"Nature Cancer": Radiotherapy + immunity, better effect! Scientists have also discovered new prognostic markers

Potential synergies between radiotherapy and immunotherapy have long been considered
.
There is a large body of preclinical evidence that radiotherapy can affect immune regulation, thereby enhancing the therapeutic response of immunotherapy [1].

Correspondingly, PD-1 and CTLA-4 immune checkpoint inhibitors (ICBs) have been shown to enhance both local and distant radiotherapy responses [2].

Based on these potential synergistic effects, more than 500 clinical trials have investigated the anti-tumor efficacy of radiotherapy plus ICB in different malignancies, however, no positive interaction
between radiotherapy and immunotherapy has been observed in the vast majority of trials.
Although ICB has been effective in the consolidation phase after radiotherapy [3], ICB has also been successful in the adjuvant phase after surgery [4].

Therefore, taken together, it is unclear
whether there is a particular synergy between radiotherapy and ICB.

Therefore, a key question that urgently needs to be answered is whether radiotherapy and ICB can be combined in some form to further improve clinical outcomes, and whether biomarkers are available to select people
who benefit from combination therapy.

To this end, a team led by Professor Sean P.
Pitroda from the Division of Radiation and Cell Oncology at the University of Chicago designed and conducted a investigator-initiated Phase 1/2, randomized clinical trial (COSINR study) evaluating the safety and efficacy
of concurrent or sequential radiotherapy in combination with ICB (ipilimumab and nivolumab) as first-line treatment for metastatic non-small cell lung cancer (NSCLC).

Recently, the interim analysis of a key secondary objective of the trial was published in the prestigious journal Nature Cancer [5].

In this secondary objective, the team led by Professor Sean P.
Pitroda systematically analyzed the molecular characteristics of paired biopsy samples before and during treatment of the same lesion, showing treatment-specific intratumor immunogenetic panorama changes
between radiotherapy alone and concurrent radiotherapy immunotherapy combination therapy, respectively.

The trial found that concurrent radiotherapy immunotherapy can enhance both intralesional and non-irradiated intralesional anti-tumor immune responses, while surprisingly, radiotherapy alone reduces intratumor cytotoxic T cells and adaptive immune responses
.

More importantly, the analysis also found that concurrent radiotherapy immunotherapy significantly improved the survival prognosis
of patients with immunocold tumors with increased baseline aneuploidy compared with sequential radiotherapy immunotherapy combined therapy.
Thus, tumor aneuploidy may be used as a biomarker of benefit from concurrent radiotherapy immunotherapy combination therapy
.

Screenshot of the first page of the paper

Let's take a look at how the interim analysis of the secondary goal of the COSINR trial was conducted
.

This interim analysis is part 1 of the COSINR trial
.
In this Phase 1 trial, a total of 37 patients with metastatic NSCLC were randomized to simultaneous or sequential multilesion (2-4) stereotactic body radiotherapy (SBRT) plus ipilimumab and nivolumab (ipi/nivo) immunotherapy, starting immunotherapy within 7 days of completion of SBRT in the sequential group, with a median follow-up of 17.
0 months
.

The results of the analysis of the main objectives have been published in the prestigious journal J Thorac Oncol [6].

The results showed that multi-lesion SBRT combined with immunotherapy was well tolerated, and the synchronous toxicity was lower than that of sequential.
The efficacy of the combination therapy was also encouraging, with a median progression-free survival (PFS) of 5.
8 months, and an objective response rate of 44 versus 36 percent, 1-year overall survival 84 versus 62%, and 2-year overall survival 62 versus 40%
compared with the ipi/nivo group in the CheckMate 227 study, which was also first-line therapy for advanced NSCLC.

According to the design of this secondary objective, the team performed pre-treatment and intra-treatment paired biopsies of the same irradiated lesion before the start of immunotherapy within 1 week of completion of SBRT in the sequential group and 1 week after completion of SBRT and one cycle of ICB in the synchronous group
.

Figure 1.
Schematic diagram of COSINR study design (Phase 1 part)

The research team performed DNA whole exon sequencing (WES) on fresh tumor tissues obtained from biopsy for human leukocyte antigen (HLA) typing, mutation and copy number variation (CNV) analysis, neoantigen prediction and tumor cell proportion (CCF) calculation, and total RNA sequencing (RNA-seq) for T cell receptor (TCR) clonal typing, transcript abundance analysis, cell pathway analysis and immune signature analysis
.

