NTRK fusion-positive cancers and TRK inhibitor therapy

1. NTRK fusion-
positive cancers
and TRK inhibitor
therapy
Dr. Arjun
DM ONCOLOGY RESIDENT
Moderator : Dr. Santosh

2. 1. Introduction.
2. TRK biology and oncogenesis
3. TRK in development and physiology
4. TRK activation in cancer
5. NTRK fusions
6. NTRK fusion frequency and diagnosis
7. TRK inhibitor therapy
8. Acquired resistance to TRK inhibition
9. Consequences of TRK inhibition
10.Take home Message

3. What is NTRK ???
• Neurotrophic tyrosine receptor kinase (NTRK) genes.
• Provide Instructions for TRK proteins.
• NTRK gene fuses with an unrelated gene.
• Produces an altered TRK fusion protein.
• active and causes a cancerous tumor to grow.

4. Introduction
• NTRK gene fusions (NTRK1, NTRK2 or NTRK3) are
oncogenic drivers.
• Historically been histology- specific.
• Therapy can either be histology- dependent or
histology- independent

5. Intro. Cont..
Treatment - NTRK fusion- positive cancers is a/w high
response rates (>75%); regardless of tumor
histology.
Histology
Dependent
lower ORRs are achieved with BRAFV600E- mutant
colorectal cancers (0% ORR with vemurafenib) v/s
with BRAFV600E- mutant
melanoma (48% ORR with vemurafenib) or
NSCLC (42% ORR with vemurafenib).
Histology Independent NTRK1, NTRK2 or NTRK3 (encoding TRKA, TRKB and
TRKC, respectively; collectively referred to hereafter as
TRK) –
Histology independent

6. Intro. Cont..
• Alterations are found at high frequencies (up to or
greater than 90%) in rare cancer.
Secretory breast carcinoma, mammary analogue
secretory carcinoma(MASC), cellular or mixed
congenital mesoblastic nephroma and infantile
fibrosarcoma.
• At lower frequencies (commonly <1%) in a range of
other tumour types.

7. TRK Biology and Oncogenesis
• NTRK1 - first identified as an oncogene in 1982.
• In 1989, isolated the cDNA of the NTRK1 proto-
oncogene.
• Protein of 790 amino acids with features
characteristic of cell surface receptor tyrosine
kinases.

8. TRK biology and oncogenesis ctd..
• In 1991, evidences suggested that TRKA was
expressed in the nervous system.
• Became phosphorylated in response to stimulation with
the neurotrophin nerve growth factor (NGF).
• Thus demonstrating the role of TRKA as a receptor
for NGF.

9. TRK biology and oncogenesis ctd..
• Receptors are capable of binding with high affinity to
the following ligands:
NGF- TRKA
brain- derived neurotrophic factor (BDNF) or neurotrophin4
(NT-4) –TRKB
neurotrophin 3 (NT-3) – TRKC

10. TRK biology and oncogenesis ctd..
• Most abundant isoform of TRKA - TRKAII.
• Expressed in most non- neuronal tissues - is
efficiently activated by both NGF and NT-3.
• TRKAI is efficiently activated only by NGF.

11. TRK biology and oncogenesis ctd..
• A third variant, TRKAIII- constitutively active in a
ligand- independent manner –
• Expressed by normal pluripotent neural stem and
neural crest progenitor cells;
• But was originally identified as an oncogenic driver in
neuroblastoma.
• a novel human NTRK2 that encodes a truncated
isoform of the TRKB protein (TRKB- T-TK) -
predominantly in CNS.

12. TRK biology and oncogenesis ctd..
• More recent studies- TRK proteins can function as
active signaling molecules by recruiting scaffolding
proteins, including
 GRP1-associated scaffold protein and
 Rho GDP- dissociation inhibitor 1.
• Together, can activate a wide variety of signaling
pathways- MAPK, PI3K and PKC pathways

13. TRK activation
Autophosporylation of intracellular
tyrosine residues
Activation of down stream signaling pathway
survival and differentiation of neurons,
synapse formation and plasticity,
membrane trafficking,
axon and dendrite formation

15. AKT=v-akt murine thymoma viral oncogene homologue;
DAG=diacyl-glycerol;
ERK=extracellular signal-regulated kinase;
MEK=mitogen-activated protein kinase;
mTOR=mammalian target of rapamycin;
NTRK=neurotrophic tyrosine receptor kinase;
PI3K=phosphatidylinositol-4,5-bisphosphate 3-kinase; PKC=protein kinase C;
PLCy=phospholipase C gamma;
RAF=rapidly accelerated fibrosarcoma kinase;
Ras=rat sarcoma kinase;
TRK=tropomyosin receptor kinase.

