【Clinical Data】

Egfr-KDD has been reported in patients without any additional EGFR mutations, supporting preclinical evidence that replication of kinase domains alone can serve as a oncogenic driver of non-small cell lung cancer (Table 5). Despite limited clinical data on this rare mutation event, the evidence reported to date suggests that EGFR-KDD is sensitive to targeting EGFRI. In a case report submitted by Baik et al., a lung cancer patient carrying EGFR-KDD was treated with gefitinib for 6 years until disease progression.

The patient used erlotinib again as a fourth-line treatment, resulting in a tumor response lasting for another 3 years after short-term chemotherapy and pemetrexed (discontinued due to disease progression). In another report, they confirmed a non-small cell lung cancer patient with EGFR-KDD who had partial response after 2 cycles of afatinib treatment. After 7 cycles of afatinib treatment, acquired resistance occurred and amplification of EGFR-KDD was detected - suggesting an increase by amplification of EGFR-KDD

Oncogene dosage is one of the mechanisms of resistance to afatinib treatment. In this largest multi-center study, the clinical results of EGFR-KDD for targeted therapy were mainly focused on. Wang et al. reviewed 10,759 patients with East Asian non-small cell lung cancer treated with NGS, and identified 13 patients with EGFR-KDD positive patients, of which 5 patients received targeted therapy. Two of the 5 patients were ineffective for EGFR-KDD treatment, including gefitinib, erlotinib and ossitinib, and experienced disease progression, with PFS shorter than 3

Months. The remaining three patients either had partial responses to EGFR, and the progression of the disease had not been reached within at least 4 months, or the condition was stable for 11 months (see Table 5). In summary, these studies showed that despite the certain heterogeneity of the patient's response, the EGFR-KDD mutation was the target of EGFR. In addition, T790M mutation and EGFR-KDD amplification were reported in the biopsy tissue of the EGFR-KDD tumor specimens after EGFR treatment, which indicated the same resistance mechanism for targeted treatments as the classic EGFR mutation.

Picture [Compound Mutation]

【Preclinical data】

Kohsaka et al. performed EGFR exon sequencing of a set of NSC samples. It was reported that in the g719x mutation, more than 90% of the mutations (n=15) exist in the form of complex mutations. In addition, they reported that more than 75% of the e709x mutations (n=102) in the cosmic database also exist in the form of complex mutations. Droplet     digital      pcr(ddpcr) results show that all complex mutations exist in the cis alleles. koh

Saka et al. described that in the nih-3t3 and BA/F3 cell models, L858R, G719A, G719C or Ex19del alone or in combination with trans or cis expressed a rare set of mutations. They found that nih-3t3 cells expressing complex mutations in cis genes form more lesions than nih-3t3 cells expressing single or complex mutations in trans genes. Combined with the DDPCR data, this finding suggests that the transformation potential of complex mutations is greater than that of rare egfr mutations.

The conversion potential of individual mutations is higher. High-throughput screening is used to evaluate the response of single and complex EGFR mutations to gefitinib, erlotinib, afatinib and ossitinib. In high-throughput screening, BA/F3 cells expressing different mutations are encoded and pooled together by unique barcodes, and the detection of barcodes uses deep sequencing to estimate relative cell counts. For gefitinib, erlotinib and ossitinib treatment, the I50 value of each compound EGFR mutation is between each corresponding single EGFR mutation. For example, GI

Feninib alone treated the IC50 value (4.4 nm) of l858r was lower than the IC50 value (259 nm) of l858re709a, but the composite mutation was more sensitive to drug treatment than the single rare mutation of E709a (785.8 nm). Interestingly, these differences were not observed in the irreversible second-generation EGFRI    Afatinib, which effectively inhibited cellular activity at a similar level in all the single and complex EGFR mutations tested.

【Clinical Data】

EGFR complex mutations include a wide range of different mutation combinations, and therefore, a wide range of patients are expected to respond to EGFR. However, besides known drug-resistant mutations, such as T790M, complex EGFR mutations have produced more favorable patient outcomes compared to single rare mutations. In particular, the coexistence of rare EGFR mutations with classic L858R or Ex19del mutations may be a strong indicator of EGFR sensitivity. Baek et al.

It is reported that after EGFRI treatment, the median PFS for complex classical and rare mutation combinations were 7.4 months and the median PFS for complex rare and rare mutation combinations were 5.1 months. In these two cases, the median PFS was significantly prolonged compared to the patient group with a single rare exon 18 point mutation or exon 20 insertion (1.3 months and 2.6 months, respectively). This supports preclinical data, suggesting that complex mutations are more sensitive to EGFRI than a single rare mutation.

