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Brain metastasis (brms) in lung cancer is associated with significant morbidity and mortality. Traditional treatment models focus on local control, but have limited survival benefits. Over the past decade, the immune system has become a strong prognostic and therapeutic target for cancer, leading to the development of immune checkpoint inhibitors. Drugs targeting programmed cell death 1 (pd-1) and programmed death ligand 1 (pd-l1) have shown efficacy in a variety of cancer subtypes. The anti-pd-1 drug pembrolizumab is very effective in the treatment of melanoma and non-small cell lung cancer (nsclc). In some cases, the persistent response of immunotherapy changes the treatment model for treatment of Nsclc. Although the results of these immunotherapies have shown efficacy in only a small number of patients, this emphasizes the need to optimize response predictors and identify new therapeutic targets.

With the success of drugs targeting pd-l1/pd-1, there has been increasing focus on alternative co-inhibitory receptors as therapeutic targets, such as T-cell immunoglobulin mucin receptor 3 (TI-3) and lymphocyte activation gene 3 (LAG-3). Upregulation of these receptors was initially observed in chronic viral infection models, reflecting the activation and dysfunction of T-cells under sustained T-cell stimulation. These “depleted” T-cells have reduced their ability to perform cytotoxic effectors, resulting in dysregulation of immune responses.

In lung tumors, the co-expression of immunosuppressive receptors in tumor-infiltrating lymphocytes (Til) can also be interpreted as indicators of sustained antitumor responsiveness. The spatial resolution and single-cell analysis of our team demonstrated that in tiles that express pd-1, lag-3 and tim-3 in lung cancer, the significant expression of activation markers supports that they may still perform antitumor function. Therefore, recovery effect function and memory status are of great significance to enhancing antitumor immunity.

Phylogenetic genomic analysis shows that Brms has obvious somatic genetic changes relative to matched primary tumors, biological differences in diseases within S) and opportunities for selective intracranial targeted therapy.

Although immune checkpoint inhibitors have become active and effective drugs in the central nervous system, basic elements of the tumor immune microenvironment, including the composition and functional characteristics of adaptive immunomarkers in BM, have been largely unexplored. Here, we used multiple quantitative immunofluorescence (QIF) tissue analysis to evaluate the differential expression, functional characteristics and clinical significance of the major til subpopulations in lung cancer-related BM and primary tumors.

We also used qif to measure pd-l1 for local measurement of tumor and stromal compartment. Compared with tumor compartment, pd-l1 expression in stromal compartment was significantly reduced (proportion score p=0.023, qif    p=0.018), but there was a strong correlation between compartments.

In primary lung tumors, cd4 expression (r=0.432) and cd8 expression (r=0.418) were positively correlated with pd-l1 (Supplementary Figure 1b online). These correlations were not observed in Brms (Supplementary Figure 1c online). In primary lung tumors and Brm, the correlations of foxp3 and pd-l1 were similar (foxp3: r=0.395 in lung, r=0.256 in Brm).

Similar tumor pd-l1 expression was observed between primary lung tumor and Brm by TPS and QiF measurements (Figure 1C and Supplementary Figures 2A and 3A online). A similar trend was also found in the paired sample subset.

Compared with primary lung tumors, brms patients had significantly reduced til density (p<0.0001; Figure 2a,b and Supplementary Figures 2 and 3), comparing the microenvironment of lung and brain tissue.

Granzyme B was selectively determined as a marker of T cell activation in cd3T cells or Ki67 as a marker of cell proliferation, showing lower T cell gzb (p=0.019) and similar Ki67 levels in Brms.

Using QiF to simultaneously measure immunosuppressive receptors on cd3t cells (Figure 3a), we observed that pd-1 (p=0.013), tim-3 (p=0.021) and lag-3 (p=0.008) in Brms were lower than those in primary lung tumors (Figure 3b and Supplementary Figures 2,3 online).

A relatively high correlation was observed between pd-1, tim-3 and lag-3 expression in cd3t cells in primary lung (r=0.62–0.79) and brms (r=0.64–0.80).

The expression of tim-3 (pulmonary r=0.568, brain r=0.433) and lag-3 (pulmonary r=0.549, brain r=0.393) in cd3t cells was correlated with pd-l1 in both tissues, although the correlation intensity in Brms was lower.

pd-l1 and pd-1 are related in lung tumors (r=0.405, p=0.009), but are not related in Brms.