16. TRK in development and physiology
• NTRK genes are predominantly - Nervous system
• The types and amounts of neurotrophins and
neurotrophin receptors - crucial - maintaining normal
neuronal homeostasis.
• Indeed, neurotrophins were initially - survival
molecules for neurons;
• However, their functions are now known to be more
diverse and complex.

17. TRK activation in cancer
A. NTRK mutations, splice variants and TRK
overexpression :
Three mechanisms for activation of TRK protein:
1. Somatic NTRK mutation:
Ex: NTRK2 mutations affecting TRKB at two different
kinase domain sites
• T695I and D751N – colorectal cancer and
• within extracellular region (L138F) in the NCI- H2009
lung adenocarcinoma

18. TRK activation in cancer ctd..
2. Activation of splice variant
EX: The NTRK1 splice variant TRKAIII - human
neuroblastoma and
a genomic inframe deletion mutant (ΔTRKA)- AML
specimens.
3. TRK overexpression:
• variety of cancers : breast, cutaneous (for example,
basal cell carcinoma) and lung cancers,
neuroblastoma, cylindroma and others.

19. TRK activation in cancer ctd..
• In Neuroblastoma, TRKA and TRKC overexpression is
strongly predictive of favorable outcomes;
• While TRKB is mainly expressed in higher- grade
tumours that also harbour MYCN amplification.
• In Breast cancer models, ectopic overexpression of
TRKA - MAPK and PI3K pathway activation s/o TRKA
overexpression correlates with tumour
aggressiveness.

20. TRK activation in cancer ctd..
• TRKB and/or TRKC overexpression –
Cylindroma, a tumour that can develop in patients
with germline mutations in the tumour- suppressor
gene CYLD, and
sporadic basal cell carcinomas.

21. B. NTRK fusion:
• NTRK1, NTRK2 or NTRK3 are the most common
mechanisms of oncogenic TRK activation.
• In 1998, the ETV6–NTRK3 gene fusion was
discovered in congenital fibrosarcoma.
ETV6–NTRK3 expression - activation of - MAPK and
PI3K pathways.

22. NTRK fusion ctd..
• Activation of EGFR signaling provides an adaptive
survival mechanism in NTRK1 fusion- positive lung
and colorectal cancer cell lines upon TRK inhibition;
• suggesting potential crosstalk between TRKA fusion
proteins and receptor tyrosine kinases in these
malignancies.

23. NTRK fusion ctd..
• The patient- derived acute promyelocytic leukaemia
(APML) cell line AP-1060 - harbors the ETV6–NTRK3
fusion & is sensitive to TRK inhibition;
• this is the only hematological cancer model driven by
an endogenous NTRK translocation reported in the
literature to date.
• Thus, only limited data on the potentially histology-
dependent signaling downstream of TRK fusion
proteins are currently available from primary cancer
cell lines.

24. NTRK fusion frequency and
diagnosis
• Two general categories according to the frequency at which
these fusions are detected.
first category:
rare cancer types are highly enriched for NTRK fusions.
For example, the ETV6–NTRK3 fusion is considered
practically pathognomonic in
• Secretory breast carcinoma,
• MASC (mammary analogue secretory carcinoma)
• Congenital mesoblastic nephroma(cellular or mixed subtypes)
• Infantile fibrosarcomas,
with a prevalence of >90%

25. NTRK fusion frequency and diagnosis
d..
Second category:
• NTRK fusions are found at much lower frequencies
(5–25% or <5%) - more common tumors
• Breast, Lung and Colorectal cancers, and Melanoma.
• Papillary thyroid cancers,
• Spitzoid neoplasms,
• GIST lacking canonical KIT, PDGFRA or RAS
alterations,
• and certain paediatric gliomas are- NTRK fusions with
frequencies of 5–25%.

