Colorectal cancer (CRC) is one of the most common malignancies of the digestive tract and is ranked third for incidence and second for cancer-related mortality worldwide (1). It has been reported that 20% of patients with CRC have metastatic disease at the time of presentation, and a further 25% who initially present with in situ tumors develop metastases during follow-up (2). Notably, 5% of metastatic CRCs harbor alterations in Erb-B2 receptor tyrosine kinase 2 (ERBB2), also known as human epidermal growth factor receptor 2 (HER2). HER2 is a known oncogene (3) that can trigger carcinogenesis via the upregulation of HER2 protein expression, gene amplification of ERBB2, or point mutations, which occur mainly in patients with breast or gastric cancer (4).

HER2 positivity is considerably less common in CRC than in breast or gastric cancer, affecting only ~5% cases overall, but represents an important focus of research (5). First-line chemotherapeutic agents currently approved for KRAS/NRAS wild-type (WT) metastatic CRC include conventional cytotoxic agents such as fluorouracil, oxaliplatin and irinotecan, vascular endothelial growth factor inhibitors such as bevacizumab, and epidermal growth factor receptor (EGFR) inhibitors, including cetuximab and panitumab (2,6,7). However, there is no standard treatment for HER2-positive metastatic CRC. Although several recent studies have found anti-HER2 therapy to be effective in HER2-positive CRC (8–10), the evidence is not conclusive enough to warrant the inclusion of anti-HER2 agents in the relevant diagnostic and treatment guidelines. The present case report describes a patient who was diagnosed with rectal adenocarcinoma and multiple metastases to the liver in whom next-generation sequencing (NGS) results suggested positive HER2 expression. As two cycles of an intensive folinic acid (leucovorin) + fluorouracil + oxaliplatin (FOLFOX) exhibited poor treatment efficacy, anti-HER2 therapy was introduced. The clinical findings for this case are described to highlight the important role of anti-HER2 therapy in HER2-positive CRC and underscore the importance of further investigating personalized treatment strategies for this disease.

A 60-year-old woman with complaints of abdominal pain and passage of bloody stools was admitted to Hebei General Hospital (Shijiazhuang, China) in October 2023. Contrast-enhanced scans of the abdomen and pelvis revealed wall thickening in the sigmoid colon and rectum, enlarged lymph nodes in the surrounding area suggestive of malignancy, and low-density shadows in the S2, S3 and S4 segments of the liver that were thought to be metastases (Fig. 1). Colonoscopy suggested rectal cancer, and a tissue biopsy revealed moderately and well-differentiated adenocarcinoma with necrosis (Fig. 2). Magnetic resonance images revealed liver metastases in the S2, S3 and S4 segments and two enlarged lymph nodes, one anterior and one posterior to the portal vein (Fig. 3). Digestive tract tumor marker levels, including carcinoembryonic antigen, carbohydrate antigen (CA)242 and CA724, were all higher than normal (Fig. 4). NGS of a biopsy specimen obtained by colonoscopy suggested that KRAS, NRAS and BRAF were WT (Figs. S1 and S2). It also revealed that ERBB2 (HER2) had a mutation abundance 8.3-fold higher than the reference level, suggesting possible resistance to panitumumab and cetuximab. In addition, TP53 exon5c.399del had a mutation abundance of 31.61%, suggesting microsatellite stability. The NGS was performed by Novogene Bioinformatics Technology Co., Ltd. DNA extraction and library preparation was performed using the Qiagen QIAamp DNA FFPE Kit (Qiagen, GmbH) and Agilent SureSelect XT HS2 (Agilent Technologies, Inc.), respectively. Sample quality was assessed by pathological analysis of tumor cell content, nucleic acid quality assessment (total DNA amount, DNA degradation degree and total pre-library amount) and sequencing quality assessment (average sequencing depth, coverage uniformity, genome alignment rate and base quality Q30 proportion). The hybridization capture method was used, and the read length and sequencing direction were double ends of 150 bp and double-end sequencing, respectively. The sequencing platform and sequencing kit were the Illumina NextSeq 550 and Illumina NextSeq 550 High Output kits (Illumina, Inc.), respectively. Final library loading concentration was 1.2–1.8 pM. The software used for the analysis included CNVkit (version 0.9.9; University of California), GATK Mutect2 (version 4.1.8.1; Broad Institute of MIT and Havard) and PierianDx (version 7.3; Velsera, Inc.).

