ASCO 2022: Circulating tumor DNA (ctDNA) in HER2 exon 20 insertion mutations and responses in NSCLC HER2 exon 20 insertion treated with poziotinib

Circulating tumor DNA (ctDNA) in HER2 exon 20 insertion mutations predict responses in NSCLC	3051
HER2 exon 20 insertion treated with poziotinib

Arunthi Thiagalingam1, Sribalaji Lakshmikanthan1, Allysia J. Mak2, Scott A. Shell2, Sharon Leu1, Sophie Sun3, Erin Bertino4, Eric Haura5, Rocky Washington1, Gajanan Bhat1, Francois Lebel1 and John A Barrett1

1Spectrum Pharmaceuticals, Boston, MA; 2Guardant Health, Redwood City, CA; 3BC-Cancer Vancouver, Canada; 4The Ohio State University, Columbus, OH; 5H. Lee Moffitt Cancer Center & Research Institute, Tampa, FL

Abstract

Introduction: ctDNA levels in plasma samples permits temporal assessment of tumor mutational status and tumor burden during therapy. Poziotinib is an oral pan-ErbB TKI in development for NSCLC patients harboring HER2 exon 20 insertion mutations. We assessed serial plasma samples for changes in HER2 exon 20 insertion mutations and other driver mutations in first- and second-line patients comparing to clinical response per RECIST1.1.

Methods

Patients from SPI-POZ-202 Cohorts 4 & 5 with tumors harboring HER exon20 insertion mutations received poziotinib 16mg QD were studied. In this sub analysis, 5/7 patients met the following inclusion criteria.

o	Plasma samples at baseline and longitudinal beyond C3D1
o	Tumor size assessment at C3D1 was partial response (PR)

Here we report the longitudinal changes in ctDNA beyond C3D1during poziotinib treatment and changes in the target HER2 exon 20 insertion mutation, the %VAF of the variants at longitudinal timepoints and patient tumor response (RECISTS1.1).

Results	Summary of baseline demographics of the 5	Tumor response to poziotinib treatment
	responding patients	at longitudinal time points

Methods: NSCLC patients harboring HER2 exon 20 insertion mutations were enrolled into the poziotinib ZENITH20 using tumor tissue based NGS. Serial plasma samples were collected at baseline, at C3D1, at Day 1 of every other cycle until disease progression. The Guardant360 74-gene liquid biopsy assay was used to assess changes in tumor-associated somatic variants including the target variant HER2 exon20 insertion as well as other emergent driver mutations in ctDNA as expressed as percent variant allele frequency (%VAF).

Results: 23 first- and second-line NSCLC patients were evaluable with tumor tissue confirmation of HER2 exon 20 insertion mutations. 22 of 23 (96%) had baseline plasma samples with detectable ctDNA. 21 of 22 samples had detectable HER2 exon 20 insertion mutations (mean % VAF 20±5) resulting in a concordance of 95% versus tissue based NGS. 7 patients had serial testing through C7D1 permitting assessment of ctDNA dynamics and comparison to clinical responses. 5 of 7 (71%) serially tested patients treated with poziotinib at 16mg QD had undetectable HER2 exon 20 insertion at C3D1 which was associated with a tumor response PR. Tumor escape (PD) was observed in 2 of the 5 patients which correlated with increases in target HER2 exon 20 insertion VAF in the plasma with the remaining 3 patients ≥PR. Notably, the rise in HER2 exon 20 in ctDNA occurred prior to tumor escape. In one patient treated with poziotinib at 16 mg QD we observed undetectable levels of the HER2

o Sufficient cfDNA and ctDNA to analyze samples

		Variant calling was done by a validated
Plasma isolation, cfDNA extraction,
library construction and sequencing, and		custom bioinformatics pipeline that uses
quality-control assessments were		molecular barcoding and double-stranded
performed using Guardant360 as		consensus sequencing to achieve >99.99%
previously described.		analytic specificity per sequencing position
[Odegaard, 2018].		(Mak (Talasaz), 2021)

• Tumor tissue genotyping correlated with plasma ctDNA with a concordance of 95% at baseline.
• Poziotinib treatment resulted in reduction of the target HER2 exon 20 insertion mutation %VAF in plasma ctDNA at C3D1 compared to baseline and correlated to
  patient tumor response (RECISTS1.1). (Thiagalingam A, et al., 2022; AACR, abstract #3400)

