370fs	N/A	8
PIK3CD	p.Gly245Ser	Pathogenic	15
PIK3R2	p.Val54Met	Pathogenic	5
PTEN	p.His272fs	Pathogenic	1
PTEN	p.Asn323fs	N/A	10
TGFBR2	p.Trp529Ter	N/A	16
TP53	p.Met246Arg	Pathogenic	1
TP53	p.Pro72Arg	Neutral	11
TP53	p.Cys176Tyr	Pathogenic	15

^*Based on the FATHMM, SIFT, PolyPhen scores predicting functional consequences of coding variants.

None of ten NTRK fusion colon cancers harbored mutations in the typical colorectal cancer drivers BRAF, K-RAS, N-RAS, and PIK3CA. However, eight mutations in different components of the PI3K-AKT/MTOR signaling pathway were identified in 56% (5/9) analyzed tumors. These mutations affected AKT1(n=1), MTOR (n=1) PTEN (n=2) and genes (PIK3CD and PIK3R2, and PIK3C2B) encoding different subunits of Class I and Class II Phosphoinositide 3-kinases (PI3Ks).

In five tumors, mutations in APC (n=3), AMER1 (n=3) and CTNNB1 (n=2) components of the Wnt/β-catenin signaling pathway were identified. TP53 was mutated in two colon cancers. Two other tumors contained subclones with mutated FBXW7, another cancer related tumor suppressor gene. Mutations in ACVR2A and TGFBR2, genes playing a role in the TGFβ (transforming growth factor beta) signaling pathway, were identified in two tumors. However, there was no SMAD4 mutations in all analyzed 10 NTRK fusion colon cancers.

DISCUSSION

This study analyzed clinicopathologic and genetic features of 16 colon cancers characterized by Trk expression. Tumors that were immunohistochemically positive with the pan-Trk antibody comprised 0.23% of the screening cohort of 7008 cases. All cases, except one with unsatisfactorily preserved RNA, were shown to harbor NTRK gene fusions. This suggests a high specificity of pan-Trk immunostaining in colon cancer. Previous studies using different antibodies and automation platforms showed high specificity of TrkA (clone EP1058Y/Ab 76291) or pan-Trk (EPR 17341, C17F1) immunohistochemistry in detecting colorectal carcinomas harboring NTRK1 fusions.^{8,9,11,12,22–24} However, previously described perinuclear/nuclear membrane staining in tumors harboring LMNA-NTRK1 fusions²² was not seen in our study. Instead, two tumors with the LMNA fusion involving e10 and e11 displayed focal nuclear immunoreactivity.

The lack of Trk staining has been occasionally reported in colorectal carcinomas harboring TPM3-NTRK1 and ETV6-NTRK3 fusions.^22–23 In this study, colon cancer with the EML4-NTRK3 fusion displayed confluent but weak Trk staining. Little is known about Trk immunoreactivity of colorectal carcinomas harboring NTRK3 fusions, because all such tumors were identified by the RNA sequencing.¹⁶ However, negative or suboptimal Trk immunoreactivity has been reported in pediatric sarcomas and gliomas harboring NTRK3 fusions.^22,25 One group of investigators using TrkA antibody (clone OTI5B6) described strong Trk expression in 6 and 15% of colorectal tumors, respectively, in Chinese and in Korean populations.^13,26 A high frequency of pan-Trk positive tumors was also reported in a study using a cocktail of ALK/pan-Trk and ROS1 antibodies to screen colorectal carcinomas for several different fusions.²⁷ High frequencies of immunopositivity in those studies most likely included false positive results, probably due to incomplete specificity of TrkA antibody (clone OTI5B6) or other technical factors. Previous studies applying Trk immunohistochemistry to search for colorectal tumors harboring NTRK fusions are summarized in supplemental data Table S4A.

