Objective:To investigate the variations and co-alteration of KRAS and HER-family genes in the patients with ampullary carcinoma.Methods:A total of 37 formalin-fixed paraffin-embedded primary ampullary carcinoma specimens, which were collected at Peking Union Medical College Hospital from April 2019 to October 2024 were analyzed for KRAS and HER-family gene mutations using next-generation sequencing (NGS). Immunohistochemistry (IHC) was performed for HER2 protein expression in HER2 mutation cases and fluorescence in situ hybridization (FISH) for further gene status in HER2 IHC 2+cases.Results:In our cohort (22 males, 15 females; 31-82 years old), KRAS gene mutations were detected in 51.4% (19/37) of cases, with G12D being the most frequent abnormality (7/19), followed by G12V (5/19) and Q61R (3/19). Other variants of KRAS gene included G12C, A146T, N116H, and Q61H (each 1/19). In this cohort, 27.0% (10/37) of cases harbored HER-family gene alterations with most frequently in HER2 (6/10) and HER3 genes (missense mutations mainly). Notably, 3 cases (8.1%, 3/37) with coexistence of KRAS and HER-family genes mutations were recognized in our series, including KRAS p.G12D/HER2 p.V842I/HER2 p.V777L (c.2329 G>T)/HER3 p.Asp581Asn, KRAS p.Q61R/HER4 p.D1018H and KRAS p.G12C/HER2 p.R678Q. Additionally, a mutation of HER3 p.V104L (c.310 G>C) was identified in our population. Moreover, 4 novel mutations including HER3 p.V296E, HER3 p.V920L (c.2758 G>T), HER3 p.Asp581Asn, and HER4 p.D1018H were detected. In 6 tumors with HER2 gene changes (16.2%, 6/37), 5 variants with the high proportion of HER2 p.S310Y (3/6) were revealed. A tumor (HER2 IHC 2+) with HER2 p.S310Y presented HER2 gene amplification confirmed by NGS and FISH, and another one (also HER2 IHC 2+) with HER2 p.L755S possessed HER2 gene amplification determined by FISH assay.Conclusion:In ampullary carcinoma, co-alteration of KRAS and HER-family genes is observed, and HER2 gene mutations account for more than half of HER-family gene abnormities, which may be accompanied by gene amplification.

Objective:To investigate the characteristics of RET gene rearrangement revealed by fluorescence in situ hybridization (FISH) in lung cancer.Methods:A total of 616 formalin-fixed paraffin-embedded surgical samples from lung adenocarcinomas with wild-type EGFR gene and no ALK protein expression by immunohistochemistry obtained at Peking Union Medical College Hospital, Beijing, China between December 2019 and April 2022 were included. Thirty-three tumors with RET gene rearrangement determined by imbalanced-based reverse-transcription droplet digital PCR (RT-ddPCR) were analyzed using break-apart FISH. The results were confirmed, and RET gene fusion variants were identified through next generation sequencing (NGS).Results:RET gene rearrangements were found in all 33 RET RT-ddPCR positive cases via NGS, including 27 cases of KIF5B::RET, 3 CCDC6::RET, 2 ERC1::RET and 1 CCDC186::RET rearrangements. Moreover, 32 RET positive and 1 RET negative cases were defined using FISH. Among the RET FISH-positive cases, 25 (78.1%, 25/32) showed break-apart FISH signal pattern in 52%-100% of tumor cells with the rearrangement and 7 cases (21.9%, 7/32) presented isolated 3′ signal type in 38%-88% positive tumor cells. There was no RET-positive case with single 5′ pattern in the cohort. The most common partner gene was KIF5B (81.8%, 27/33). Most of the patients with RET gene rearrangement were female (72.7%, 24/33).Conclusion:RET FISH-positive lung cancer is commonly characterized by a high proportion of rearrangement cancer cells and break-apart FISH signal type.