Following discussions among the multidisciplinary team and considering the patient's preference for surgery, neoadjuvant therapy was initiated, with plans to proceed to surgery for the primary CRC lesions and liver metastases if the treatment was effective. After obtaining informed consent, the patient was treated with two cycles of a modified FOLFOX6 regimen based on a body surface area of 1.66 m², comprising oxaliplatin 140 mg + leucovorin calcium 600 mg + fluorouracil 0.625 g by intravenous injection + fluorouracil 3.75 g. This regimen was administered by continuous intravenous drip on 13 days post admission and 28 days post admission. However, after the two cycles, no reduction in the size of the tumor lesions was evident (Fig. 5) and the tumor marker levels remained elevated (Fig. 4), suggesting poor treatment efficacy.

EGFR inhibitor therapy was not considered due to the NGS result and the evidence suggesting that patients with HER2-positive CRC are highly resistant to EGFR inhibitors (11,12). Furthermore, targeted bevacizumab therapy was not started because hemorrhage is one of the most serious adverse effects of bevacizumab and the patient had persistent hematochezia. In light of the recent developments in CRC research and the financial circumstances of the patient, single-agent anti-HER2 therapy combined with a modified FOLFOX6 regimen was selected as the subsequent treatment, in which the doses of chemotherapeutic agents were adjusted and administered on a 3-week cycle. The patient received six cycles of trastuzumab 450 mg + FOLFOX, with the latter comprising oxaliplatin 150 mg + leucovorin calcium 600 mg + fluorouracil 0.625 g by injection + fluorouracil 4 g by continuous intravenous drip. These agents were administered on 41 days post admission, 62 days post admission, 83 days post admission, 108 days post admission, 134 days post admission, and 157 days post admission. During treatment, the tumor marker levels gradually decreased (Fig. 4), and computed tomography (CT) and magnetic resonance scans of the liver indicated a marked reduction in the size of the tumor lesions (Fig. 6, Fig. 7, Fig. 8, Fig. 9). This was evaluated as a partial response.

On April, 2024, the patient underwent laparoscopy-assisted resection of rectal cancer and abdominal lymph node dissection, followed by surgery for the liver metastases. This included dissection of the group 8a lymph nodes, complete resection of the left outer liver lobe with intrahepatic metastases, and S4 resection, including metastases. This radical resection of the rectal cancer and liver metastases provided a foundation for subsequent treatment and potential improvements in survival and quality of life. Postoperative pathological examination of the rectal specimen suggested moderately and well-differentiated adenocarcinoma penetrating the muscularis propria to the subserosa, with no intravascular cancer thrombus or nerve invasion, and negative resection margins. One positive regional lymph node was detected. Immunohistochemistry (IHC) of the rectal specimen suggested positivity for mismatch repair endonuclease PMS2 (PMS2), DNA mismatch repair protein Msh2 (MSH2), DNA mismatch repair protein Msh6 (MSH6) and DNA mismatch repair protein Mlh1 (MLH1), which indicated microsatellite stability (Fig. S3). Postoperative pathological examination of the excised liver tissue suggested moderately and well-differentiated adenocarcinoma with a negative resection margin and no lymph node involvement. For IHC, the tissues were paraffin-embedded and fixed in 10% neutral-buffered formalin for 24 h at room temperature. The tissues were section to a 3-µm thickness. 3% Hydrogen peroxide was used for blocking for 10 min at room temperature. The following ready-to-use primary antibodies supplied by Roche Diagnostics, were added at 37°C for 32 min: VENTANA anti-MLH1 (M1) Mouse Monoclonal Primary Antibody (cat. no. 08033668001), VENTANA anti-MSH2 (G219-1129) Mouse Monoclonal Primary Antibody (cat. no. 07862253001), VENTANA anti-MSH6 (SP93) Rabbit Monoclonal Primary Antibody (cat. no. 07862245001) and VENTANA anti-PMS2 (A16-4) Mouse Monoclonal Primary Antibody (cat. no. 08033692001). These primary antibodies and OptiView DAB IHC Detection Kit (cat. no. 06396500001) supplied by Roche Diagnostics were used together to detect mismatch repair protein. The OptiView DAB IHC Detection Kit includes HRP-conjugated secondary antibodies optimized for automated staining on Ventana BenchMark instruments and was added at 37°C for 30 min. Results were assessed under a light microscope.