Sensitivity of Trk immunohistochemistry in search of NTRK fusion colon cancers cannot be assessed in this investigation because negative cases were not genotyped. However, a next generation sequencing study of 1272 colorectal carcinomas estimated the frequency of NTRK fusion tumors to be around 0.2%, as found in our study.²³ Results of studies applying molecular genetic screening to search for NTRK fusions in colorectal carcinomas are summarized in supplemental data Table S4B.^{3–5,13,23,28,29}

In colorectal carcinomas, NTRK1 has been shown to form fusions with different gene fusion partners including LMNA, PLEKHAG (Pleckstrin Homology Domain Containing AC), SCYL3 (SCY1 Like Pseudokinase 3), TPM3 and TPR.^8,9,13–16 In this study, TPM3-NTRK was the most common (60%) fusion detected in colon cancers. Two other fusion types previously reported in colorectal carcinomas, LMNA-NTRK1 and TPR-NTRK1, were less common and accounted for 20 and 13% of analyzed cases. No NTRK1 fusions engaging PLEKHAG or SCYL3 were detected. All these fusions are the result of intrachromosomal rearrangements between chromosome 1q21.3 (TPM3), 1q22 (LMNA), 1q24.2 (SCYL3), 1q31.1 (TPR), and 1q32.1 (PLEKHA6) and chromosome 1q23.1, NTRK1 locus. (www.genecards.org) Variants of NTRK1 fusions reported in colorectal carcinoma in this study, as well as in previous studies, are shown in Table 6.

Table 6.

Types of NTRK1 fusions described in 33* colorectal carcinomas in this (n=15) and previously published studies.^{8–14,15,21,26}

NTRK1	LMNA					PLEKHA6	SCYL3	TPM3		TPR
	e4	e6	e9	e10	e11	e22	e11	e7	e8	e16	e21
e8								2⸸
e9									1	1
e10	1					1		6	10		2
e11		1		2	1&				1‡
e12			1				1
e13									2
Subtotal:	6					1	1	22		3
Total:	33

Excluding 16 cases harboring 11 TPM3-, 3 LMNA-, and 2 TPR-NTRK1 fusions reported without specific data on fusion breaks^15,16; e - exon;

^&two fusion transcripts, LMNA(e10)-NTRK1(e11) and LMNA(e11)-NTRK1(e11) formed due to alternative splicing;

^‡two fusion transcripts, TPM3(e8)-NTRK1(e11), TPM3(e8)-NTRK1(e12) in one tumor;

^⸸intraexonic break in one case.

NTRK1 fusions with TPM3 and other gene partners have also been reported in papillary thyroid carcinoma, spitzoid melanocytic neoplasms, intrahepatic cholangiocarcinoma, glioblastoma, pediatric high-grade glioma, non-small cell lung cancer, soft tissue and uterine sarcomas, and a low-grade sarcoma called lipofibromatosis-like neural tumor.^30–38

NTRK3 fusions in colorectal carcinoma appear very rare and only a few cases involving COX5A, EML4, ETV6 and VPS18 have been reported with ETV6(e5)-NTRK3(e15) being the most common molecular event.^4,15,16,29 ETV6-NTRK3 fusion resulting from reciprocal t(12;15)(p13;q25) translocation was first described in infantile fibrosarcoma and congenital mesoblastic nephroma.³⁹ In our study, EML4(e2)-NTRK3(e14) fusion was identified in one tumor with weak Trk staining. Previously, an identical fusion formed by the reciprocal t(2;15)(p21;q25) translocation was reported in a case of colon cancer.⁴ In vitro studies documented that EML4-NTRK3 chimeric protein leads to the oncogenic activation of the MAPK/ERK signaling pathway.⁴ Moreover, expression of EML4-NTRK3 oncoprotein in NIH 3T3 cells was sufficient for cellular transformation.⁴⁰ NTRK3 fusions involving EML4, ETV6 and a number of other gene partners were identified in other malignancies originating from different cell lineages. These include: secretory breast carcinoma, secretory carcinoma of the salivary gland, papillary thyroid cancer, radiation-associated thyroid cancer, acute myeloid leukemia, melanocytic neoplasms, glioma, infantile fibrosarcoma, and congenital mesoblastic nephroma.^{23,29,41–47} Variants of NTRK3 fusions reported in colorectal carcinoma in this and previous studies are shown in Table 7.

