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Abstract
Background Lymph nodes (LNs) are secondary lymphoid organs that surveil tissue fluids and initiate adaptive immunity. Due to the direct drainage of interstitial fluid from tumor tissue through lymphatic vessels to lymph nodes, metastatic dissemination to tumor draining lymph nodes (TDLNs) occurs in early stage of cancer, making LN metastatic burden a strong prognostic factor for patient outcome. LN surgery to prevent metastatic recurrence, including sentinel lymph node biopsy (SLNB) and complete lymph node dissection (CLND), are commonly performed in the clinic. Intriguingly, ectopically implanted murine tumors, such as MC38 (colon adenocarcinoma) and CT26 (colon carcinoma),1 2 have impaired immune checkpoint blockade (ICB) response after resection of TDLNs. However, in phase III clinic trials, patients with completely resected stage IIB, IIC, III or IV melanoma (primary tumor, sentinel LNs and disease-related LNs all removed) benefited from ICB treatment.3–5 The inconsistence between pre-clinic and clinical findings demands further investigation of how TDLN resection affects ICB efficacy.
Methods Through comparing progression-free survival, recurrence-free survival and hazard ratio reduction among multiple phase III clinical trials (EORTC18071, Checkmate066, Checkmate067, Checkmate238 and Keynote716), relative efficacy of ICB is assessed in melanoma patients with various extent of LN surgery. Breast cancer (E0771) and melanoma (B16F10) murine tumor models are implanted to orthotropic sites to evaluate impacts of TDLN dissection on response to ICB treatment and antigen transport. Computational model is developed to predict lymph flow in patients after LN surgery. The radiology images (at diagnosis and follow-up visit) of head and neck cancer patients treated by neoadjuvant durvalumab and irradiation are used to validate the presence of remaining reactive LNs.
Results LN dissection does not diminish ICB-mediated survival improvement (figures 1,2), despite the important role of TDLNs in anti-tumor responses. Mechanistically, after TDLN resection, antigen can be transported to distal LNs through remodeled lymph drainage, which extends the responsiveness to ICB (figures 3, 4). After primary tumor and TDLN resection, inflamed distant LNs (metastases-free) were detected in head and neck cancer patients treated by neoadjuvant ICB (figure 5). Strikingly, compared to systemic administration, delivery of ICB to distant LNs achieves better response, after TDLN dissection (figure 6).
Conclusions Fluid exploits alternative interstitial transport pathways to adjacent basins, which can lead to immune responses occurring distally with the disruption of local fluid drainage. Thus, ICB efficacy is not reliant solely on the presence of the original TDLNs, and ICB can be effective against locoregional recurrence even after TDLN dissection.
Acknowledgements This work was financially supported by grants from NIH (K00CA234940 to H.Z.; F32CA275298 to M.J.O.; R21AI097745, R01CA214913, R01HL128168, R01CA284372, and R01CA284603 to T.P.P.), the Rullo Family MGH Research Scholar Award (T.P.P.), Walter Benjamin Programme, Deutsche Forschunsgemeinschaft Nr.: ME5486/1–2 (L.M.).
References
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Patients with advanced melanoma benefit from immune checkpoint blockade independent of LN surgery status. (A) Progression free survival (PFS) was plotted for Checkmate 066 and Checkmate 067 trials (top), while recurrence free survival (RFS) was plotted for EORTC 18071 and Checkmate 238 trials (middle). Relative efficacy of PFS and RFS were calculated using standard care (dacarbazine or placebo) as the normalizer (bottom), relative efficacy = treatment (PFS or RFS)/standard care (PFS or RFS). Error bars are 95% confidence interval (not enough deaths were recorded to calculate upper arm for RFS in Nivolumab treated cohort) which was also adjusted proportionally to PFS or RFS. (B) Hazard ratio was plotted for Keynote 716, EORTC 18071 (top) and Checkmate 238 (middle) trials. Hazard ratio all conditions were compiled using placebo condition as the baseline (HR=1) (bottom). Hazard ratio of nivolumab to placebo was calculated as follow: HR (nivolumab to placebo) = HR (nivolumab to ipilimumab) x HR (ipilimumab to placebo). Error bars are 95% confidence interval which was also adjusted proportionally to hazard ratio. These plots need better description. What does resected vs unresected mean (LNs or primary tumor)? How is relative efficacy defined. Probably need the equation you used to calculate.
ICB response persists after LN dissection. (A) Sham surgery or SLNB was performed on C57B16 mice (day -10) prior to orthotopic tumor implantation (day 1). Mice were treated by Rat IgG control or anti-PD1 antibody (day 7) for three times at 10mg/kg. Tumor volume (left & middle) and overall survival (right) were plotted for mice bearing orthotopic E0771 tumors. (B) To evaluate the impact of more aggressive surgery, sham surgery or CLND was performed followed by tumor implantation and subsequent anti-PD1 inhibition using the same procedure described as above. Tumor volume (left) and overall survival (right) were plotted for mice bearing E0771 (bottom) tumors. (C) Frequency of complete responders from TDLN removal studies were plotted. Fisher’s exact test was used for statistical analysis. In all animal studies, ’n’ indicates number of mice used. Log-rank test with Bonferroni test was used for statistical analysis of survival data. *p<0.05, ****p<0.0001.
DLN dissection alters lymph drainage and promotes antigen access to distant lymphoid organs. FITC-conjugated 2 million Dalton (FITC-2MD) and TRITC-conjugated 4.4 kilodalton (TRITC- 4.4kD) dextran were injected to the mammary fat pad of mice have undergone sham surgery, SLNB or CLND. (A) Representative immunofluorescence (IF) images show dextran distribution in ipsilateral inguinal (IN), axillary (AX), brachial (BR) LNs as well as contralateral inguinal LNs in animals that have undergone sham, SLNB or CLND. (B) To assess the tumor-specific T cell response, B16F10 tumors overexpressing Ovabulmin257–264 (OVA) were implanted to sham and CLND mice followed by anti-PD1 inhibition. Abundance of OVA-specific CD8 T cells (CD45/CD3/CD8/Tetramer+) is plotted for various LNs. One way ANOVA with Tukey adjustment was used for statistical analysis, *p<0.05, **p<0.01, ****p<0.0001.
Mathematical modeling of antigen access to adjacent lymphosomes via dermal network after node dissection (A) Human male model with network of collecting lymphatic vessels (black), collecting points (green) and lymph nodes (red), (B) Computational mesh for network of initial lymphatic vessels in the skin, (C) Drainage basin (red) with disabled axillary node (black), four most active compensatory nodes (green).
Distant LNs are inflamed after resection of tumor and related LNs in head and neck cancer patients. (A) from phase I trial NCT03635164, CT images of patient 01–013‘s neck at diagnosis and 2- year follow-up scan are demonstrated. Arrows indicate enlarged lower neck LNS. (B) CT and PET images of patient 01–009 at diagnosis and 3-month follow-up scan. Arrows indicate PET activate cervical (diagnosis) and apex mediastinal LNs (follow up).
Distant LNs can be targeted to improve ICB response. Sham surgery or CLNB was performed on C57B16 mice (day -10) prior to orthotopic E0771 tumor implantation (day 1). Anti-PD1 and anti-CTLA4 were given intraperitoneally or intradermally to the lymphatic basin of contralateral inguinal LN (day 7) for three times at 5mg/kg in sham and CLND mice. Tumor volume (left) and overall survival (right) were plotted. In all studies, ’n’ indicates the number of mice used. Log-rank test with Bonferroni test (survival) were used for statistical analysis. *p<0.05.
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