On postoperative day 6, abdominal-pelvic CT revealed postoperative changes in the rectum and liver, along with blurring of the left abdominal fat space, and small local nodules and small lymph nodes in the hilar area and omental capsule (Fig. 10). Repeat CT examination on 27 days post surgery showed less shadowing from the small nodules (Fig. 11).

The CT results indicated that a small lymph node in the hilum and omental capsule had a short diameter of ~8 mm, could be malignant, and would be difficult to remove surgically. Therefore, seven 3-week cycles of anti-HER2 therapy (300 mg trastuzumab on day 1 of the cycle) combined with oral chemotherapy (1 g capecitabine twice daily on days 1–14 of the cycle) were administered on 28, 39, 60, 81, 102, 123 and 145 days post-surgery. Thereafter, the chemotherapy was discontinued and the patient received three 3-week cycles of anti-HER2 therapy (trastuzumab 300 mg) on 165, 186 and 207 days post-surgery. The tumor marker levels were within the normal range during the postoperative period, and the patient remained on anti-HER2 therapy for 1 year. The latest CT result, which was recorded on 210 days post-surgery, showed postoperative changes in the rectum and liver, along with no signs of recurrence (Fig. 12). The patient was found to be in good condition and discontinued anti-HER2 therapy with instructions to attend for review every 3 months. The timeline of the treatment sequences, imaging evaluations and key clinical events are shown in Fig. 13.

The incidence of HER2 positivity in CRC is relatively low, at only ~5% overall (5). HER2 is a member of the ERBB family of transmembrane receptor tyrosine kinases (RTKs), which also includes ERBB1 (EGFR), ERBB3 and ERBB4 (13,14). It is widely accepted that the overexpression, amplification or mutation of HER2 results in resistance to EGFR inhibitors and can be used as a biomarker for the prediction of treatment efficacy (11,12,15). The EGFR inhibitor cetuximab exerts an antitumor effect by inhibiting EGFR and downstream ERK activation, thereby hindering cancer cell proliferation (16). The mechanism underlying resistance to EGFR inhibitors is thought to involve HER2 amplification, leading to aberrant activation of HER2-ERK signal transduction and the bypass of EGFR-ERK signaling (16). Accordingly, ERK activation is maintained in HER2-amplified cells, conferring resistance to EGFR inhibitors in patients with HER2-positive tumors (16). The specific targeting of HER2 with small interfering RNA or HER2 inhibitors has been shown to block HER2 and ERK activation in HER2-amplified cells, thereby restoring sensitivity to cetuximab (16). These findings suggest that the HER2-mediated activation of alternative signaling pathways promotes resistance to EGFR inhibitors and that anti-HER2 therapy could potentially restore sensitivity to EGFR inhibitors and exert an anticancer effect in HER2-positive tumors. Preclinical and clinical studies have shown that the progression-free and overall survival rates achieved by EGFR inhibitors in patients with HER2-amplified CRC are significantly lower than those of their WT counterparts and comparable with those in patients with RAS or BRAF mutations (17,18). This suggests that HER2 predicts a poor response to EGFR inhibitor therapy. Therefore, we hypothesize that CRC with HER2 positivity, whether amplified, overexpressed or mutated, can lead to resistance to EGFR inhibitors and that the early initiation of anti-HER2 therapy should be considered.