Table 7.

Types of NTRK3 fusions described in 10^* colorectal carcinomas in this (n=1) and previously published studies^4,15,26 and available at The Cancer Genome Atlas.

	COX5A	ELM4	ETV6	VPS18
NTRK3	e1	e2	e5	e11
e14		2
e15	1		6
e18				1
Total:	10

^*Excluding 4 cases harboring ETV6-NTRK3 fusions reported without specific data on fusion breaks^15,16; e - exon;

Majority of colon cancers harboring NTRK fusions revealed some features characteristic of microsatellite-unstable colorectal tumors, such as female predominance, right colon location, presence of mucinous differentiation and a high level of tumor-infiltrating lymphocytes. In this study, mismatch repair (MMR)-deficiency was identified by immunohistochemistry in 81% (13/16) of analyzed cases. Similar frequency (77%) was previously recorded by a study on metastatic colorectal carcinomas with NTRK fusions.¹⁶ Loss of MLH1 and PMS2 forming MMR protein MuLα complex, lack of mutations affecting MLH1, PMS2, MSH2 and MSH6 genes and evidence of MLH1 promotor methylation suggested the nature of theses alterations was sporadic.

BRAF mutations, typically seen in colon cancer with MMR-deficiency, were not detected in the NTRK fusion tumors analyzed in this study. Additionally, other common colorectal carcinoma drivers – such as K-RAS, N-RAS and PIK3CA – were not involved. An absence of BRAF (V600E) and RAS mutations in NTRK fusion tumors has previously been reported.^8,9,11,12,16 This highlights a primary oncogenic role of chimeric Trk fusion proteins. Nevertheless, in our cohort, components of PI3K-AKT/MTOR signaling pathway other than PIK3CA were affected by mutations implicating this pathway in NTRK fusion tumors.

In contrast to the mutual exclusivity of NTRK1 fusion to oncogenic BRAF and RAS mutations, this study documented the coexistence of mutations in genes of Wnt/β-catenin and p53 pathways in a majority (7/10) of analyzed cases. β-catenin nuclear accumulation (documented by immunohistochemistry) in a tumor with APC mutation and confluent nuclear p53 expression in tumors harboring TP53 mutations supported functional modification of these pathways. Alterations of Wnt/β-catenin and p53 signaling pathways are more common in non-hypermutated tumors.⁴⁸ A recent study reported both APC and TP53 mutations in metastatic NTRK fusion colorectal carcinomas.¹⁶ In two tumors, mutations in FBXW7, a p53-dependent tumor suppressor gene, were identified. Approximately, 10% of human CRCs harbor FBXW7 mutations. Mutational inactivation of FBXW7 contributing to tumor progression is secondary to TP53 mutation.⁴⁹

In this study, mutations in ACVR2A and TGFBR2, components of TGFβ signaling pathway, were identified in two MMR-deficient NTRK fusion colon cancers. Inactivation of TGFβ signaling pathway is a common event in colorectal carcinoma. The components of this pathway are mutated in more than 85% of hypermutated tumors.⁵⁰

The NTRK fusion colon cancers described in this series revealed striking gender predilection to female patients with a 1:4.3 male to female ratio. This simply cannot be explained by a higher frequency of MMR-deficient colorectal carcinomas among female patients, since the male to female ratio is around 1:1.5 among MMR-deficient tumors.⁵¹ Furthermore, 30% of female patients were younger than 60 years old, mimicking cancers with a hereditary predisposition.⁵² Yet, no mutations affecting MMR-genes (MLH1, PMS2, MSH2 and MSH6) were identified.