Patient Characteristics	HER2 exon 20 ins
Age, median (range)	62 (53-72)
Female/Male	4 / 1
White/Asian/Black	3 / 2 / 0
Histological types
Adenocarcinomas	5
Stage (n)	IV (5)
ECOG status: 0/1	0 / 5
Smoker/nonsmoker	0 / 5
Prior therapy ≥1	1
Tumor tissue genotype	5
Guardant360 ctDNA	5

13
1
6														16 mg
													14 mg
														12 mg
4														10 mg
													8 mg
														Complete Response
														Partial Response
12														Stable Disease
														Progressive Disease
0	28	56	84	112	140	168	196	224	252	280	308	336	364	392	420	448	476

Study Day

exon 20 insertion in ctDNA at C3D1 which continued through C16. This patient's responses correlated with patient tumor response of SD at C2 which then became PR through C9 and CR through C17.

Conclusions: Poziotinib treatment resulted in reductions in HER2 exon 20 insertion mutations in ctDNA preceded and correlated with the clinical tumor response. Increases in ctDNA HER2 exon 20 insertion mutations were observed prior to confirmation of tumor escape. Serial monitoring of ctDNA is a potential predictive biomarker for treatment response and disease progression. Future evaluation in a larger population is required to confirm the impact of these findings.

Poziotinib

Poziotinib is an oral pan-ErbB TKI with activity in patients with HER2 exon 20 insertion mutated NSCLC. HER2 exon 20 insertion mutations are a rare subset accounting for approximately 2-4% in NSCLC. There is no approved therapy for either treatment-naïve or previously treated NSCLC with HER2 exon 20 insertion mutations

Poziotinib MoA

Poziotinib inhibits tyrosine kinase phosphorylation in patients harboring HER2 exon 20 mutations resulting in inhibition of the RAS/RAF pathway.

Poziotinib treatment also increases HER2 receptor expression on the surface of tumor cells harboring exon20 insertion mutations. (Cartoon)

Circulating Tumor DNA (ctDNA)

1. Circulating tumor DNA, ctDNA, is tumor- derived fragmented DNA in plasma.

1. Apoptosis and necrosis are the primary methods of ctDNA release into circulation.

1. ctDNA is isolated and sequenced for mutational analysis and is known to reflect the mutational status of the tumor genome. (Cartoon)

Summary of baseline demographics of 23

patients with HER2 exon 20 insertion mutations

Patient Characteristics	HER2 exon 20 ins
Age, median (range)	62 (43-79)
Female/Male	10 /13
White/Asian/Black	16 / 5 / 2
Histological types
Adenocarcinomas	23
Stage (n)	I (2) / IV (21)
ECOG status: 0/1	7 /16
Smoker/nonsmoker	3 / 20
Prior therapy ≥1	3
Tumor tissue genotype	23
Guardant360 ctDNA	21

Baseline ERBB2 exon 20 insertion mutation subtypes in tissue biopsy NGS and plasma ctDNA demonstrating 95% concordance

	ctDNA exon20	Tissue NGS exon 20
Patient ID	variant	variant	Concordance
2	A775_G776insYVMA	A775_G776insYVMA	✓
3	A775_G776insYVMA	A775_G776insYVMA	✓
10	A775_G776insYVMA	A775_G776insYVMA	✓
12	A775_G776insYVMA	A775_G776insYVMA	✓
14	A775_G776insYVMA	A775_G776insYVMA	✓
16	A775_G776insYVMA	A775_G776insYVMA	✓
18	A775_G776insYVMA	A775_G776insYVMA	✓
19	A775_G776insYVMA	A775_G776insYVMA	✓
20	A775_G776insYVMA	A775_G776insYVMA	✓
21	BLQ	A775_G776insYVMA
22	A775_G776insYVMA	A775_G776insYVMA	✓
23	A775_G776insYVMA	A775_G776insYVMA	✓
9	G776delinsVC	G776_delinsVC	✓
1	G776delinsVC	G776delinsVC	✓
4	G776delinsVC	G776delinsVC	✓
5	BLQ	G776delinsVC
7	G776delinsVC	G776delinsVC	✓
17	G776delinsVC	G776delinsVC	✓
15	G776delinsVV	G776delinsVV	✓
8	G778_P780dup	G778_P780dup	✓

Longitudinal changes in ctDNA HER2 exon 20 insertion in responders to poziotinib treatment.