Objective:To investigate the mutation of PIK3CA, AKT1 and PTEN genes in hormone receptor (HR)-positive and HER2-negative invasive breast cancer.Methods:A total of 44 formalin-fixed paraffin-embedded samples from HR-positive/HER2-negative female patients with breast cancer obtained between January 2020 and July 2024 in Peking Union Medical College Hospital were selected. The mutations of PIK3CA, AKT1 and PTEN genes were analyzed by next-generation sequencing (NGS), and the related clinicopathological characteristics were summarized.Results:In the cohort, 31 out of 44 cases (70.5%) exhibited alterations in the PIK3CA, AKT1 and PTEN genes. Of these, 83.9% (26/31) tumors harbored genetic abnormalities involving one gene, including 21 (47.7%, 21/44) PIK3CA, 2 (4.5%, 2/44) PTEN and 3 (6.8%, 3/44) AKT1 gene mutations. Mutations of both PIK3CA and PTEN genes were found in 16.1% (5/31) of specimens. Among the 26 cases with PIK3CA gene mutations, 13 variants were identified, including E542K, E545K, Q546K, H1047R, H1047L, G1049R, M1043I, C420R, P447_L455del, N345K, N345I, K711N and H1047L/V346G. In addition, 7 mutants of PTEN gene were determined (T319 *, T321Qfs *23, Q245 *, Q171H, L108P, Y68Ifs *6 and V343fs). For AKT1 gene mutation, only E17K was observed.Mutations of PIK3CA/AKT1/PTEN genes are more likely to occur over 40 year-old patients.In this cohort, the PIK3CA V346G mutation (co-existent PIK3CA H1047L) and the PTEN V343fs mutation were not found in previous publications. Conclusion:In addition to the predominance of common loci, PIK3CA and PTEN gene mutations also have rare loci mutations in the breast cancer, warranting further analysis with an expanded sample size.

Objective:To investigate the abnormalities of mesenchymal-epithelial transition factor (MET) gene in early-stage lung adenocarcinomas and to provide genetic bases for related clinical studies.Methods:A total of 630 cases of formalin-fixed and paraffin-embedded lung adenocarcinoma specimens with ALK and EGFR double-negativities were collected at Peking Union Medical College Hospital, Beijing, China between July 2020 and April 2022. Forty-three stage Ⅰ-ⅢA tumors with MET exon 14 skipping mutation identified by reverse transcription droplet digital PCR (RT-ddPCR) were identified and then evaluated for MET amplification using fluorescence in situ hybridization (FISH). MET amplification was determined using the ratio of MET to chromosome 7 enumeration probe (CEP7) or the mean of MET gene copy number (GCN).Results:Among the 43 samples with MET exon 14 skipping mutation, MET amplification was detected in 9 cases (9/43, 20.93%), including 1 case of MET/CEP7 ≥2 and GCN ≥5 (1/9), 8 cases of GCN≥5 (8/9), as well as 10 cases with high level of CEP7 (7.00-9.72) which included 5 cases with MET amplification. There were no significant differences in clinicopathological features between the two subgroups of tumors which harbored MET exon 14 skipping mutation with MET amplification versus those without ( P>0.05). Conclusions:Co-occurrence of MET exon 14 skipping mutation and MET amplification or high level of CEP7 is frequently observed in early-stage lung adenocarcinomas. The most common pattern of MET gene amplification is GCN ≥5.

Objective:To assess the clinicopathological characteristics of non-muscle-invasive bladder cancers (NMIBC) with high expression of human epidermal growth factor receptor 2 (HER2) and to examine the prognostic values of HER2 expression in NMIBC patients with intravesical anthracycline instillation.Methods:A total of 221 NMIBC samples diagnosed between January 1, 2017 and April 15, 2024 were collected. Their clinical, diagnostic and treatment features were analyzed. The expression of HER2 protein and the Ki-67 proliferation index were assessed using immunohistochemistry (IHC). For the patients with HER2 high-expression (IHC 3+), the clinical pathological features (age, gender, tumor grade, Ki-67 expression level, tumor size, and tumor number) were compared with those without (i.e., HER2 IHC 0/1+/2+). The impact of HER2 expression on the recurrence-free survival (RFS) of patients with intravesical anthracycline (epirubicin or pirarubicin) instillation after transurethral resection of bladder tumor (TURBT) was evaluated.Results:Among the 221 NMIBC patients, 30 (13.6%) were HER2 IHC 3+, 142 (64.3%) HER 2+, 46 (20.8%) HER2 1+, and 3 (1.4%) HER2 IHC 0. The proportion of high-grade tumors in patients with HER2 high-expression was higher than that in patients without (83.3% versus 44.5%, P<0.001). Additionally, a high Ki-67 index (≥20%) was more commonly noted in HER2 high-expression tumors ( P=0.003). In the patients treated with intravesical anthracycline instillation, HER2 high-expression was associated with a shorter RFS ( P<0.001). Conclusion:HER2 high-expression seems to be not only associated with worse clinicopathological features of NMIBC but also a poor RFS in NMIBC patients treated with anthracycline instillation after TURBT.