In the present case, the NGS results indicated that KRAS, NRAS and BRAF were all WT, while ERBB2 (HER2) was mutated and amplified with a mutation abundance elevated 8.3-fold. These findings suggest possible resistance to drugs such as panitumumab and cetuximab, based on the aforementioned findings. Accordingly, first-line chemotherapy alone was initiated for metastatic CRC; however, after two cycles, an increase in tumor marker levels was observed and no clear change in the size of the lesions was shown by imaging, indicating that chemotherapy alone was having a poor effect. Given advances in clinical trials on CRC diagnosis and treatment, as well as the patient's circumstances, the patient was subsequently treated with anti-HER2 therapy combined with chemotherapy. Notably, tumor markers gradually decreased during the treatment, and imaging revealed a reduction in the size of the lesions. After six cycles of treatment, the tumor marker levels were within the normal range and the lesions were markedly smaller. Simultaneous surgical resection of the rectal cancer and liver metastases was subsequently achieved, demonstrating that anti-HER2 therapy can be beneficial in the treatment of metastatic CRC. Although HER2 has a well-established prognostic significance in breast cancer, its prognostic role in metastatic CRC remains unclear (19). However, HER2 positivity appears to have been beneficial in the present case, converting unresectable lesions into resectable ones after anti-HER2 therapy, potentially improving the prognosis and quality of life of the patient.

HER2 positivity is more common in patients with RAS/BRAF WT disease (20), with one study reporting HER2 upregulation in only 1.0% of patients with KRAS/BRAF mutations compared with 5.2% in those with WT disease (21). In the MyPathway trial, 23% of patients had KRAS mutations, with an objective response rate (ORR) of only 8%, much lower than the ORR of 40% observed for patients with KRAS WT disease (22). An exploratory analysis of the Phase II TRIUMPH study also showed the treatment responses in patients with alterations in RTK/RAS/PI3K-related genes detected by circulating tumor DNA were poorer compared with those of patients without these alterations (23). These studies suggest that patients with KRAS WT disease may have a stronger response to anti-HER2 therapy. The microsatellite instability (MSI) status of CRC can be classified as MSI-high, MSI-low or microsatellite-stable (MS-stable) (24). Immunotherapy offers significant benefits to patients with MSI-high CRC and has been approved in this indication by the US Food and Drug Administration. However, it does not provide a significant survival advantage in patients with MS-stable CRC (25). In the present case, NGS results showed that KRAS, NRAS and BRAF were WT, the tumor microsatellite status was MS-stable, and HER2 expression was positive; therefore, anti-HER2 therapy was recommended.

HER2 overexpression or amplification is traditionally assessed by IHC to evaluate protein expression and by in situ hybridization (ISH) to evaluate gene amplification. The diagnostic criteria developed by Valtorta et al (26) for the HERACLES clinical trial established a standardized procedure for defining HER2 positivity in CRC by IHC and ISH. According to these criteria, HER2 status is considered positive if there is intense (3+) IHC staining in ≥50% of cells, or either intense (3+) IHC staining in >10 to <50% of cells or moderate (2+) IHC staining in ≥50% of cells, combined with amplification on ISH testing, defined as an ERBB2 to centromere of chromosome 17 ratio of ≥2 in ≥50% of cells (26). Studies (27,28) have shown that NGS results are closely consistent with IHC/ISH testing results, and can also be used to evaluate the amplification of HER2 in CRC. Furthermore, the assessment of circulating tumor DNA by liquid biopsy is a simple method for the detection of HER2 status that requires only a blood sample rather than a tissue biopsy, with some studies demonstrating its success in metastatic CRC (23,29,30). Therefore, the circulating tumor DNA assay can be used to complement tissue-based methods or serve as an alternative when tissue testing is not feasible. Sartore-Bianchi et al (12) proposed that HER2 status assessment should be included in the molecular diagnostic workup of all patients with metastatic CRC to allow prompt referral to clinical trials involving HER2-targeted double blockade, regardless of previous anti-EGFR treatment. Another study emphasized the importance of detecting HER2 status in patients with advanced or metastatic CRC, particularly those with RAS WT and MS-stable disease (31). The current National Comprehensive Cancer Network guidelines recommend the assessment of HER2 status in all patients with metastatic CRC using either IHC/ISH or NGS (32). This indicates that greater emphasis should be placed on the detection of HER2 expression in patients with CRC, particularly those with metastatic disease. Timely treatment based to these results may lead to enhanced clinical outcomes and an improved prognosis.