In this study, a majority of Trk positive tumors were classified as left-sided based on their occurrence up to the splenic flexure.⁵³ However, a recent molecular study indicated that classification of colorectal carcinomas by tumor location better highlights molecular differences.⁵⁴ Taking specific location into consideration, a majority (12/16) of Trk-positive tumors were diagnosed in locations designated uncommon such as hepatic flexure (n=4), transvers colon (n=2), splenic flexure (n=3), and descending colon (n=3).^52,55 The majority of previously published studies omitted information regarding specific location of tumors. Nevertheless, based on the current and previous studies, distribution of NTRK1- versus NTRK3- fusion tumors appears to be different with the latter being more equally present in left and right colon. Three of 7 (43%) NTRK3 colon cancers compared with only 4 of 15 (27%) NTRK1 fusion tumors were diagnosed in the left colon, including sigmoid colon and rectum. (supplemental data Table S1A and S1B)

Mucinous differentiation was seen in 56% (9/16) tumors. In contrast, none of recently reported 8 metastatic colorectal carcinomas with NTRK fusions had mucinous changes.¹⁶ Nevertheless, mucinous differentiation could be seen in 3 of 4 histological images attached to NTRK3 fusion colorectal carcinomas available at The Cancer Genome Atlas (https://www.cancer.gov/tcga).

The mechanisms underlying better prognosis of some colon cancers are incompletely understood. Several positive and negative prognostic markers have been implicated. The high level of tumor infiltrating lymphocytes has been considered a positive prognostic indicator, especially if coupled with deficient MMR status.⁵⁶ In this study, eight of 13 MMR-deficient tumors revealed high levels of CD4 and CD8 positive tumor-infiltrating lymphocytes. These tumors were saturated with CD68 positive macrophages and PD-L1 positive immune cells. However, tumor cells revealed variable focal positivity with no tumor being confluently and strongly positive. Expression of PD-L1 and other immune-checkpoints in NTRK fusion colorectal carcinomas has not been well studied. One study reported TPM3-NTRK1 tumor with PD-L1 amplification and strong diffuse (100%) PD-L1 expression.²³ A durable response to anti PD-1 treatment has been reported in a case of MMR-deficient colon cancer harboring tyrosine kinase fusion (AML4-ALK) with partial PD-L1 positivity.¹⁶

In our cohort, colon cancers with NTRK1 or NTRK3 fusions commonly showed aberrant immunophenotypes with a frequent loss of CK7 and CK20 and MUC2 expression, occasionally accompanied by the loss of CDX2. Such antigenic patterns constituted a highly aggressive subgroup of poorly differentiated colorectal carcinomas with early recurrences and shorter overall survivals.^57–59 Despite these unfavorable prognostic markers, some patients had longer survival – from 45 months to 17 years. However, conclusions of overall survival are hampered here due to limited follow-up data.

Several previous reports on NTRK fusion colorectal cancers presented both disseminated tumors and tumors with no evidence of disease after 4- and 5-years follow-up and no adjuvant chemotherapy.¹¹ A more recent study reported NTRK fusion colorectal carcinomas with synchronous and metachronous lymph node, liver and peritoneal metastases in 100% (8/8) of analyzed case and concluded extremely poor prognosis for these tumors.¹⁶ In our cohort, synchronous metastases were documented in 37.5% (6/16) of NTRK fusion colon cancers, mostly collected from regional and university hospitals. This might include a bias toward more advanced tumors in studies based on cases from cancer centers. Additional multicenter studies are necessary to better define biological potential of NTRK fusion colon cancers.

In order to identify patients who could benefit from TRK inhibitor therapy, two-step screening – employing NTRK immunohistochemistry and followed by molecular genetic testing of positive cases – should be considered in all advanced and/or metastatic BRAF/RAS wild type colorectal carcinomas.

In summary, our study presents the clinicopathologic and molecular genetic profile of the rare primary colon cancers harboring NTRK gene fusions. Although these tumors displayed some phenotypic and genetic features typically seen in the MMR-deficient colon cancers, the separation of NTRK fusion tumors from the MMR-deficient tumors into a new molecular subtype seems to be indicated, especially considering targeted treatment inhibiting oncogenic Trk fusion proteins.