A Novel, Personalized Drug-Screening System for Platinum-Resistant Ovarian Cancer Patients: A Preliminary Clinical Report

Figures & data

Figure 1 The protocol for establishing the mini-PDX models. Ovarian cancer cells were collected and transferred to capsules that had been washed in HBSS (Hank’s Balanced Salt Solution). The capsules were subsequently implanted subcutaneously into BALB/c nude mice. Four to six chemotherapy regimens (and normal saline as a control) were then used to treat the mice. After 7 days of treatment, the capsules were removed and analyzed by the OncoVee^® MiniPDX Assay for the rapid systemic detection of drug sensitivity.

Table 1 Baseline Patient Characteristics

Number	Age	Tumor Staging	Pathological Types	Sites of Recurrence	CA125 (U/mL)	Regimen of Last Chemotherapy
1	49	IIIC	High-grade serous carcinoma	Pelvic cavity	>5000	Non-platinum-based
2	51	IIIC	High-grade serous carcinoma	Pelvic cavity	251	Platinum-based
3	62	IV	High-grade serous carcinoma	Pelvic cavity, liver lesions	488	Non-platinum-based
4	62	IV	High-grade serous carcinoma	Lymph nodes	>5000	Platinum-based
5	51	IIIC	High-grade serous carcinoma	Pelvic cavity	321	Platinum-based
6	43	IV	High-grade serous carcinoma	Liver lesions	128	Non-platinum-based
7	54	IIIC	High-grade serous carcinoma	Pelvic cavity	215	Non-platinum-based
8	54	IIA	High-grade serous carcinoma	Pelvic cavity	1526	Non-platinum-based
9	56	IIIC	High-grade serous carcinoma	Pelvic cavity	146	Non-platinum-based
10	63	IIIC	High-grade serous carcinoma	Malignant ascites	510	Platinum-based
11	48	IIIA	Clear cell carcinoma	Pelvic cavity	348	Platinum-based
12	40	IIIC	High-grade serous carcinoma	Pelvic cavity	364	Non-platinum-based

Notes: Tumors were staged according to NCCN guidelines for ovarian cancer (2020–03). Sites of recurrence were defined by the location of the main recurrent lesions.

Table 2 Medical History of the Patients Included in This Study

Number	Primary Onset	Primary Surgery	Primary Chemotherapy History	Recurrence Recorded	Type of Platinum-Resistance	Treatment Before Enrollment (of This Recurrence)
1	2016–02	2016–02 Cytoreduction (R0)	TC*6, CR Docetaxel: 120 mg, C: 650 mg	2018–06	B	TC*2→G*1 (Docetaxel:120 mg, C: 650 mg) G: 1500 mg (Gemcitabine 1200 mg)
2	2016–02	2016–02 Cytoreduction (R0)	TC*6, CR Docetaxel: 120 mg, C: 600 mg	2018–07 2018–12	A	2018-12-26 Secondary cytoreduction (R0)
3	2015–04	2015–04 Cytoreduction (R0)	TC*4, CR Docetaxel: 120 mg, C: 600 mg	2017–01 2018–04 2018–09	A	TC*5(Docetaxel: 120 mg, C: 650 mg) →Olaparib*3 months (Olaparib: 300 mg bid PO.)
4	2017–10	2017–11 Cytoreduction (R2)	TC*6, CR Docetaxel: 120 mg, C: 600 mg	2018–10 2019-02	A	Cisplatin injection: 40 mg pleural perfusion*2 (this patient had malignant pleural fluid)
5	2016–08	2016–08 Cytoreduction (R0)	TC*6,CR Paclitaxel: 210 mg C: 600 mg	2017–02 2018–07	A	Paclitaxel: 150 mg weekly*4→ TC*5 (Docetaxel: 120 mg, C:600 mg)→ Secondary Cytoreduction (R0)
6	2015–08	2015–08 Cytoreduction (R0)	TC*8 Paclitaxel: 210 mg, C: 600 mg	2017–02 2018-04	A	Liposomal Dox+Nedaplatin*3 (Day 1: Liposomal Dox 20 mg Day 1–Day 2: Nedaplatin 50 mg)→Apatinib+Olaparib maintenance treatment for 1 year (Olaparib: 300 mg bid PO. Apatinib: 750 mg qd PO.)
7	2018–05	2018-07 Cytoreduction (R0)	TC*5, CR Docetaxel: 120 mg, C: 600 mg	2019–06	B	TC*3 (Day 1: Paclitaxel 210 mg+Day 2: Lobaplatin 40 mg)→Secondary cytoreduction (R0)→G*3(Gemcitabine: 1200 mg)
8	2018-04	2018–05 Cytoreduction (R0)	Primary treatment result:PD	/	C	TC*3 (Day 1: Paclitaxel 210 mg+Day 1–3: Cisplatin 30 mg)→Gemcitabine (Day 1–3:1400 mg)*2→Paclitaxel+ Bevacizumab*3 (Day 1: Paclitaxel 90 mg+Bevacizumab 400 mg, Day 8+Day 15: Paclitaxel 90 mg)
9	2016–10	2016–10 Cytoreduction (R0)	TC*8, CR Docetaxel: 120 mg, C: 600 mg	2018–01 2018-12 2019–10	A	Etoposide (Etoposide 50 mg qd PO.)*20 days→Bevacizumab 500 mg once
10	2015–10	2015–11 Cytoreduction (R0)	TC*6 Paclitaxel: 210 mg, C: 600 mg	2017–08 2018–06 2019–04 (recurrence during Olaparib treatment)	A	Olaparib*4 months (Olaparib: 300 mg bid PO.)→Paclitaxel+ Bevacizumab*4 (Day 1: Paclitaxel 120 mg+Bevacizumab 400 mg, Day 8+Day 15: Paclitaxel 120 mg)→Topotecan (Day 1, 8, and 15: Topotecan 4 mg)→Pemetrexed 800 mg+Cisplatin 100 mg once
11	2018–06	2018–06 Cytoreduction (R0)	TC*6, CR Docetaxel: 90 mg, C: 500 mg	2019–05	A	Paclitaxel weekly treatment*3 (Day1, 8, and 15: Paclitaxel 90 mg)→Gemcitabine+Oxaliplatin (Day 1 and 8: Gemcitabine 1200 mg, Day 1: Oxaliplatin 120 mg)→Irinotecan+Cisplatin*1 (Day 1: Irinotecan 70 mg, Day 1 and 8: Cisplatin 70 mg)
12	2013–11	2018-06 Cytoreduction (R0)	TC*8 Paclitaxel: 210 mg, C: 700 mg	2016–04 2018-10	B	TC+Bev*4 (Day 1: Paclitaxel 210 mg+Bevacizumab 400 mg, Day 2: Oxaliplatin 130 mg)→Bevacizumab*4 (Day 1: Bevacizumab 400 mg)→Cytoreduction (R1)