High T cells in Brms were also associated with longer survival (Figure 3c). No differences in overall survival were found based on the expression of individual immunomarkers (Supplementary Figure 6 online).

In Brms, tumors with neuroendocrine characteristics had higher tumor cell Ki67 and lower tim-3 compared to other histology (Supplementary Table 3 online). Tim-3 in primary lung tumors at the time of diagnosis was also significantly higher in patients with IV stage than in early stages. Cd3 (p=0.022), tim-3 (p=0.003) and lag-3 (p=0.031) were also significantly lower in Brm tissues that had received any treatment prior to tissue sampling.

pd-1 was higher in previously treated Brms, but not statistically significant (p=0.051). Compared with Brms that were not treated with radiotherapy, Pd-l1 was significantly increased (p=0.023) and tim-3 was significantly reduced (p=0.044).

Using multiple direct qif and spatial resolution to measure protein markers, this study objectively described the differential expression of pd-l1, major til subpopulation, T cell activity and co-inhibitory receptors pd-1, tim-3 and Lag-3 markers in lung cancer-related Brm and primary tumor samples. Compared with primary lung tumors, the immune infiltration and T cell activation of Brms were significantly reduced.

We did not find significant differences in pd-l1 expression between brain and lung tumors. We observed a significant decrease in levels of all T-cell subpopulations (including regulatory T cells) in Brms, as well as decreased levels of T-cell pd-1, TI-3 and Lag-3. However, high levels of cd3til in Brms were associated with longer overall survival.

In addition, the expression of TIM-3 and LEG-3 in high T cells in Brms is related to prolonged survival. This study is the first to report the quantitative protein level expression, functional characteristics and clinical significance of TIM in Brms related to primary lung tumors.

The retrospective and small sample of this study limits the scope of our results. Due to the relative lack of tissue sampling for lung cancer BM, we included several lung cancer histology, including small cell lung cancer. Most primary lung cancer tissue samples were from patients with early stage disease, probably partly due to differences between primary lung cancer and BM. However, results from our cohort of matched primary lung tumor and BM patients support the overall results of this study. Furthermore, treatment characteristics vary in our patient cohort. Most patients in this historical cohort did not receive immunosuppressive blockade, which limits our ability to evaluate the predictive value of immunomarkers for immunotherapy response.

This study also relies on analyzing TMAs in small tumour fragments. We have included at least two to three samples from each specimen, although this may still not capture the overall spatial complexity of each tumor. However, our research group has published papers extensively using TMAs and shows consistency and significant correlations with clinicopathological variables and patient outcomes. The results from this single institution cohort are exploratory in nature and need to be validated in a larger independent cohort.

On November 18, the State Anti-Monopoly Administration was officially listed in the office building of the State Administration for Market Regulation. On the same day, the "Anti-Monopoly Guidelines of the Anti-Monopoly Committee of the State Council on the Field of Raw Materials" (hereinafter referred to as the "Guidelines") was also released on the website of the State Administration for Market Regulation.

The establishment of the National Anti-Monopoly Administration is regarded as an important upgrade of the anti-monopoly law enforcement system, and the law enforcement power will be further strengthened. The pharmaceutical industry has always been a key area of ​​anti-monopoly supervision, especially in the field of raw materials. The release of the "Guidelines" will help promote the healthy development of the raw materials industry, maintain fair competition in the raw materials field, protect consumer interests and social public interests, prevent and stop monopoly behavior in the raw materials field, and promote scientific and effective anti-monopoly supervision.

The Guide has six chapters and 29 articles, which are closely linked to the structure of the Anti-Monopoly Law. In response to the most prominent monopoly problems in the raw material field that all aspects reflect, it clarifies the basic principles, ideas and methods of anti-monopoly supervision, and refines the standards for identifying monopoly behaviors, which mainly stipulate five aspects of content.

(I) General Provisions. First, clarify the purpose and basis of the guideline and define relevant basic concepts. Second, it is proposed that anti-monopoly law enforcement agencies should adhere to the principle of protecting fair competition in the field of raw materials for anti-monopoly supervision, scientific and efficient supervision in accordance with the law, focus on protecting consumer interests, and continuously strengthening the regulatory principles of legal deterrence. Third, considering the characteristics of the wide coverage of the industrial chain and many business types in the field of raw materials, the definition ideas of related commodity markets and related regional markets will be explained in detail.

