Classification of Tumor Drug Resistance Tumors can be drug resistant in a number of ways and can also be classified into different categories. One way to classify tumors is based on drug responsiveness, which can be divided into two categories, primary resistance and acquired resistance. Primary resistance means that the tumor does not respond to the drug from the beginning to the end, and acquired resistance means that the patient responds well to the targeted therapy in the early stage of the treatment, but the response decreases in the later stage. In another classification, tumor resistance is classified into three major categories based on the mechanism of tumor resistance to targeted therapy. Pathway redundancy: the signaling pathway can remain activated during targeted therapy. Evasive pathway: After the signaling pathway is blocked by targeted therapy, the cell can turn on another alternative signaling pathway. Pathway reactivation: After the signaling pathway is blocked by an inhibitory therapy, the cell can reactivate the signaling pathway by mutating the downstream receptor. Biopsy is the only test of ‘truth’ Before exploring tumor drug resistance, one point needs to be emphasized: biopsy plays an important role in tumor drug resistance control. Only by serial histological analysis of tumor samples at the time of resistance can we better understand the clinical mechanism of action of acquired resistance to targeted therapy. Biopsy analysis makes it possible to determine the mechanisms of resistance in progressive lesion tissue, so that targeted solutions can be designed for patients. Resistance to anti-HER2 therapy in breast cancer Human Epithelial Growth Factor Receptor 2 (HER2) is an important molecular target for targeted therapy in breast cancer. Nearly 15-20% of breast cancer samples have high expression of HER2, a receptor that activates downstream signaling pathways and alters transcription and translation levels to promote cell proliferation and metastasis, and resistance to apoptosis. Therefore, HER2 is a common target for breast cancer treatment. The first approved anti-HER2 drug was a monoclonal antibody, trastuzumab, which binds to the extracellular structural domain of HER2. Based on its theoretical mechanism of action, Trastuzumab was approved primarily for the treatment of metastatic HER2-positive breast cancer, but also in combination with chemotherapy. A pairwise analysis of the NSABP B31 and NCCTG N9831 clinical trials showed that patients receiving trastuzumab and standard chemotherapy had significantly longer disease-free survival compared to patients receiving standard chemotherapy. In addition, several other studies have validated the clinical benefit of trastuzumab. However, some patients experienced tumor recurrence after receiving trastuzumab. As a result, researchers have begun to investigate the mechanisms of resistance to anti-HER2 therapy. Trastuzumab is an effective inhibitor of HER2/HER2 homodimerization, but not of heterodimerization. 1. Resistance due to redundancy in the HER2 signaling pathway Incomplete receptor closure may be a mechanism of anti-HER2 resistance. The results of numerous preclinical trials have shown that multipoint inhibition of the HER2 pathway improves the efficacy of anti-HER2 therapy. The CLEOPATRA trial has brought dual inhibition of HER2 (homo-/heterodimerization) to the forefront of treatment for HER2-positive metastatic breast cancer. In the study, breast cancer patients were randomized to trastuzumab + docetaxel (Pertuzumab), trastuzumab + docetaxel + patuzumab (Pertuzumab). The results of the study showed that trastuzumab + patuzumab treatment significantly improved patient survival compared to trastuzumab, extending overall survival by 16 months. This study provides strong evidence that combining HER2 dual inhibition with chemotherapy is the treatment of choice for metastatic cancer. The NEOSPHERE trial also validated the clinical benefits of anti-HER2 dual inhibition therapy. In this study, patients with HER2-positive breast cancer were randomized to four groups: standard chemotherapy + trastuzumab, standard chemotherapy + patuzumab, standard chemotherapy + trastuzumab + patuzumab (dual-inhibitory treatment group), and trastuzumab + patuzumab. The results showed that the complete remission rate of patients in the dual-inhibition treatment group was twice as high as that in the single-inhibition treatment group (standard chemotherapy + trastuzumab or patuzumab). The complete remission rate in the trastuzumab + patuzumab group was nearly 17%, indicating that both targeted agents produced an anti-tumor effect by blocking the HER2 pathway in some patients. The NeoALTTO trial randomized breast cancer patients into 3 groups: paclitaxel + lapatinib, paclitaxel + trastuzumab, and paclitaxel + palatinib + trastuzumab. The same results were found in this trial, with patients in the dual-inhibitory treatment group experiencing twice the rate of complete remission as those in the monotherapy group. The TBCRC 006 trial enrolled