Neoadjuvant PD-1 Inhibitor combines with Chemotherapy in Resectable Squamous Cell Non-Small Cell Lung Cancer

Single blockade combine platinum-based doublet 21 patients with squamous cell lung accepted neoadjuvant therapy followed by surgery in Beijing Cancer Hospital were involved. 8 patients accepted two cycles of neoadjuvant platinum-based doublet chemotherapy combine with anti-PD-1 therapy, while 13 patients accepted two cycles of neoadjuvant platinum doublet chemotherapy. Besides baseline tumor staging, chest CT was repeated two to three weeks before surgery. Adverse events were monitored. Peripheral lymphocytes counting was tested during whole treatment. The residual viable tumor cells were counted after surgery to decide a major pathological response (MPR) rate. Selected specimen was sent for immunohistochemical, multiplex immunofluorescence analyses, and T-cell receptor DNA sequencing. Comparing with neoadjuvant alone, combination with PD-1 and chemotherapy increased the pathological complete response rate (37·5% Vs. and MPR rate (50% The evaluation is not consistent with The special Multiplexed among


Backgroud
The past two decades witnessed the rapid progress on treatment of lung cancers. The widely use of targeted therapy greatly improved the overall survival of patients with lung cancers, especially adenocarcinoma with driver gene mutations. However, the driver gene mutation-based treatment is still difficult to help patients with lung squamous cell carcinoma which closely relates to smoking and contributes around 30% cases of lung cancer. Since 75% of these patients are diagnosed relatively late and the squamous carcinoma often locates in proximal bronchus, the complete dissection (R0 dissection) is very hard to be achieved. Thus, the choice of treatment is usually limited to chemotherapy and radiotherapy, although the outcome is still unsatisfactory.
The PD-1 blockade immunotherapy has made great improvement on treatment of late stage squamous non-small cell lung cancer 1 . Pembrolizumab combine with traditional platinum-base doublet chemotherapy has been recommended as first line therapy for late stage squamous cell lung cancer. Single agent of PD-1 or PD-L1 inhibitor has also been explored recently for resectable patients before surgery. Nivolumab and atezolizumab have shown impressive effect as single agent neoadjuvant therapy regimen 2,3 . However, the effectiveness and safety of PD-1 blockade combine with platinum-based doublet chemotherapy have not been fully investigated.

Patients
From October 2018 through June 2019, 21 consecutive patients with squamous cell lung cancer accepted neoadjuvant therapy followed by surgery after the discussion and approval of multiple discipline team (MDT) of Center of Thoracic Tumor, Beijing Cancer hospital, including 8 patients with stage IIB or IIIA squamous cell lung cancer who accepted two cycles of neoadjuvant platinum-based doublet chemotherapy combine with anti-PD-1 therapy (patient No. IM-1 to IM-8) and 13 patients accepted two cycles of neoadjuvant platinum doublet chemotherapy (patient No. C-1 to C-13). All the patients were current or former smokers. Informed consent was received from all patients before treatment. All the patients planned to accepted surgery five to seven weeks after the second cycle of neoadjuvant therapy by same surgeon. The characteristics of the involved patients are shown in Table 1.

Neoadjuvant therapy regimen
Neoadjuvant chemotherapy was given every three weeks (21 days) for two cycles. PD-1 inhibitor (Pembrolizumab or Toripalimab) was given one dose before infusion of chemotherapy agents on first day of each cycle. Detailed information on neoadjuvant therapy regimen for each patient are shown in Table S1 in Supplementary Appendix.

Pathology
Pathological assessments The size of primary lung cancer, involved lymph nodes, and metastases were evaluated according to The number of each tumor was recorded, and major pathological response (MPR) is defined as no more than 10% residual viable tumor cells (8) .

Immunologic analysis and T-Cell Receptor Sequencing
The immunohistochemical and multiplex immunofluorescence analyses of tumors are described in the Methods section in Supplementary Appendix 1.
The T-cell receptor DNA sequencing was used to define T-cell clonal distribution and functional specificity for mutant tumor antigens. The detailed methods were described in Supplementary Appendix 1.

Statistical Analysis
The institutional review board at the Peking University Cancer Hospital approved this observational study. The requirement of patient consent was waived because of the retrospective nature of this study.
Side effects, adverse events were continuously monitored. The response was evaluated by RECIST 1·1 and pathologically. The difference of response rate was compared by Chi-square test. P value for differences were calculated with a significance level of P 0·05. SPSS software was used for all analysis.  Although there were 13·4% residual tumor cells, the squamous carcinoma component is less than 10%. Thus, in this group, a major pathological response occurred in four patients (50%).

Results
Of the 13 patients who accepted neoadjuvant chemotherapy only, a major pathological response occurred in five patients (38·46%), including one patient (7·69%) with complete response. The median degree of pathological regression in the primary tumor was − 69·30% (rang, -100 to -8·55, mean − 63·28%). (Fig. 1) Then comparison between RECIST and pathological assessment indicates the discordance of these two methods. The result of image test is not the indicate of pathological regression. (Table 2) Peripheral lymphocytes counting was recorded before and around day-7 of both cycle of neoadjuvant therapy, as well as in seven days before surgery. Figure 2 shows the change of the median of peripheral lymphocytes counting during the treatment. After neoadjuvant agents infusion of each cycle, the lymphocytes counting dropped, and then restored before next cycle or before surgery.