Objective:To investigate the variations and co-alteration of KRAS and HER-family genes in the patients with ampullary carcinoma.Methods:A total of 37 formalin-fixed paraffin-embedded primary ampullary carcinoma specimens, which were collected at Peking Union Medical College Hospital from April 2019 to October 2024 were analyzed for KRAS and HER-family gene mutations using next-generation sequencing (NGS). Immunohistochemistry (IHC) was performed for HER2 protein expression in HER2 mutation cases and fluorescence in situ hybridization (FISH) for further gene status in HER2 IHC 2+cases.Results:In our cohort (22 males, 15 females; 31-82 years old), KRAS gene mutations were detected in 51.4% (19/37) of cases, with G12D being the most frequent abnormality (7/19), followed by G12V (5/19) and Q61R (3/19). Other variants of KRAS gene included G12C, A146T, N116H, and Q61H (each 1/19). In this cohort, 27.0% (10/37) of cases harbored HER-family gene alterations with most frequently in HER2 (6/10) and HER3 genes (missense mutations mainly). Notably, 3 cases (8.1%, 3/37) with coexistence of KRAS and HER-family genes mutations were recognized in our series, including KRAS p.G12D/HER2 p.V842I/HER2 p.V777L (c.2329 G>T)/HER3 p.Asp581Asn, KRAS p.Q61R/HER4 p.D1018H and KRAS p.G12C/HER2 p.R678Q. Additionally, a mutation of HER3 p.V104L (c.310 G>C) was identified in our population. Moreover, 4 novel mutations including HER3 p.V296E, HER3 p.V920L (c.2758 G>T), HER3 p.Asp581Asn, and HER4 p.D1018H were detected. In 6 tumors with HER2 gene changes (16.2%, 6/37), 5 variants with the high proportion of HER2 p.S310Y (3/6) were revealed. A tumor (HER2 IHC 2+) with HER2 p.S310Y presented HER2 gene amplification confirmed by NGS and FISH, and another one (also HER2 IHC 2+) with HER2 p.L755S possessed HER2 gene amplification determined by FISH assay.Conclusion:In ampullary carcinoma, co-alteration of KRAS and HER-family genes is observed, and HER2 gene mutations account for more than half of HER-family gene abnormities, which may be accompanied by gene amplification.

Objective:To investigate the characteristics of RET gene rearrangement revealed by fluorescence in situ hybridization (FISH) in lung cancer.Methods:A total of 616 formalin-fixed paraffin-embedded surgical samples from lung adenocarcinomas with wild-type EGFR gene and no ALK protein expression by immunohistochemistry obtained at Peking Union Medical College Hospital, Beijing, China between December 2019 and April 2022 were included. Thirty-three tumors with RET gene rearrangement determined by imbalanced-based reverse-transcription droplet digital PCR (RT-ddPCR) were analyzed using break-apart FISH. The results were confirmed, and RET gene fusion variants were identified through next generation sequencing (NGS).Results:RET gene rearrangements were found in all 33 RET RT-ddPCR positive cases via NGS, including 27 cases of KIF5B::RET, 3 CCDC6::RET, 2 ERC1::RET and 1 CCDC186::RET rearrangements. Moreover, 32 RET positive and 1 RET negative cases were defined using FISH. Among the RET FISH-positive cases, 25 (78.1%, 25/32) showed break-apart FISH signal pattern in 52%-100% of tumor cells with the rearrangement and 7 cases (21.9%, 7/32) presented isolated 3′ signal type in 38%-88% positive tumor cells. There was no RET-positive case with single 5′ pattern in the cohort. The most common partner gene was KIF5B (81.8%, 27/33). Most of the patients with RET gene rearrangement were female (72.7%, 24/33).Conclusion:RET FISH-positive lung cancer is commonly characterized by a high proportion of rearrangement cancer cells and break-apart FISH signal type.