27. NTRK fusion frequency and diagnosis
d..
• Hematological malignancies:
Interestingly, some hematological malignancies,
such as ALL and AML- harbor NTRK fusions at low
frequencies.
Importantly, a partial response to the TRK inhibitor
larotrectinib has been observed in a patient with AML
harboring an ETV6–NTRK2 fusion - suggesting that
TRK inhibition is a valid alternative.

28. NTRK fusion frequency and diagnosis
d..
• Clinical detection – predominantly - on NGS.
• Care must be exercised, however, in selecting NGS
platforms that enable reliable detection of NTRK
fusions;
• Because not all assays - even the most advanced
DNA based NGS platforms might not enable the
identification of all NTRK fusions, especially those
involving NTRK2 and NTRK3.

29. NTRK fusion frequency and diagnosis
d..
Other methods:
1. FISH and RT- PCR-
relatively quickly.
at lower costs.
limited to the detection of a single driver alteration.
2. plasma- based cell-free DNA (cfDNA) testing.
3. Immunohistochemistry (IHC)- potential presence of
a NTRK fusion.
In a series of solid tumors harboring NTRK fusions, IHC using a
pan- TRK antibody revealed positivity for TRK expression in 20

30. TRK inhibitor
therapy

31. TRK inhibitor therapy
• Several TKIs with varying degrees of activity against
TRKA, TRKB and/or TRKC are available.
• Broadly be grouped into multi- kinase inhibitors -
against - TRK or more- selective TRK
• Multi- kinase inhibitor group includes
Entrectinib, Crizotinib,
Cabozantinib, Lestaurtinib,
Altiratinib, Foretinib,
Ponatinib, Nintedanib,
Merestinib,
MGCD516, PLX7486, DS-6051b and TSR-011.

32. TRK inhibitor therapy ctd..
• Larotrectinib is currently the most specific; potent
and selective inhibitor of all three TRK proteins.
• Entrectinib is an orally available pan- TRK inhibitor
with additional activity against ROS1 and ALK.
• Treatment of TRK fusion- containing tumor models with
entrectinib or larotrectinib results in the inhibition of
the MAPK, PI3K–AKT, PKC and STAT3 pathways

33. Larotrectinib in clinical trials
explored in three clinical trials
Phase I in adult Phase I/II in peds Phase II adult
&adolescents
NCT SCOUT NAVIGATE
(02122913)

34. • first 55 pts. treated with larotrectinib on these trials
have been analyzed.
Primary end point of this combined analysis:
The median time to response was 1.8 months.
Independent radiology
review
Investigator assessed
ORR 75% 80%
CRR 13% 16%

35. • Patients with a total of 17 unique cancer types
harboring NTRK fusions were included in this analysis.
• Importantly, responses were observed in a histology-
agnostic fashion; regardless of
-fusion type
-upstream partner
-independent of age .
55% of the patients remained progression- free after 1
year of treatment.
 71% of the responses were ongoing.

36. Entrectinib in clinical trials
• Tested in 4 clinical trials:
1. Phase I Study In Adults With NTRK, ROS1 Or ALK
Rearrangements (ALKA, Eudract).
2. Phase I Trial In Adults With Various Ntrk, Ros1 Or
Alk Aberrations (STARTRK-1)
3. Phase I/Ib Study In Children Or Young Adults
(Aged 2–22 Years) With Or Without NTRK, ROS1 Or
ALK Fusions (STARTRK- NG).
4. A Phase II Basket Trial In Adults With Ntrk, Ros1
Or Alk Fusions (Startrk-2).