Histological features of lung squamous cell carcinoma treated by neoadjuvant chemotherapy with or without anti-PD-1 inhibitor
The patients underwent neoadjuvant chemotherapy combine with anti-PD-1 inhibitor or not share similar gross morphological changes of the tumor. The features described in "Pathology-Pathological assessment" present in patients of both groups. (Fig. 3A and 3B).
The presence of immune infiltrates in the patients accepted neoadjuvant chemotherapy only indicates the traditional platinum-base doublet chemotherapy may also induce some degree of immune response to the tumor cells. The lymphocytes seem more densely infiltrated in the primary/residual tumor (or regression bed) in IM group than that in the C group, which may indicate that the combination with PD-1 blockade may induce somehow more lymphocytes proliferation and recruitment. However, the tumor infiltrating lymphocytes are not dense as expected or as reported in former study 6 , and we also found the tumor infiltrates are more likely be found in the nonresponders (SD), rather than responders (PR or CR) (Fig. 3B).
Besides the features observed above, in the specimen of pathological partial responders, immune exclusion happens in both groups. The immune exclusion is defined as immune cells in the immediate peritumoral stroma, but not infiltrate into the tumor parenchyma. Even in the patients who accepted anti-PD-1 inhibitor, the T cells are still can not penetrate the "barrier" of the residual tumor cells. On immunohistochemical staining, the immune cells infiltrate in the peritumoral stroma are mainly composed by CD4 + T cells and CD20 + B cells, but not CD8 + T cells ( Figure S1 and S2 in Supplementary Appendix). In the multispectral immunofluorescence on the metastased lymph node, the infiltrated cells in or around the tumor cells including dense FOXP3 + Treg cells. However, in the normal lymph node (subcarinal lymph node), Treg cells can be seen with much less density (Fig. 4).

T-Cell Receptor Sequencing
Specimen from six patients (three patients for each group, detailed information is show in Supplementary Appendix) were tested through T-cell receptor sequencing.
In the amino acid clonotype and Shannon entropy analysis, the diversity of T-cell receptors is different between patients and specimen. However, in each single patients, the number of amino acid clonotype consistently presents with primary tumor metastased lymph node normal lymph node ( Figure S3 in Supplementary Appendix). The diversity of T cells in primary tumor is statistically less than that in normal lymph node (p = 0·003). (Fig. 5) Regarding to the shared amino acid clonotype, we analyzed the top 100 used amino acid of each specimen. The results revealed that in single patient, some amino acids were shared among primary tumor, normal lymph node and metastased lymph node. Nevertheless, different patients share almost no same amino acids ( Figure S4 and S5 in Supplementary Appendix).

Discussion Clinical Analysis
In this research, comparing with platinum-based doublet neoadjuvant chemotherapy, we observed that the combination of neoadjuvant chemotherapy and anti-PD-1 immunotherapy was associated with few additional adverse events, did not delay the planned surgery, and led to the improvement of both radiological and pathological evaluation ( Table 2). Although the safety of neoadjuvant PD-1 blockade have been reported, we still observe one patient with immune-related pneumonia after surgery. Surgeons still need to be aware of the serious adverse events, especially those potentially lead to cancellation of the planned surgery, that could radically change the treatment of the resectable lung cancers.
The relationship between pathological response and long-term survival has been explored by many studies. Researches have investigated and indicated the correlation between complete pathological response and overall survival 9-12 . Other researches also have proved the validity of major pathological response as a surrogate of survival 13-16 . In this study, the complete response is different between IM and C group (37·5% vs. 7·69%). The small P-value (0.098) may suggest potential statistical different with increasing of sample size. However, the rate of major pathological response in the chemotherapy only group is 38.46%, which is much higher than reported before (26%) 17  We also observed the inconsistency of radiologic and pathologic assessment. One major reason is that the interval between the second CT scan and surgery was usually more than 10 days. The continued shrinkage of tumors after neoadjuvant therapy could have happened in this period. Another reason may be the repaired tissue (fibrosis) may also be shown on CT. Although not observed in our study, pseudo progression should also be considered during the radiological assessment. We advocate the modification of RECIST 1·1 to fit the "golden standard" of pathological assessment.

Histological Findings
The

Availability of data and materials
The datasets supporting the conclusions of this article are included within the article and its supplementary appendix. Figure 1 The residual viable tumor cell after surgery in each patient The residual viable tumor cell after surgery in each patient Change of the peripheral lymphocyte counting during neoadjuvant therapy Figure 3 Pathological features of the patients.    The diversity (Shannon Index) analysis of the specimen The diversity (Shannon Index) analysis of the specimen

Supplementary Files
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