A775_G776insYVMA	G778_S779insCPG	G776delinsVC	G776_V777delinsCVG	G776delinsVC
Patient ID: 12, 1L	Patient ID: 13, 1L	Patient ID: 1, IL	Patient ID: 6, 1L	Patient ID: 4, 2L

16mg QD

	30	16mg QD			1.0		SD	PR	CR	50						5			PR	PD	100
				0.8											16mg QD
								40						4
	20			PR						16mg QD							(%)	75
(%)			(%)	0.6				(%)	30	PR	PD	(%)							PR			PD
			16 mg QD		3				50
VAF					VAF	0.4		VAF	20					VAF					VAF
											2
10				0.2						14mg QD					25	14mg QD
									10					1	14-10mg QD
						0.0
	0							#		0						# #		#		0		#	#
					-0.2									0
	0	56	112	168	0	56			0	56	112 168 224 280 336		0	56	168 224 308	0	56	224	280
			112 224 252 308 420				112	168
			Days					Days					Days						Days				Days
		HER2 (A775_G776insYVMA)		HER2 (G778_S779insCPG)			HER2 (G776delinsVC)			HER2 (G776_V777delinsCVG)		HER2 (G776delinsVC)
		TP53 (C277F)								TP53 (R333fs)			TP53(R273C)
					CCND2 (N256S)				TP53 (K132N)
								HRAS (G13S)														AR (P37S)
								KRAS (Q22R)
								MET (T733K)

HER2 exon20 insertion mutation	HER2 exon20 insertion mutation	HER2 exon20 insertion mutation was	HER2 exon20 insertion mutation	HER2 exon20 insertion mutation
decreased >95% at Day 56 with low	decreased >95% at Day 56 and	decreased >95% between Day 56 -168	decreased >95% at Day 56 which was	decreased >95% on Day 112 which was
expression continuing through Day	persisted through Day 420 which was	which was associated with patient tumor	associated with patient tumor response	associated with a PR. Dose was reduced
168. This decrease in expression was	associated with tumor response of	response of PR. At Day 113, patient was	of PR. Multiple dose reductions	and HER2 exon 20 insertion mutation
associated with patient tumor	PR/CR. # denotes 23-day drug holiday.	dose reduced to 14mg QD, and was	beginning on Day 23 including 8mg QD	rapidly increased was associated with
response of PR through Day 168.		associated with increased HER2 exon 20	on Day 247 was associated with	PD. # denotes 7-day drug holidays.
		mutation and PD later.	increased HER2 exon 20 mutation and
			PD. # denotes 8-9 day drug holidays.

Conclusion

• In poziotinib treated patients with advanced NSCLC harboring HER2 exon20 insertion mutations, baseline ctDNA presence was associated with the tumor tissue genotyping with a concordance of 95%.
• In responders, ctDNA reduced level were associated with tumor response.
• Serial monitoring of ctDNA is a potential predictive plasma biomarker for poziotinib treatment response and disease progression.
• Future evaluation in a larger population is required to confirm these findings.

[Green et al., 2021 https://doi.org/10.1016/j.euo.2021.04.005]

1. In NSCLC patients treated with Poziotinib, we assessed changes in ctDNA.

1. Changes in variant allele fraction (VAF%) of HER2 exon20ins somatic alterations at baseline and longitudinal plasma samples beyond C3D1 (Cycle 3 Day 1) in responders were associated with tumor response per RECIST1.1.

13	G778_S779insCPG	G778_S779insCPG	✓
6	G776_V777delinsCVG G776_V777delinsCVG	✓
11	G776_V777delinsVCD	G776_V777delinsVFD	x

BLQ= below limit of quantification; x Discordant.

5/7 patients had PR and sufficient plasma to perform longitudinal analysis

References

1. ZENITH20 study publication-https://clinicaltrials.gov/ct2/show/NCT03318939
2. Odegaard JI, et al. Clin Cancer Res. 2018;24(15):3539-3549
3. Mak (Talasaz) et al. 2021 Cancer Res 81 (13_Supplement): 401https://doi.org/10.1158/1538-7445.AM2021-401
4. Thiagalingam A, et al., 2022; AACR, abstract #3400.

Acknowledgement

We thank all the patients and their families

Poziotinib is an investigational drug that has not been approved by the Food and Drug Administration

Presenting author email: [e-mail]

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This study and publication were sponsored by Spectrum Pharmaceuticals, Inc.