patients with estrogen receptor (ER)-positive breast cancer in the study for 12 weeks of lapatinib + trastuzumab + estrogen deprivation therapy. The results showed that patients had a complete remission rate of only 27%, similar to the results of the targeted monotherapy arm of the NEOSPHERE trial, suggesting that effective pathway blockade can completely inhibit tumor growth. Of course, not all clinical trial results support the conclusion that dual-inhibition therapy is superior to single-inhibition therapy. For example, the ALTTO trial randomized 8,000 HER2-positive breast cancer patients into three groups: chemotherapy + trastuzumab, trastuzumab + lapatinib, and chemotherapy + trastuzumab + lapatinib. Interestingly, there was no significant difference in the complete remission rates of patients in the three different treatment groups. However, these conclusions are again supported by the results of the ExteNET trial, which randomized trastuzumab-treated breast cancer patients into 2 groups: lenatinib and placebo.The opposite results of the ALTTO trial and the ExteNET trial may be due to differences in the patient populations or the types of medications, or perhaps to the fact that lenatinib was significantly more potent than lapatinib. The above clinical trials of targeted therapies suggest that pathway redundancy is a mechanism of drug resistance in tumors, and that effective pathway inhibition can still enhance efficacy or reduce drug resistance. 2. ER signaling pathway is an evasive pathway for HER2 resistance Nearly 50% of HER2-positive breast cancer samples are positive for estrogen receptor (ER) expression, which may be another mechanism of tumor resistance: the evasive pathway. When cells are ER-positive, signals can be transmitted through the ER pathway even when other signaling pathways are inhibited, ultimately producing changes in transcript levels that promote cell growth. Clinical trials have shown that ER-positive breast cancer patients have significantly lower complete remission rates than ER-negative patients. This hypothesis was tested in the TBCRC023 trial, which randomized patients with HER2-positive breast cancer into two groups: a 12-week or a 14-week course of lapatinib + trastuzumab + estrogen deprivation (for ER-positive patients). The results showed that for ER-positive patients, the 12-week treatment group had a complete remission rate of only 9%, while the 24-week treatment group had a complete remission rate of 33%. However, for ER-negative patients, there was no difference between the two treatment groups. This suggests that long-term targeted therapy may be effective only in ER-positive patients. Despite the lack of evidence, the results of the above clinical trials suggest that the ER pathway plays an important role in the drug resistance pathway in HER2-positive breast cancer. 3. PI3K Molecules, HER2 Mutations, and Reactivation of Pathways A third mechanism of resistance is pathway reactivation, which is the removal of the “brakes” from signaling pathways involved in cell growth regulation. Several studies have shown that phosphatidylinositol-3-kinase (PI3K) is the “brake” of the resistance-associated reactivation pathway. Patients with wild-type PIK3CA cancers have a higher rate of complete remission compared to PIK3CA mutants. This suggests that activation of the PI3K pathway may lead to resistance to anti-HER2 therapy. Similarly, HER2 mutations can produce resistance through reactivation of the pathway. In the TBCRC003 trial, biopsies of in situ HER2-positive breast cancers were compared with treated metastatic tumor lesions, and the rates of HER2 mutations were found to be significantly different in the two tissues. These studies contribute to our further understanding of the mechanisms of drug resistance. Regarding pathway reactivation, PI3K is a very attractive therapeutic target. Therefore, HER2 treatment is not as simple as expected, and it is necessary to classify HER2-positive breast cancers into different subtypes in order to give the appropriate treatment regimen for different types. Resistance to tyrosine kinase inhibitors in lung cancer There are two main categories of resistance to targeted therapy in lung cancer, resistance to epithelial growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) and resistance to mesenchymal lymphoma kinase (ALK) tyrosine kinase inhibitors (TKIs). 