In rare and rare combinations of complex EGFR mutations, sensitivity to EGFR may be affected by specific symbiotic partner mutations. For example, Chiu et al. observed differences between G719xl861q and G719xs768i RR (88.9%) and G719xs768i (50%). After EGFR treatment, the L858 Rex19del complex mutation obtained a median 9.5 month PFS, which is similar to the median PFS in patients with L858R or Ex19del single mutations, indicating that such complex mutations have equal sensitivity to the corresponding single mutations. Larger needs to be done through in vitro preclinical data

Clinical studies at scale to predict the combination of mutations most sensitive to EGFRI. While individual patients with classical EGFR mutations have the best clinical response to EGFRI, these studies show that in most cases, patients with complex mutations have better clinical response to EGFRI than patients with single rare EGFR mutations. By contrast, clinical responses to complex mutations including primary mutations (such as T790M) associated with first-generation or second-generation EGFRI resistance were not as good. Undesirable RRs were reported in patients with complex EGFR mutations of 8.3% and median PFS was 1.4 months, with these mutations containing exon 20 insertion or T790M mutations.

【Future prospects】

Structural data and preclinical models can elucidate the activation mechanisms of rare EGFR mutations and have been used to predict the sensitivity of different mutations to EGFR. When sufficient patient populations are recruited and evaluated in clinical trials, this information can be translated into the identification of therapies that significantly improve survival than traditional chemotherapy, such as the successful approval of AFATINIB treatment for patients carrying rare G719x, S768i or L861q. As novel EGFR, immunotherapy and reasonable drug combinations develop, the treatment landscape of classic EGFR mutations is rapidly evolving to determine the most effective and durable therapeutic strategies. However, there are several open questions to be addressed to ensure that research on rare EGFR mutations keeps pace with this rapidly developing field.

Rare mutations in epidermal growth factor receptors account for 15% of epidermal growth factor receptor mutations in non-small cell lung cancer, with approximately 30,000 diagnosed cases per year due to the high prevalence of lung cancer. Although many rare EGFR mutations are associated with poor response to first-generation EGFR mutations compared to classic EGFR mutations, more effective alternatives to EGFR mutations have been found for several rare EGFR mutations, such as the insertion of exon 20.

Therefore, in future clinical practice, it is necessary to increase the absorption of improved detection methods (such as NGS) in order to identify rare EGFR mutations and specify the most effective EGFR mutations on the basis of mutation specificity. The question of whether different rare EGFR mutations differ in their interactions or downstream signaling networks remains unanswered. To fully address this biological problem, it is necessary to develop new preclinical models of rare EGFR mutations. A better understanding of the basic biology of different rare EGFR mutations may determine the specific dependencies of mutations that can be utilized therapeutically.

To address the lack of clinical data associated with rare EGFR mutations, future studies cannot rule out patients with rare EGFR mutations, and PFS and Os data for each rare EGFR mutation should be reported separately for post-combination analysis. Clinical trials specifically targeting rare EGFR mutations can greatly improve the treatment options for patients carrying rare EGFR mutations. The EGFR mutant NSCLC is also evaluating EGFR alternative strategies. Immunotherapy has shown efficacy in patients carrying rare EGFR mutations, which may open up new therapeutic strategies for those with poor mutations to available EGFR mutations.

However, this exciting prospect is hampered by the small sample size and lack of predictive biomarkers in the current study. Larger cohort clinical trials targeting rare EGFR mutations and identifying reliable predictive biomarkers are critical for the clinical development of these therapies. Finally, gefitinib combined with chemotherapy can prolong the median survival of patients with typical EGFR mutations. Future clinical trials evaluate whether similar benefits can be achieved in patients with rare EGFR mutations who are sensitive to afatinib, which may significantly improve the treatment of these mutations.

Checkpoint receptors are crucial to prevent hyperimmune and autoimmune responses. L numerous experiments have proved that blocking immune checkpoint signals produces a significant anti-tumor immune response.

Patients with non-small cell lung cancer (NSCLC) can benefit from inhibiting pd-l1 and ctla-4 using antibodies, however, simple and effective prediction of the biomarkers of the treatment response are still missing.

To predict response to ICI treatment, histological quantification of pd-l1 expression in tumors was usually performed. However, it was found that the correlation between pd-l1 expression and the overall response rate (ORR) was insufficient in tumor biopsy.