Objective:To investigate the mutation of PIK3CA, AKT1 and PTEN genes in hormone receptor (HR)-positive and HER2-negative invasive breast cancer.Methods:A total of 44 formalin-fixed paraffin-embedded samples from HR-positive/HER2-negative female patients with breast cancer obtained between January 2020 and July 2024 in Peking Union Medical College Hospital were selected. The mutations of PIK3CA, AKT1 and PTEN genes were analyzed by next-generation sequencing (NGS), and the related clinicopathological characteristics were summarized.Results:In the cohort, 31 out of 44 cases (70.5%) exhibited alterations in the PIK3CA, AKT1 and PTEN genes. Of these, 83.9% (26/31) tumors harbored genetic abnormalities involving one gene, including 21 (47.7%, 21/44) PIK3CA, 2 (4.5%, 2/44) PTEN and 3 (6.8%, 3/44) AKT1 gene mutations. Mutations of both PIK3CA and PTEN genes were found in 16.1% (5/31) of specimens. Among the 26 cases with PIK3CA gene mutations, 13 variants were identified, including E542K, E545K, Q546K, H1047R, H1047L, G1049R, M1043I, C420R, P447_L455del, N345K, N345I, K711N and H1047L/V346G. In addition, 7 mutants of PTEN gene were determined (T319 *, T321Qfs *23, Q245 *, Q171H, L108P, Y68Ifs *6 and V343fs). For AKT1 gene mutation, only E17K was observed.Mutations of PIK3CA/AKT1/PTEN genes are more likely to occur over 40 year-old patients.In this cohort, the PIK3CA V346G mutation (co-existent PIK3CA H1047L) and the PTEN V343fs mutation were not found in previous publications. Conclusion:In addition to the predominance of common loci, PIK3CA and PTEN gene mutations also have rare loci mutations in the breast cancer, warranting further analysis with an expanded sample size.

Objective:To investigate the abnormalities of mesenchymal-epithelial transition factor (MET) gene in early-stage lung adenocarcinomas and to provide genetic bases for related clinical studies.Methods:A total of 630 cases of formalin-fixed and paraffin-embedded lung adenocarcinoma specimens with ALK and EGFR double-negativities were collected at Peking Union Medical College Hospital, Beijing, China between July 2020 and April 2022. Forty-three stage Ⅰ-ⅢA tumors with MET exon 14 skipping mutation identified by reverse transcription droplet digital PCR (RT-ddPCR) were identified and then evaluated for MET amplification using fluorescence in situ hybridization (FISH). MET amplification was determined using the ratio of MET to chromosome 7 enumeration probe (CEP7) or the mean of MET gene copy number (GCN).Results:Among the 43 samples with MET exon 14 skipping mutation, MET amplification was detected in 9 cases (9/43, 20.93%), including 1 case of MET/CEP7 ≥2 and GCN ≥5 (1/9), 8 cases of GCN≥5 (8/9), as well as 10 cases with high level of CEP7 (7.00-9.72) which included 5 cases with MET amplification. There were no significant differences in clinicopathological features between the two subgroups of tumors which harbored MET exon 14 skipping mutation with MET amplification versus those without ( P>0.05). Conclusions:Co-occurrence of MET exon 14 skipping mutation and MET amplification or high level of CEP7 is frequently observed in early-stage lung adenocarcinomas. The most common pattern of MET gene amplification is GCN ≥5.

Objective:To assess the clinicopathological characteristics of non-muscle-invasive bladder cancers (NMIBC) with high expression of human epidermal growth factor receptor 2 (HER2) and to examine the prognostic values of HER2 expression in NMIBC patients with intravesical anthracycline instillation.Methods:A total of 221 NMIBC samples diagnosed between January 1, 2017 and April 15, 2024 were collected. Their clinical, diagnostic and treatment features were analyzed. The expression of HER2 protein and the Ki-67 proliferation index were assessed using immunohistochemistry (IHC). For the patients with HER2 high-expression (IHC 3+), the clinical pathological features (age, gender, tumor grade, Ki-67 expression level, tumor size, and tumor number) were compared with those without (i.e., HER2 IHC 0/1+/2+). The impact of HER2 expression on the recurrence-free survival (RFS) of patients with intravesical anthracycline (epirubicin or pirarubicin) instillation after transurethral resection of bladder tumor (TURBT) was evaluated.Results:Among the 221 NMIBC patients, 30 (13.6%) were HER2 IHC 3+, 142 (64.3%) HER 2+, 46 (20.8%) HER2 1+, and 3 (1.4%) HER2 IHC 0. The proportion of high-grade tumors in patients with HER2 high-expression was higher than that in patients without (83.3% versus 44.5%, P<0.001). Additionally, a high Ki-67 index (≥20%) was more commonly noted in HER2 high-expression tumors ( P=0.003). In the patients treated with intravesical anthracycline instillation, HER2 high-expression was associated with a shorter RFS ( P<0.001). Conclusion:HER2 high-expression seems to be not only associated with worse clinicopathological features of NMIBC but also a poor RFS in NMIBC patients treated with anthracycline instillation after TURBT.