Study design and GI cancer patient characteristics
The study enrolled 304 GI cancer patients with peritoneal metastasis at diagnosis or during disease progression (Table 1). The median diagnosis age was 57 (range 20-93), and 145 (47.7%) were female. The major cancer type was colorectal cancer (CRC), and gastric cancer (GC), followed with appendix cancer, pancreatic cancer, small intestinal and ampullary carcinoma, and esophageal cancer. One hundred and sixty-nine (55.6%) patients had distant organ metastasis except peritoneal. Two hundred and five (67.4%) patients had previous system treatment. A total of 390 specimens were collected to analyze the efficacy of mutation detected ability among different samples in the real world, included 188 plasma, 156 tissue, 45 ascites supernatant, and 1 pleural effusion supernatant.
ASC supernatant in the real world
We retrospectively analyzed 304 GI cancer patients with peritoneal metastasis to further analyze the efficacy of ASC supernatant in the real world. Targeted NGS of 1021 or 59 cancer-related genes was applied to 398 specimens for genomic profiling. We compared the maximum somatic allele frequency (MSAF) in the different samples first. The MSAF were significantly higher in ASC supernatant (50.18% ± 32.03%) than plasma ctDNA (12.31% ± 19.90%, p < 0.0001) and tissues (27.41% ± 19.67%, p < 0.0001) (Figure 1A). ASC supernatant (100%, 45/45) had a higher frequency of somatic alterations detected than plasma (88.3%, 166/188) and tissues (98.7%, 154/156). Among 304 patients, 169 had distant organ metastasis while 125 had only peritoneal metastasis. We compared the MSAF of different samples of the distant organ metastasis patients to only peritoneal metastasis to explore the impact of the metastatic site on MSAF. The MSAF of plasma in patients of only peritoneal metastasis was significantly lower than that of distant organ metastasis (18.23% ± 23.66% vs 3.63% ± 5.95%, p < 0.0001). However, the MSAF were similar between distant organ metastasis and only peritoneal metastasis among ASC supernatant (53.65% ± 32.21% vs 51.95% ± 27.90%, p = 0.70) and tissue (29.44% ± 21.33% vs 25.19% ± 17.35%, p = 0.33) (Figure 1B). Actionable mutation rates of plasma, ASC supernatant, tissue was 55.86%, 83.33%, 75.29% in distant organ metastasis group and 41.67%, 61.54%, 64.29%, respectively. ASC supernatant had a higher actionable mutation rate than plasma in both two groups (Figure 1C & 1D). It was significant different in distant organ metastasis (83.33% vs 55.86%, p = 0.01) (Figure 1C). ASC supernatant had a higher or comparable actionable mutation rate than tissue.
ASC supernatant in paired samples among 26 patients
Twenty-six patients had more than one sample, 26 paired ASC supernatant and plasma ctDNA, 7 tissues, and 1 PE supernatant. All samples had detectable somatic alterations. In the different subtype groups, 69.2% (18/26) of ASC supernatant samples, 50% (13/26) of plasma ctDNA samples, 57.1% (4/7) of tissues, and 100% (1/1) PE supernatant samples had detectable actionable alterations (Table 2). ASC supernatant had a higher detectable rate in actionable alterations although there was no statistically significant difference. In the paired ASC supernatant and plasma ctDNA samples, 7 patients had actionable alterations detected only from ASC supernatant, 2 patients had actionable alterations detected only from plasma ctDNA. ASC supernatant accounts for more actionable alterations than plasma ctDNA. One CRC patient (P23) had paired ASC supernatant and PE supernatant when disease recurrence, and the actionable alterations in two samples had high consistency. Six patients had no distant organ metastasis, three patients (P01, P09, P10) had the same discovery in actionable alterations. However, actionable alterations were more detected from ASC supernatant compared with plasma ctDNA in the other three patients (P14, P15, P20). We then compared the MSAF of ASC supernatant and plasma ctDNA in the 26 patients. The MSAF in the ASC supernatant was significantly higher than plasma ctDNA (p=0.003) (Figure 2). We speculated that the higher MSAF may explicate the superior detecting efficacy of ASC supernatant compared to plasma ctDNA, especially in the patient who had no distant organ metastasis.
Study design and lung cancer patient characteristics
The study enrolled 407 lung patients with pleural metastasis at diagnosis or during disease progression (Table 3). The median diagnosis age was 60 (range 28-94), and 202 (49.6%) were female. The major histology subtype was adenocarcinoma (84.8%). One hundred and twenty-four (30.5%) patients had only pleural metastasis with stage M1a and 227 patients had the other organ metastasis with stage M1b/c. Three hundred and twenty-one (78.9%) patients had previous system treatment. A total of 1002 specimen were collected to analyze the efficacy of mutation detected ability among different samples in the real world, included 389 plasma, 122 tissue, 446 pleural effusion (PE) supernatant, and 45 pleural effusion sediment.
PE supernatant in the real world
We retrospectively analyzed 407 lung cancer patients with pleural metastasis to further analyze the efficacy of PE supernatant in the real world. Targeted NGS of 1021 or 59 cancer-related genes was applied to 1002 specimens for genomic profiling. Firstly, we compared MSAF in the different samples. The PE supernatant MSAF (33.74% ± 28.34%) was higher than plasma ctDNA (6.28% ± 12.17%, p < 0.0001), tissues (31.29% ± 23.31%, p = 0.88) and PE sediment (13.20 ± 16.64, p < 0.0001) (Figure 3A). Plasma MSAF were higher in M1b/c than M1a patients (8.18 ± 13.82 vs 2.33 ± 4.12, p = 0.0034) (Figure 3B). PE supernatant MSAF were higher in M1b/c than M1a patients too (37.35 ± 29.07 vs 30.71 ± 26.94, p = 0.04). Tissue MSAF and PE sediment MSAF were comparable in stage M1b/c and M1a patients, respectively.
Sequentially, we evaluated the targetable variations detected ability in different samples, including EGFR, ALK, BRAF V600E, KRAS, MET ex14 skipping, RET, ROS1, ERBB2, which had high evidence in lung cancer. Among stage M1a patients, PE supernatant, PE sediment, tissue had higher total actionable mutation rate than plasma, especially in PE supernatant (80.3% vs 48.4%, p < 0.05) and tissue (80.0% vs 48.4%, p < 0.05) (Figure 3C). EGFR had a higher mutation rate in the other three samples than plasma, especially in PE supernatant (37.4% vs 63.2%, p < 0.05). These results were observed in stage M1b/c patients too (Figure 3D). PE supernatant had comparable mutation detected ability with the tissue. EGFR uncommon mutation, BRAF V600E, MET ex14 skipping, RET, ROS1, ERBB2 were classified as uncommon variations. PE supernatant had a higher frequency of uncommon variations than plasma no matter had distant organ metastasis (Figure 3C & 3D).
PE supernatant in paired samples among 139 patients
One hundred and thirty-nine patients had simultaneously paired plasma and PE supernatant samples. About 89.2% (124) plasma and 93.5% (130) PE supernatant had mutation detected. PE supernatant had higher MSAF (31.47 ± 28.90 vs 6.23 ± 13.63, p < 0.0001) (Figure 4A) and more mutations detected (6.67 ± 5.12 vs 3.91 ± 4.60, p < 0.0001) (Figure 4B) than plasma ctDNA. PE supernatant had higher detectable rate of targetable alterations than plasma (79.1% vs 56.1%, p < 0.05) (Figure 4C). And it’s significantly different about EGFR mutation (Figure 4C). PE supernatant also had a higher frequency of uncommon variations than plasma (Figure 4C).