Quality control screening
was performed on 37 patients by histopathological and sequencing analysis.
In the end, the sequential group vs synchronous group were: 18 vs 16 qualified at baseline histopathology→ 12 vs 10 qualified for pre-treatment biopsy DNA sequencing→ 8 vs 10 biopsy DNA sequencing after treatment→ 8 vs 7 people qualified for pre-treatment and in-treatment RNA sequencing
.

Figure 2.
Consort flowchart of patient selection and data analysis framework

Professor Sean P.
Pitroda's research team observed no difference
in PFS and overall survival (OS) between the sequential and synchronized groups over the current follow-up period, regardless of all 37 patients, or in the 22 patients who passed the molecular test.

In this case, the team sought to explore whether there was a specific molecular profile of patients who showed different survival benefits
for sequential or concurrent radiotherapy immunotherapy combination therapy.

First, the research team compared mutation changes
in 18 patients who were eligible for DNA sequencing before and during treatment.
After simultaneous SBRT+ipi/nivo treatment, all or almost all of the mutant clones disappeared, indicating complete pathological response or near-complete remission, which coincided with a significant reduction in tumor cells in TTF1+/CK5+ in the corresponding pathological sections
.
In contrast, after SBRT treatment alone in the sequential group, a large number of mutant clones still existed, and the tumor cell reduction in the corresponding pathological sections was not obvious
.

At the same time, tumor mutation burden (TMB) and aneuploidy score (proportion of chromosomal arms amplified or deleted) were significantly reduced after concurrent SBRT+ipi/nivo treatment, reflecting diploidization of tumor tissue and a decrease
in the proportion of tumor cells.
That is, the reduction in the type and frequency of mutations is caused by tumor cells being eliminated and immune cells entering and enriching in tumor tissue, rather than weakening the genetic instability of tumor cells
.

It can be seen that there is a significant anti-tumor immune response in tumor tissues after simultaneous SBRT+ipi/nivo treatment, while no anti-tumor immune response
is observed after SBRT treatment alone in the sequential group.

Figure 3.
Simultaneous mutation clonal changes before and after SBRT+ipi/nivo treatment and before and after SBRT treatment alone in sequential groups

Next, the team compared the results of RNA-seq sequencing before and during treatment
.
Simultaneous SBRT+ipi/nivo significantly upregulated immune-related pathways
such as antigen presentation, interferon response, IL-6/JAK/STAT3, cytokine and chemokine inflammatory signaling, and effector T cell function.
SBRT alone significantly downregulates these immune-related pathways
.
Correspondingly, the mesenchymal cell score (ESTIMATE algorithm) and immune cell score (xCell algorithm) increased
significantly in the simultaneous SBRT+ipi/nivo treatment group.
The analysis also showed that concurrent SBRT+ipi/nivo treatment significantly reduced the proliferative activity of remaining tumor cells, while the proliferative activity of remaining tumor cells was not weakened
after SBRT treatment alone.

Figure 4.
Transcriptome changes were performed with simultaneous SBRT+ipi/nivo therapy and sequential SBRT alone

Interestingly, TCR analysis (MiXCR algorithm) found that although the TCR diversity of the two biopsies before and after did not change overall compared with the two groups, and the vast majority of TCR was cleared
before treatment in both groups (SBRT 94.
5% in the sequential group and 95.
5% in simultaneous SBRT + ipi/nivo).
。 However, the new TCRs after SBRT treatment in the sequential group were less than 2/3 of the pre-treatment (401 new / 641 before treatment), while the new TCRs after simultaneous SBRT+ipi/nivo treatment were more than twice as high as before treatment (764 new / 398 before treatment).

This suggests that ICB prompts a large number of new T cells to enter the tumor microenvironment, while SBRT alone cannot do
.

The results of RNA-seq sequencing analysis were confirmed
by multiplex immunohistochemical staining of the corresponding pathological sections.
The density of intratumoral CD8+ T cells after simultaneous SBRT+ipi/nivo treatment remained consistent or increased compared with before treatment, while the density of intratumoral CD8+ T cells decreased in most patients after sequential SBRT treatment
.