In lung cancer, evaluating smoking history, tumor mutation load (tmb), microsatellite instability (msi), high expression of CTLA4, low expression of CX3cl1 and infiltration of CD8T cells in TME appear to be superior in predicting responses to anti-PD-1/PD-11 treatments compared with histopathological PD-11 quantification, however, so far, these markers have not been converted into a reliable and clinically easy-to-use biomarker feature.

In this study, quantitative proteomic analysis was used to study the global changes in protein expression at distant sites, and the impact of local rt on distant sites was observed, and non-invasive probes were developed.

Research Results 1-Tumor cells transfer pd-l1 to platelets

Pdl1 positive is defined as tumor cells with ≥5% expression of pd-l1 in cells. Pd-l1 expression on platelets was observed after coincubation with NCI-H226 and NCI-H460 cells expressing pdl1, but not after coincubation with NCI-H23 and A549 of pd-l1 low/negative cell lines.

Live cell imaging can observe the interaction between tumor cells and platelets.

The interaction between tumor cells and platelets transports the protein of pd-l1, which is not mRNA. Because actinomycin inhibits protein translation in platelets does not lead to a decrease in pd-l1-gFP expression in platelets.

The expression level of adhesion molecules may determine the effect of protein transfer from tumor cells to platelets.

The pd-l1 transfer rate is positively correlated with Fn expression.

The transfer rate of pd-l1 is related to Fn expression, integrin α5β1, and gpibα.

Study Results 2-Detection of functional pd-l1 in platelets in patients with nsclc

A large number of pd-l1-positive platelets were observed in tissue sections of patients with pd-l1-positive non-small cell lung cancer.

Adding αpd-l1 will inhibit the inhibitory effect of pd-l1-positive platelets on immune cells.

Adding αpd-l1 will inhibit the inhibitory effect of pd-l1-positive platelets on antigen-specific immune cells. It is related to T cell infiltration in TME

Research Results 3-The regulation of platelet pd-l1 during platelet activation

Pd-l1 protein level increases after platelet activation in patients with nsclc

Study Results 4-Adjusted platelet-derived PD-L1 can be used as a prognostic and predictive indicator of NSCLC

Since neutrophils were previously associated with immune and radiotherapy responses, we next wondered whether the lack of glut1 in these cells could enhance this treatment. Although glut1   Ko neutrophils had reduced PD-L1 expression, they failed to sensitize KP tumors against PD1 (Figure 7A-B). In contrast, the anti-tumor response of radiation therapy was significantly enhanced, multiple (62.5%) tumor regression was monitored after two weeks of radiation, and tumor growth period (at least 28 days) loss in glut1   Ko tan-irradiated mice (Figure 7A). Thus, glut1 deletion in neutrophils increased the sensitivity of KP lung tumors to radiotherapy, resulting in lasting growth impairment.

Neutrophils are the most abundant leukocytes in the circulation and are crucial for natural immunity. In cancer, tumor-associated neutrophils (tans) have promotonic or antitumor properties. Here, we focus on the accumulation of tan in lung tumors and determine that glut1 is a necessary glucose transporter that supports tumor behavior. Compared with normal neutrophils, glut1 and glucose metabolism are increased in tan in the mouse model of lung adenocarcinoma. To clarify the effect of glucose uptake on tans, we used two recombinant enzymes.

The strategy is to isolate tumor initiation from neutrophil-specific glut1 deletion. The loss of glut1 accelerates the turnover of neutrophils in tumors and reduces the tan subset of siglecf expression. In the absence of glut1 expression in tan, tumor growth is weakened and radiotherapy is enhanced. Our results demonstrate the importance of glut1 in tan, which may affect its antitumor behavior. These results also show that for metabolic vulnerability, it is beneficial to antitumor neutrophils.

A combined evaluation of platelet CD62P and PPD-L1 levels predicted Os in 128 patients with NSCLC.

ppd-l1adj has a predictive effect on Os and a predictive effect on the response to PD-1 antibody treatment.

Highlights:

1. Prove that platelets interact with tumor cells and transport the pd-l1 protein through fibronectin, integrin, and gpibα-dependent methods.

2. Pd-l1 on platelets can inhibit immune cells.

3. An algorithm was developed to calculate activated platelet pd-l1 payload predicts the therapeutic efficacy of ICI. It is different from traditional pathological pd-l1 quantification.

4. Platelet pd-l1 reflects the overall pd-l1 expression of the tumor and plays an important role in tumor immune escape. It overcomes the histological quantitative limitation of heterogeneity of pd-l1 expression in the tumor.
Chapter completed!