Table 2.

Overview of the main alterations detected in the samples examined (surgical, bioptic, blood), at least two for each case except for Case 4. Comparison among samples collected at different times in the same patient was made in order to better understand disease evolution and the behavior of its counterparts: shared alterations were highlighted in red, while new ones were reported.

	First NGS	MS	TMB	VUS	Sample	Diagnosis	Second NGS	MS	TMB	VUS	Sample	Diagnosis
Case 1	AKT2 amplification AXL amplification CCNE1 amplification MYCL1 amplification MAP2K4 loss RB1 loss exons 1–2 TP53 R273H	MSS	Low (4 Muts/Mb)	Common: ERBB3 L1177I JAK3 E113K and I63V MED12 L1348F SDHC L14F Not shared: CD22 amplification CD79A amplification CEBPA amplification CIC amplification DOT1L T1460K TET2 F1597L	Surgical	NEC of unknown origin Revision: Presence of focal epithelial high-grade dysplasia	TP53 R273H DNMT3A R676W—subclonal	MSS	Low (1 Muts/Mb)	Common: ERBB3 L1177I JAK3 I63V MED12 L1348F SDHC L14F	Surgical	Metastases of colon adenocarcinoma
Case 2	KRASG12V FBXW7W673* PTEN N276K RICTOR amplification—equivocal APC D1486fs*21 TP53 R175H	MSS	Intermediate (13 Muts/Mb)	Common: BCORL1 N1473I DOT1L P1354L FANCC D306E KDR A379V MAP3K1 S939C MED12 Q2119_Q2120insHQQQ PIK3C2G F89Y SMAD4 Y353H RICTOR I253V Not shared: NTRK3 H521D	bioptic	NEC of colon Revision: Presence of high-grade dysplasia in a context of neuronendocrine cells	KRAS G12V FBXW7 W673* PTEN N276K APC D1486fs*21 TP53 R175H	MSS	Intermediate (12 Muts/Mb)	Common: BCORL1 N1473I DOT1L P1354L FANCC D306E KDR A379V MAP3K1 S939C MED12 Q2119_Q2120insHQQQ PIK3C2G F89Y SMAD4 Y353H RICTOR I253V + amplification Not shared: FGF10 amplification SDHA amplification RICTOR amplification	Surgical liver	Colorectal adenocarcinoma
Case 3	KRAS G12D APC R876* I1557fs*1 FAM123B R601* TP53 R196* C242F Not shared: SMAD4 P356L MYC-N amplification	MSS	8 Muts/Mb	Common ATR D331G BRIP1 R106C CD79A T75M HGF W329L LTK V113L Not shared: DIS3 amplification FGF14 amplification FGF6 G187R IRS2 amplification	Surgical	MINEC (colorectal adenocarcinoma G2+ NEC)	KRAS G12D APC R876*, I1557fs*1 FAM123B R601* TP53 R196* Not shared: ASXL1 R1415* DNMT3A W440*, N552fs*99 ERBB2 S310Y STAG2 splice site 2924+2T>G	MSS	8 Muts/Mb	Common: ATR D331G BRIP1 R106C CD79A T75M HGF W329L LTK V113L Not shared: DNMT3A I369F EPHB1 P844S MAP3K13 R474Q ATM F2485G PDGFRA H425R RICTOR V120fs*2	Blood	Colorectal MINEC
Case 4	NF1 loss exons 1–36 CTNNB1 D32N TP53 K382fs*40 IRS2 amplification	MSS	8 Muts/Mb	CDH1 D587N DIS3 K923N ERBB4 M887I FGF12 G82* NTRK1 G714S NTRK3 I533L PIK3C2B D777A PIK3C2G I232T STK11 S240L TEK I591V	Surgical	MINEC (mucinous adenocarcinoma + NEC) of unknown primary origin (gastric, colon?)	Not available