1. EGFR tyrosine kinase inhibitors Erlotinib, Gefitinib, and Afatinib were the first EGFR tyrosine kinase inhibitors to enter the clinical treatment of lung cancer, and patients with EGFR-mutant phenotypes of lung cancer have responded well to these drugs. However, acquired resistance is a major challenge for EGFR tyrosine kinase inhibitors, and what measures can be taken to delay or overcome this resistance.One cause of resistance to EGFR tyrosine kinase inhibitors is EGFR target modification, a two-point mutation in the structural domain of the EGFR tyrosine kinase (T790M). This mutation is present in nearly 60% of samples from patients with erlotinib-, gefitinib-, and afatinib-resistant lung cancers, and is a major cause of resistance to first- and second-generation EGFR tyrosine kinase inhibitors. Mutation-specific EGFR tyrosine kinase inhibitors are a new class of irreversible inhibitors that target EGFR mutations. Erlotinib, gefitinib, and afatinib mainly target wild-type EGFR, while mutation-specific EGFR tyrosine kinase inhibitors mainly target activating mutations, such as the T790M mutation. Currently there are two mutation-specific inhibitors that are close to entering the clinic, CO-1686 (Rociletinib) and AZD9291. A recent study published in the New England Journal of Medicine included 46 patients with erlotinib, gefitinib, and afatinib-resistant lung cancers in a clinical trial. The results of the study showed a 59% response rate to Rociletinib in T790M-positive patients. Interestingly, second-line treatment with a drug targeting T790M in lung cancer patients who had received first-line therapy resulted in response rates approaching 60 percent and disease control rates as high as 90 percent. In contrast, if a lung cancer patient does not develop a T790M mutation during first-line treatment, the response rate is very low. AZD9291 is another third-generation irreversible mutation-specific EGFR inhibitor. One study reported a response rate to AZD9291 of nearly 60% in patients with T790M-positive cancers and a very low response rate in patients with T790M-negative cancers, which is the same effect of Rociletinib described above. In 2014 a team evaluated the efficacy of an irreversible second-generation inhibitor in combination with afatinib (a wild-type EGFR-targeted inhibitor) and cetuximab (an EGFR monoclonal antibody) in patients with lung cancer. The study patients were all EGFR-mutant lung cancer patients who began treatment with afatinib and cetuximab after developing acquired resistance to a first-generation EGFR inhibitor. Results showed an overall response rate of 29% in lung cancer patients, including both T790M-positive and negative patients. This study suggests that mutation-specific EGFR inhibitors hold promise as first-line therapeutic agents for patients with EGFR-mutant lung cancer, but they are not effective in progressive T790M-negative patients. However, combining mutation-specific EGFR inhibitors with afatinib and cetuximab may be one solution to this dilemma. What if a third-generation EGFR tyrosine kinase inhibitor fails? Targeted therapies are generally considered effective, but even with third-generation EGFR tyrosine kinase inhibitors, the issue of resistance has to be considered. A recent trial enrolled 12 patients with T790M-positive cancers to evaluate the efficacy of Rociletinib. The results showed that six patients had a significant clinical benefit during Rociletinib treatment, but the response rate decreased and T790M disappeared later in the course of treatment. In other words, the allele in the T790M mutation in the patient’s tumor, which was the main cause of resistance to the first-generation EGFR tyrosine kinase inhibitors, was lost during treatment. Similarly, another trial in 2015 looked at acquired resistance to AZD9291. The study included 15 patients with T790M-positive EGFR-mutated lung cancer who developed resistance after treatment with AZD9291. They were categorized according to the mechanism of resistance: six patients had the C797S mutation (another mutation in the structural domain of the EGFR kinase); five patients had the T790M mutation but were negative for the C797S mutation; and four patients lost the T790M allele. This study suggests that although mutation-specific EGFR tyrosine kinase inhibitors hold promise as a first-line therapy for patients with EGFR-mutant lung cancer, attention needs to be paid to how to deal with the emergence of drug resistance. 2. ALK tyrosine kinase inhibitors Another class of targeted drugs for lung cancer are the mesenchymal lymphoma kinase tyrosine kinase inhibitors (ALK TKIs). Crizotinib (Crizotinib) is an FDA-approved ALK inhibitor primarily used to treat patients with metastatic ALK-rearranged lung cancer. Lung cancer patients respond well to crizotinib, but acquired resistance can occur, particularly in patients with EGFR mutant lung cancer. For patients with EGFR-mutant lung cancer, T790M-mediated resistance occurs in nearly 50-60% of patients. However, fewer than 30% of patients with ALK-rearranged lung cancer will develop a two-point mutation in the structural domain of ALK kinase, ultimately resulting in crizotinib resistance. In addition, there is a wide range of mutations that can trigger resistance to ALK tyrosine kinase inhibitors, and any site in the ALK structural domain can be mutated. Currently, several strategies have emerged to address crizotinib resistance, such as second-generation ALK inhibitors, ALK inhibitors in combination with HSP-90 inhibitors, and ALK tyrosine kinase inhibitors in combination with chemotherapy. Currently, there are two second-generation ALK inhibitors, LDK378 (Ceritinib) and Alectinib. A recent study evaluated