The findings from the Trastuzumab for Gastric Cancer trial, published in 2010, revealed that the mean overall survival of patients with HER2-positive (IHC 3+ or overexpression on fluorescence ISH) recurrent or metastatic gastric adenocarcinoma or gastric-esophageal junction cancer was prolonged when trastuzumab was added to first-line chemotherapy. In addition, the combination of trastuzumab and chemotherapy exhibited a safety profile comparable with that of first-line chemotherapy alone (33). This discovery marked a shift from traditional therapy to a new era of molecular targeted therapy for gastric cancer. Trastuzumab combined with chemotherapy is now considered the standard first-line treatment for HER2-positive gastric cancer (34,35). Furthermore, the 2023 Chinese Society of Clinical Oncology gastric cancer guidelines emphasize that HER2 status testing is necessary in all patients with a confirmed pathological diagnosis of gastric adenocarcinoma (36).

Patients with gastric cancer are tested early for HER2 status, and those who are positive receive first-line anti-HER2 treatment. However, in CRC, the testing of anti-HER2 treatment typically occurs later. Insights from the present case and a review of the literature on anti-HER2 therapy for gastric cancer suggest some notable similarities between the diagnosis and treatment principles of gastric cancer and those of CRC (33). This leads to the suggestion that the early detection of the HER2 expression status of patients with CRC, particularly those with metastatic CRC, could allow anti-HER2 therapy to be brought forward into the first-line setting, potentially improving patient outcomes and quality of life. Similarly, another study proposed that anti-HER2 therapy for HER2-amplified CRC should be initiated as soon as possible rather than reserved for last-line application (37). However, further research is necessary to determine whether the earlier application of anti-HER2 therapy should be included in clinical guidance on the diagnosis and treatment of CRC.

At present, anti-HER2 therapy mainly includes monoclonal antibodies such as trastuzumab and pertuzumab, tyrosine kinase inhibitors such as lapatinib and tucatinib, and antibody-drug conjugates, including trastuzumab deruxtecan and trastuzumab emtansine. Advances in molecular pathology and detection methods have led to a series of studies in patients with HER2-positive CRC, including MyPathway, TRIUMPH, MOUNTAINEER, DESTINY-CRC and HERACLES (22,38–40). Most treatments in these studies involved a dual-targeted approach, such as a combination of two anti-HER2 antibodies or a monoclonal antibody combined with a tyrosine kinase inhibitor. Significant improvements have been reported in the overall prognosis for these treatments compared with standard treatment or best supportive care in the aforementioned trials, particularly with regard to the ORR, suggesting that patients with HER2-positive CRC can benefit from anti-HER2 therapy. Based on these promising outcomes, the National Comprehensive Cancer Network guidelines recommend four HER2-targeted regimens, namely, trastuzumab combined with pertuzumab, lapatinib or tucatinib, or trastuzumab deruxtecan (DS-8201) alone for patients with CRC, HER2 amplification and WT RAS/BRAF (32). In the present case, a dual-targeted regimen was not feasible due to financial constraints. Therefore, single-agent trastuzumab was used as a targeted therapy and achieved favorable results. Nevertheless, a dual-targeted approach is recommended when financial resources allow.

