Background: The antibody-drug conjugate T-DM1 is widely used for the treatment of HER2-positive metastatic breast cancer. Its activity in other advanced solid tumors harboring HER2 alterations identified by comprehensive genomic profiling (CGP) needs further characterization. Methods: The MoST program provides a precision oncology framework for screening patients (pts) by CGP to identify genomic alterations with potential to match to biomarker-directed clinical trials. This multi-center, single-arm phase 2 trial (ACTRN12619001265167) enrolled adult pts with refractory solid tumors (excluding breast and gastroesophageal cancers) with HER2 alterations. This trial recruited 2 groups of 16 pts (group 1: mutations, group 2: genomic amplification, defined as copy number ≥ 6 copies). The primary composite endpoint was objective tumor response (OTR) assessed by RECIST 1.1 and time-to-progression (TTP) ratio (defined as TTP on T-DM1: TTP1 on prior line of systemic therapy). Anti-tumor activity of T-DM1 was confirmed if ≥3 of each 16 pt group achieved an OTR. Key secondary endpoints were progression-free and overall survival (PFS and OS) and safety as assessed by CTCAE 5.0. Results: 32 pts had a median age of 64 years (interquartile range [IQR] 53 to 69); 94% an ECOG PS ≤1; 53% female; and median 2 (IQR 1 to 3.5) prior lines of systemic therapy. The cohort included non-small cell lung cancer (NSCLC, n=12), colorectal (CRC, n=5), salivary gland (SGC, n=5), ovarian (n=3), uterine (n=2), gallbladder (n=2), and 1 each of bladder, pancreas and cervix cancers. After a median follow-up of 17 months (mo), the primary endpoint was met with 7 pts (22%) achieving an OTR, including 3 pts (19%) in group 1 (2 NSCLC and 1 SGC) and 4 pts (25%) in group 2 (3 SGC, and 1 uterine serous carcinoma [USC]). Fourteen of 29 TTP-evaluable pts (48%) achieved a TTP ratio ≥1.3, including 10 pts without an OTR: equating to 53% (n=17) deriving clinical benefit. The median PFS and OS were 4.5 (95% CI 2.1 to 7.0) and 18.23 mo (95% CI 8.11 to not reached) respectively and 6 mo PFS rate 44% (95% CI 26 to 60%). The (NSCLC) responders in group 1 harbored exon 20 insertions (Y772_A775dup and G778_P780dup) and the SGC, a L755S mutation. None of the S310Y/F mutations (n=5, including 2 NSCLCs) had an OTR. The USC was ERBB2 amplified (18 copies) on CGP, discordant with in situ hybridisation (ISH) testing results. Overall, 4 pts had CGP-amplified, ISH negative tumors, of whom 2 achieved an OTR. No responses were seen in CRC pts (all RAS/BRAF-wildtype and IHC 2-3+). No new safety concerns for T-DM1 were reported. Conclusions: T-DM1 has anti-tumor activity across a heavily treated, diverse range of cancer types with HER2 alterations identified by CGP, including several unidentified by standard ISH testing. We await results from our partner trial in the first line advanced NSCLC setting to corroborate the group 1 findings. Clinical trial information: ACTRN12619001265167.