the efficacy of Ceritinib in patients with ALK-rearranged lung cancer. The response rate to crizotinib was 56% in patients with crizotinib-treated lung cancer and 58% in patients with crizotinib-naïve lung cancer. It can be seen that crizotinib-resistant lung cancer patients have a good response to second-generation ALK inhibitors. Alectinib is a second-generation ALK inhibitor that is close to clinical ALK rearrangement therapy in lung cancer. A recent study enrolled 47 ALK-positive lung cancer patients treated with crizotinib, who had an overall response rate to Alectinib of nearly 55%, which is the same as the response rate for crizotinib in the study described above. In addition to second-generation ALK inhibitors, heat-stimulating protein 90 (HSP90), a protein that stabilizes oncogenic proteins such as ALK fusion proteins and the EGFR, has also been a therapeutic strategy in response to crizotinib resistance. In vitro studies have found that crizotinib-resistant cells are very sensitive to treatment with HSP90 inhibitors, suggesting that HSP90 may be a breakthrough in dealing with crizotinib resistance. In addition, the team found that crizotinib-resistant patients with ALK-rearranged lung cancer showed a favorable clinical response after one cycle of treatment with an HSP90 inhibitor (Ganetespib). Several studies are currently evaluating the efficacy of ALK inhibitors in combination with HSP90 inhibitors in lung cancer patients, which will help to understand the mechanism of action of HSP90 inhibitors in alleviating crizotinib resistance. ALK tyrosine kinase inhibitors in combination with chemotherapy is another treatment option to address crizotinib resistance. A retrospective study found that pemetrexed treatment significantly improved progression-free survival in patients with ALK-rearranged lung cancer compared to patients with other subtypes of metastatic lung cancer. However, the molecular mechanism of action of this clinical response is not clear. In conclusion, the development of targeted therapeutics has revolutionized the treatment of lung cancer patients. Currently, these drugs are mainly used in the treatment of metastatic lung cancer, and research in the field of breast cancer is significantly lagging behind. Resistance to Anti-EGFR Therapy in Colorectal Cancer This section will discuss the mechanisms of primary and acquired resistance to EGFR inhibitors in metastatic colorectal cancer, as well as the drugs currently being developed to counteract resistance to EGFR inhibitors.The EGFR pathway plays an important role in the development of a variety of tumors. Multiple mutations downstream of the EGFR in colorectal cancer have been found to inhibit signaling activation by monoclonal antibodies and tyrosine kinase inhibitor drugs The development of EGFR inhibitors for metastatic colorectal cancer has shown that EGFR pathway mutations are a therapeutic target. For example, in 2013 the FDA approved RAS testing for colorectal cancer patients. A review of the data from anti-EGFR inhibitor studies reveals that for patients with RAS wild-type colorectal cancer, an EGFR inhibitor (e.g., Panitumumab Panitumumab) + conventional chemotherapy (e.g., Oxaliplatin Oxaliplatin) significantly improves patients’ overall and progression-free survival. The results may be even better if cetuximab is switched. The risk ratio for overall survival was 0.75 for patients with KRAS exon 2 wild-type and 0.69 for patients with KRAS exon 2 wild-type + RAS mutations. it is clear that patients with KRAS or NRAS mutations do not benefit from anti-EGFR therapy. 1. Inherited Mutations and Anti-EGFR Resistance Polygenic mutations are drivers of resistance to anti-EGFR therapy, and again are a key part of the overlap between innate and acquired resistance mechanisms. First, with respect to innate resistance in first-treatment patients, the most common single-gene mutations include KRAS (30%), NRAS (7%), and BRAF (7%). Nearly 10-15% of patients had KRAS+PIK3CA or BRAF+PIK3CA double mutations, and 10% had PIK3CA or PTEN mutations. Similarly, nearly 12% of patients with congenital resistance stem from non-genetic mechanisms. In the case of anti-EGFR therapy, nearly 15% of patients have an innate response. Theoretically, when a patient is treated with an EGFR inhibitor, only 15% of patients will respond to the drug. Obviously, this response rate is even higher when combined with chemotherapy. What is acquired resistance? A patient who previously responded to an anti-EGFR inhibitor is now not responding. The primary mechanism for this resistance is RAS mutations (e.g., KRAS, NRAS.) BRAF mutations are also responsible for acquired resistance (7%), in addition to double mutations in the RAS pathway and PIK3 pathway (12%). Interestingly, the proportion of patients with acquired resistance with HER2 or MET overexpression was significantly higher after cetuximab or panitumumab treatment, approaching 10-12%. As with congenital resistance, nearly 12% of patients with acquired resistance develop disease from non-genetic