37. • ALKA 372–001 and STARTRK-1 trials have been
published:
• 4 Pts were treated with entrectinib in NTRK fusion +ve
cancers:
confirmed partial
responses
4. Glioneural tumour- Disease regression by an
exploratory volumetric
assessment
1. Colorectal
carcinoma
2. MASC
3. Lung
adenocarcinoma

38. Harboring ORR in TKI treatment
NTRK 100%
ROS1 86%
ALK 57%

39. Other agents
• TKIs with varying degrees of inhibitory activity against
TRK - approved for indications outside of the
treatment of patients with NTRK fusions:
• Crizotinib (ALK- Rearranged And Ros1-rearranged
NSCLC)
• Cabozantinib (RCC And Medullary Thyroid
Carcinoma)
• Ponatinib (Chronic Myelogenous Leukaemia)
• Nintedanib (Idiopathic Pulmonary Fibrosis)

40. Other TKIs are in various stages of
early phase clinical testing :
• Lestaurtinib, - targets- TRK, JAK2 and FLT3, has
been explored in patients with neuroblastoma.
• Altiratinib binds TRK proteins with low nanomolar
affinity, in addition to MET, TIE2 and VEGFR2.
• Merestinib - suppresses MST1R, FLT3, AXL, MERTK,
ROS1, DDR1, DDR2 and TRK at nanomolar
concentrations.

41. Acquired resistance to TRK
inhibition Resistance mechanisms :
• Acquired resistance mediated by on- target and off- target
mechanisms:
• To date, the acquisition of on- target mutations in the
NTRK kinase domain of the oncogenic fusion is the only
mechanism of secondary resistance.
• The resistance –
1. mutations in amino acid substitutions involving the
solvent front
2. Activation loop xDFG motif or so- called gatekeeper
residue of TRKA or TRKC.
• No mutations in TRKB have been identified in clinic to
date.

42. Acquired resistance ctd..
• The first case of acquired resistance to TRK inhibition
was reported in 2016- colorectal cancer after 4
months of treatment with entrectinib.
• two mutations resulting in substitutions in the kinase
domain of TRKA — G595R and G667C
paralogue of the ALK
• Located in the solvent front region of the TRK kinase
domain is the paralogue of the ALK and ROS1 – Acq.
Resitance.

43. Next- generation TRK inhibitors
• Fortunately already in development.
• In particular, loxo-195, tpx-0005 & ono-539055.
• Developed – parallely the early phases of the clinical
characterization of larotrectinib.
• In fact, studies had predicted mechanisms of
acquired resistance - enabled the preclinical
validation of loxo-195 as a second- generation trk
inhibitor before acquired resistance to larotrectinib was
observed in the clinic.

44. Next- generation TRK inhibitors ctd..
• One adult and one paediatric patient
Developed TRK solvent front mutation- mediated
acquired resistance to larotrectinib.
Subsequently, treatment with LOXO-195 was
administered on compassionate use, single- patient,
first- in-human protocols that resulting in confirmed
objective responses in both patients

45. Consequences of TRK inhibition
• One Must Be Cognizant Of The Fact That,
• NTRK1 expression seems to be limited to visceral
sensory ganglia of neural crest origin.
• NTRK2 and NTRK3 are widely expressed in both the
central and peripheral nervous systems.
• Not exclusively confined to the nervous system-
roles in non- neuronal tissues, such as the
vasculature, ovaries and immune system.

46. Consequences of TRK inhibition
ctd..
• Loss- of-function NTRK1 mutations have been
identified in patients with :
congenital insensitivity to pain with anhidrosis
(CIPA)- a hereditary sensory and autonomic
neuropathy.
• Larotrectinib and entrectinib - favourable overall
safety profile compared with other tkis.
• Grade ≥3 with the use of other tkis (such as fatigue,
nausea, diarrhoea, constipation, vomiting and
increased serum transaminase levels)
• Mostly grade 1 or 2 in severity in the trials of
entrectinib and larotrectinib.

48. Take home message
• NTRK fusions are drivers of a wide variety of adult and
paediatric cancers.
• Alterations are either highly enriched in select tumour
types or infrequently found in subsets of other cancers,
including common tumours.
• Detection by - Comprehensive nucleic acid- based
profiling and complementary IHC assays.

49. Take home message
• The larotrectinib and entrectinib have demonstrated
histology- agnostic and age- independent activity
diverse cancers harbouring NTRK fusions.
• Acquired resistance to TRK inhibitor therapy remains
an ongoing challenge, but on- target resistance can be
abrogated by the use of second- generation TRK
inhibitors.
• The toxicological consequences of TRK inhibition
include on- target neurological adverse events.

50. Take home message
• Larotrectinib and entrectinib have a favourable overall
safety profile compared with many other tkis and are,
therefore, amenable to chronic dosing.

51. Thank you..!