The above results show that synchronous SBRT+ipi/nivo can more effectively remove tumor cells and amplify the adaptive anti-tumor immune response
compared with SBRT alone.
SBRT alone may lead to the disappearance of CD8+ T cells in the tumor at baseline, while SBRT+ipi/nivo causes the infiltration and activation of new CD8+ T cells, which constitutes a trade-off balance
with the CD8+ T cells in the baseline tumor.

Figure 5.
Multiplex immunohistochemistry shows intratumoral CD8+ T cell density before and during treatment

Finally, combining these DNA and RNA sequencing analyses, Prof.
Sean P.
Pitroda's research team explored whether specific populations could benefit more from SBRT+ipi/nivo combination therapy
.

They found that the known biomarkers of ICB efficacy, PD-L1, TMB, neoantigen volume, T cell γ interferon pathway activity, etc.
, could not predict PFS or OS
in all 37 patients with SBRT+ipi/nivo and in sequential and synchronized patients.
At the same time, there are no specific mutations, specific gene levels, or chromosomal arm levels of CNV associated
with PFS or OS.

The research team noted that recent studies have reported that tumor aneuploidy increases are associated with weakened efficacy of ICB [6], and the survival analysis of this study also showed that the increase in tumor aneuploidy before treatment was independently associated
with shorter PFS and OS in the sequential SBRT+ipi/nivo group 。 However, the biopsy results showed that the increase in aneuploidy before treatment was significantly associated with a decrease in the proportion of tumor cells biopsied in the treatment group (Spearman's correlation coefficient = -0.
80, p=0.
017), and the higher the aneuploidy score, the more the
proportion of tumor cells decreased.
However, the proportion of tumor cells decreased significantly after SBRT alone in the sequential group, and there was no correlation with aneuploidy score before treatment (p=0.
53).

Figure 6.
Association between pretreatment aneuploidy scores and a decrease in the proportion of tumor cells after concurrent SBRT+ipi/nivo or SBRT alone

Therefore, the research team decided to explore in detail the efficacy
of aneuploidy score before treatment and sequential or simultaneous radiotherapy + ICB.
In pretreatment tumor biopsy samples from all patients, aneuploidy increase was negatively correlated with activation of multiple immune signaling pathways, mesenchymal cell score, immune cell score, and aneuploidy score was higher
in patients with low PD-L1 expression.

The research team classified all patients with high and low aneuploidy scores
based on a median aneuploidy score (0.
54).
Among patients with high aneuploidy score, the synchronous group had a significant PFS and OS survival advantage over the sequential group, with a 12-month OS rate of 100% vs 17%, and log-rank p=0.
025
.
In patients with low aneuploidy scores, there was no significant difference
in PFS and OS between the two groups.
At the same time, in patients with high aneuploidy scores, the synchronous group had a lower risk of non-irradiated lesion progression than the sequential group, that is, there may be a stronger distantness effect
.

Figure 7.
Explore tumor aneuploidy scores as biomarkers for SBRT and ICB combination therapy
.

At this point, Professor Sean P.
Pitroda's research team has finally answered the question
of whether and how to combine radiotherapy and immunotherapy.
For patients with increased aneuploidy, concurrent radiotherapy combined with ICB is more effective than sequential radiotherapy plus ICB, and these patients have many baseline features of poor immunotherapy prognosis, and concurrent radiotherapy combined with ICB can maximize
the clinical benefit of these patients.
The research team validated this conclusion
using two other independent cohorts.

Overall, this study is the first to find that patients with increased aneuploidy may benefit additionally from concurrent radiotherapy plus ICB, and systematically compares the immunogenetic panorama of
tumor immunity between radiotherapy alone and concurrent radiotherapy plus immunotherapy.
However, the small sample size somewhat dampened the statistical power
of the study.
Nevertheless, the findings of this study may provide a guiding framework for the development of individualized radiotherapy and ICB combination protocols for NSCLC patients, and may be generalized to other tumors
.

It has been observed that concurrent radiotherapy immunotherapy can enhance both intralesional and non-irradiated intralesional anti-tumor immune responses, and surprisingly, radiotherapy alone reduces intratumor cytotoxic T cells and adaptive anti-tumor immune responses
.
More clinical data and biological evidence from patients are expected to optimize the combination of radiotherapy and ICB, including radiation dose, fractionation, timing, choice of target organ, type and dose of ICB, etc
.

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[6].
Davoli T, Uno H, Wooten EC, Elledge SJ.
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