mechanisms. Note that patients with innate resistance to EGFR inhibitors often have mutations in KRAS exon 2. Similarly, patients with acquired resistance to EGFR inhibitors also have mutations in KRAS exon 2, which are much more frequent than in patients with innate resistance. The Johns Hopkins team examined mutations in the RAS and PIK3 signaling pathways before, during, and after treatment with anti-EGFR inhibitors, and showed that the downstream effectors, KRAS and BRAF, were mutated. The results showed that the downstream effectors KRAS and BRAF were mutated, as were multiple exons of KRAS. Another team found that the frequency of mutations in several genes increased during cetuximab treatment. A total of 37 patients were enrolled in the study, and nearly 81% of the biopsies from patients who progressed had mutations. Interestingly, nearly 33% of the patients had multiple mutations (2-5) in their biopsies, suggesting that multiple genes are involved in the development of acquired resistance. The most commonly mutated genes were the RAS gene, as well as the PIK3CA, BRAF and EGFR genes. When pre-treatment biopsies of patients were examined, 24% had varying degrees of mutations. It is also important to note that the percentage of patients with mutated alleles increased significantly during the course of treatment. Once anti-EGFR therapy is discontinued, the frequency of mutations decreases to below detectable levels. 2. Tumor microenvironment and anti-EGFR resistance In addition to the previously mentioned genetic mutations, overexpression of ligands in the tumor microenvironment is also a cause of resistance, such as amphiregulin (amphoteric regulator protein), epiregulin (epithelial regulator protein), heregulin (regulator protein), and transforming growth factor-alpha (TGF-alpha). By detecting the expression of these molecules, it is possible to predict a patient’s response to treatment. The Khambata-Ford team found that patients with epithelial regulatory protein and dystrophin overexpression colorectal cancer responded better to cetuximab compared to other patients. the Tabernero team found that patients with epithelial regulatory protein overexpression responded better to treatment with cetuximab, and that up-regulation of TGF-alpha was associated with resistance to cetuximab treatment. Another team found that exposing cells to TGF-alpha resulted in significantly higher cellular resistance. In addition to mutations and ligands, the therapeutic response to EGFR inhibitors is also influenced by intrinsic molecular subtype. de Sousa’s team conducted a comparative study of epithelial and mesenchymal colorectal cancers. The results showed that for wild-type RAS tumors, only patients with epithelial colorectal cancer responded well to cetuximab, whereas mesenchymal patients did not respond to cetuximab. 3. Response options for anti-EGFR resistance Based on this information, a variety of effective therapeutic options for colorectal cancer can be developed, such as EGFR inhibition by novel monoclonal antibodies, the combination of monoclonal antibodies with kinase inhibitors, and the combination of the HER receptor family and other signaling pathway inhibitors. Dual inhibition of the receptor can be achieved by targeting both the extracellular and intracellular domains of the kinase. A team of Australian researchers evaluated the therapeutic effects of cetuximab and erlotinib in EGFR-negative and chemotherapy-resistant patients. Results showed a 41% response rate to treatment and a mean progression-free survival of 5.6 months in patients with wild-type RAS.The HERACLES trial identified HER2 overexpression in patients resistant to cetuximab or panitumumab. The overall response rate in this patient group was as high as 34% when treated with trastuzumab and lapatinib. Sym004 is a novel compound (a mixture of two chimeric antibodies) that binds to different antigenic epitopes of the EGFR.Sym004 binds to the receptor to produce a large molecular weight compound that down-regulates the receptor’s activity level. Data from studies have shown that cells are more sensitive to Sym004 than cetuximab when exposed to high concentrations of the ligand. Another treatment strategy is EGFR inhibitor activation-discontinuation therapy, second-line chemotherapy, and EGFR inhibitor retreatment. This treatment plan is based on the premise that the tumor patient’s resistance to anti-EGFR therapy decreases with drug withdrawal. The rationale for this treatment is that most tumor cells are wild-type cells at the beginning of treatment, so the tumor is very sensitive to treatment. During treatment with EGFR inhibitors, the cells begin to develop some resistance mutations. In the later stages of treatment, the tumor has become a fully drug-resistant phenotype. Therefore, if EGFR inhibitor therapy is stopped, the tumor may return to the state it was in at the beginning of treatment. This provides the rationale for tumor activation therapy, which is currently being evaluated in two clinical trials in cancer patients.