(II) Monopoly agreement. First, analyze the basic ideas of anti-monopoly law enforcement of monopoly agreements, clarify the form of monopoly agreements, and make specific explanations for other collaborative behaviors. Second, combine the characteristics of the raw materials field, list common ways for operators to reach or implement monopoly agreements and law enforcement considerations, clarify that the regional restrictions or customer restrictions imposed by the dealer counterpart may constitute vertical monopoly agreement behavior, and explain the competitive damage effect of vertical monopoly agreements. Third, the analysis ideas for operators to implement collaborative behaviors were refined. Fourth, the specific forms of the shaft and spoke agreements were clarified, and the analysis ideas for the application of the Anti-monopoly Law were proposed. Fifth, the exemption and leniency system were explained, and regulations were stipulated for the regulation of the behavior of industry associations.

(III) Abuse of market dominance. First, combine the characteristics of the raw materials field and clarify the basic ideas of anti-monopoly law enforcement for abuse of market dominance, and clearly confirm or presumes that the operator has market dominance, including the operator's market share, relevant market competition conditions, the operator's actual production capacity and output, the operator's ability to control the raw materials sales or procurement market, the operator's financial and technical conditions, the degree of dependence of the counterparty, the number of real and potential counterparty, the ability of the counterparty to check and balance, and the difficulty of the market entering the market

et al. and clarify the method of calculating market share. Second, focus on refine the manifestations of common abuse of market dominance behavior, including unfair high prices, refusal of transactions, limited transactions, tie-ups, additional unreasonable transaction conditions, differentiated treatment, etc., respond in a targeted manner to monopolistic price increase, controlling sales and supply cuts, and requiring repurchasing of preparations and other monopoly behaviors in the field of raw materials, enhance the operability of the legal system, and provide clear compliance guidance for market entities. Third, combine the business characteristics and law enforcement practices in the field of raw materials, clarify the identification ideas and considerations for the behavior of jointly abused market dominance.

(IV) Concentration of operators. First, for some raw material drug varieties, the market size is relatively small, and the annual turnover of operators may not meet the declaration standards in the "Regulations on the Application Standards of the State Council on the Application of Concentration of Operators", but the evidence shows that the concentration implemented by operators has or may have the effect of excluding and restricting competition. It is clearly stipulated that the anti-monopoly law enforcement agencies of the State Council shall conduct investigations in accordance with the law. Second, for operators who meet the above circumstances, operators in the raw material drug field are encouraged to negotiate with anti-monopoly law enforcement agencies as soon as possible before implementing concentration.

(V) Abuse of administrative power to exclude and restrict competition. The Guidelines mainly refine the manifestations of abuse of administrative power to exclude and restrict competition in the field of raw materials, including restriction on transactions, hindering the free circulation of goods, restriction on bidding, restriction on investment or establishment of branches, forcing operators to engage in monopoly behavior, formulate regulations containing exclusion and restriction of competition content, etc., and clearly state that regulations, normative documents and other policy measures involving the economic activities of market entities in the field of raw materials should be reviewed fairly to maintain a unified national market.

In recent years, there have been cases in the field of raw materials for related companies using their market monopoly position to manipulate prices to make huge profits, or attach unreasonable trading conditions to engage in unfair competition. The State Administration for Market Regulation has successively investigated and dealt with cases of monopoly of multiple raw materials such as glacial acetic acid, chlorine, calcium gluconate and batrectin in accordance with the law, and many companies have received huge fines.

On November 18, the WeChat official account of "Shanghai Market Supervision" showed that Nanjing Ningwei Pharmaceutical Co., Ltd. was administratively punished by the Shanghai Market Supervision Administration for suspected high-price sales of chlorolytic acid raw materials, with a total fine of 6.58 million yuan. Not long ago, the official website of the State Administration for Market Regulation announced that Shangqiu Xin Pioneer Pharmaceutical Co., Ltd. was fined about 11.0484 million yuan for abuse of its market dominance and selling phenol raw materials at an unfair price. After investigation, during the period of Shangqiu Xin Pioneer Monopoly, the average sales price of phenol raw materials once increased by 9.2 times compared with the historical price.

Since the beginning of this year, the pharmaceutical industry has issued several antitrust fines. According to incomplete statistics, as of September 2021, among the 18 cases of investigation by the pharmaceutical industry that can be found, 13 were cases of drug monopoly of raw materials (one was suspended from investigation), 5 were cases of drug monopoly of finished drugs (one of which involved both drug and drug), and 1 was case of medical device monopoly. In addition, there was another judicial case of drug monopoly of raw materials.
Chapter completed!