Trastuzumab deruxtecan is an antibody-drug conjugate consisting of trastuzumab linked to a topoisomerase I inhibitor. The Phase II DESTINY-CRC01 trial assessed the activity of this agent in patients with HER2-positive, WT RAS/BRAF and previously treated metastatic CRC at a dosage of 6.4 mg/kg every 3 weeks (40). In contrast with the aforementioned trials (22,38,39), the DESTINY-CRC01 trial allowed the inclusion of patients who had previously received anti-HER2 therapy (40). This trial found no significant reduction in the response rate in patients who had previously received anti-HER2 therapy, suggesting that trastuzumab deruxtecan is effective in anti-HER2-pretreated patients (40). The subsequent DESTINY-CRC02 trial explored the efficacy of two dose levels of trastuzumab deruxtecan (6.4 and 5.4 mg/kg) administered every 3 weeks (41). Unlike DESTINY-CRC01, DESTINY-CRC02 enrolled patients with both RAS-WT and RAS-mutant CRC (41). No significant difference in treatment response was observed between the two dose levels (41). However, due to toxicity concerns, the 5.4 mg/kg dosage was preferred, supporting its use in future treatments. This trial demonstrated a promising treatment effect in patients with RAS-mutated disease (n=20; ORR 20.0%) and those who had previously received anti-HER2 therapy (n=27; ORR 40.1%), suggesting that trastuzumab deruxtecan could be considered for the subsequent treatment of HER2-positive and RAS-mutated metastatic CRC (41). In addition, both trials showed that the response of patients with IHC 3+ disease was improved compared with that of patients with IHC 2+/ISH+ disease, indicating that high HER2 expression may be important for an effective response to anti-HER2 therapy (40,41).

Most anti-HER2 therapy regimens used in clinical trials for HER2-positive CRC have demonstrated good tolerability. However, cardiotoxicity associated with anti-HER2 agents, and interstitial lung disease or pneumonia associated with trastuzumab deruxtecan warrant careful monitoring (42–44). In the present case, the patient tolerated anti-HER2 therapy well, with no adverse effects observed. The results of regular electrocardiograms and measurements of left ventricular ejection fraction were within the normal ranges. Cardiac function should be assessed before, during and after the completion of HER2-targeted therapy, and treatment should be withheld or discontinued if a significant reduction in ejection fraction is observed (45,46). Grade ≥3 treatment-related adverse events have been reported in clinical trials of trastuzumab deruxtecan for breast cancer, gastric cancer and CRC (47–49). Therefore, patients treated with this agent should be promptly evaluated for any signs or symptoms of pulmonary toxicity. The aforementioned studies (40,41) have investigated a variety of anti-HER2 agents, including some that remain under investigation. Further clinical research is necessary to determine the optimal anti-HER2 regimen and the appropriate sequencing of subsequent anti-HER2 therapies.

Currently available anti-HER2 therapies have provided clinical benefits to certain patients with HER2-positive CRC. However, more than half of patients do not experience any benefit, suggesting that anti-HER2 therapy has some limitations. A meta-analysis found a median progression-free survival of only 4.89 months in patients with advanced CRC receiving anti-HER2 therapy (50). The relatively lower efficacy of anti-HER2 therapy in patients with HER2-positive CRC in comparison with that in patients with other types of cancer may be associated with HER2 resistance (51). While a number of studies have shown that the overexpression or mutation of genes involved in the RTK/RAS and PI3K pathways may contribute to resistance to anti-HER2 therapy (51–53), additional studies are necessary to explore the mechanisms of this resistance. This may facilitate the identification of strategies that can overcome drug resistance and enhance the clinical benefits of this therapy for patients with HER2-positive CRC.

In conclusion, anti-HER2 therapy has not been thoroughly investigated in CRC, and the relationship between HER2 positivity and the prognosis of patients with CRC remains inconclusive. The present case provides evidence that patients with HER2-positive CRC could significantly benefit from anti-HER2 therapy, and such therapy may confer a good prognosis for patients. Moreover, given that HER2 positivity is associated with resistance to EGFR inhibitors, early anti-HER2-containing combination therapy is warranted. Therefore, it is important to assess HER2 status promptly and accurately in patients with CRC, particularly those with metastatic disease. Further studies are required to determine whether anti-HER2 therapy can be moved into the frontline setting for patients with HER2-positive CRC and to identify the optimal anti-HER2 regimen.