Notes: “*n” refers to the number of cycles of this regimen. R0: R0-section, section with no visible lesions. R1: R1-section, section with a lesion diameter ≤1cm; R2: R2-section, section with a lesion diameters >1cm. Treatment results of primary onset followed the RECIST1.1 standard. Type of platinum-resistance: A. Conventional platinum-resistant recurrence of ovarian cancer: the recurrence occurred within 6 months from the last platinum-based chemotherapy; B. The recurrence occurred after 6 months from the last chemotherapy, but the tumor was actually platinum-resistant; C. Primary ovarian cancer but resistant to platinum-based regimens. The latest recurrence recorded was when the patient was enrolled. If not mentioned otherwise, all drugs were used intravenously. The interval between each cycle was usually 3 weeks if not mentioned otherwise. Detailed doses are given as follows: TC: Day 1 Docetaxel injection/Paclitaxel injection+Day 2 Carboplatin injection(C); G: Day 1+Day 8 Gemcitabine hydrochloride for injection.

Table 3 CtDNA Results for the 12 Patients

Number	Pathological Mutations	Mutation Frequency	Mutation Type	Matched Targeted Drug	Microsatellite Status
1	None	/		/	Unreported
2	BRCA1	45.63%	Germline mutation	Olaparib	MSS
3	TP53	9.43%	Somatic mutation	AZD1775 (clinical trial)	MSS
4	TP53	6.78%	Somatic mutation	AZD1775 (clinical trial)	MSI-H
5	None	/		/	MSI-L
6	MYC	Gene amplification	Somatic mutation	TAS-119, Roniciclib (clinical trial)	MSS
7	None	/		/	MSI-L
8	None	/		/	MSI-L
9	None	/		/	MSS
10	BRCA1	49%	Germline mutation	Olaparib	MSS
11	ATM	48.4%	Germline mutation	Olaparib	MSI-L
	KRAS	5.4%	Somatic mutation	Cobimetinib, Binimetinib, Trametinib
12	BRCA1	49.5%	Germline mutation	Olaparib	MSI-L

Notes: Microsatellite status represents a measure of microsatellite stability. Results are reported as MSI-High (MSI-H), MSI-Low (MSI-L), and MSI-Stable (MSS). AZD1775 and some other targeted drugs were still involved in clinical trials and were not available in China at the time of this study; these particular drugs were not selected for mini-PDX models.

Table 4 Mini-PDX Drug Sensitivity Results for the 12 Patients

Number	Chemotherapy Regimen	Inhibition Rate (1-T/C%)	Weight Loss of Mice>15%	Source of Cancer Cells
1	1. Liposomal Dox	88%	-	Malignant ascites
	2. Irinotecan	86%	-
	3. Docetaxel	78%	-
	4. Olaparib	54%	-
	5. Oxaliplatin	49%	-
	6. Bevacizumab	46%	-
2	1. Docetaxel	68%	-	Ovarian cancer tissues from surgery
	2. Liposomal Dox	53%	-
	3. Olaparib	55%	-
	4. Topotecan	65%	+
	5. Apatinib	50%	_
3	1. Nab-paclitaxel	86%	-	Liver metastases puncture biopsy tissues
	2. Gemcitabine+Oxaliplatin	62%
	3. Liposomal Dox	51%	-
	4. Crizotinib	47%	+
4	1. Gemcitabine+Oxaliplatin	91%	-	Malignant pleural fluid
	2. Etoposide	77%	-
	3. Nab-paclitaxel	72%	-
	4. Olaparib	41%	-
5	1. Topotecan	89%	+	Ovarian cancer tissues from surgery
	2. Liposomal Dox	70%	-
	3. Cyclophosphamide	46%	-
	4. Olaparib	7%	-
6	1. Apatinib+Etoposide	62%	-	Liver metastases puncture biopsy tissues
	2. Gemcitabine+Oxaliplatin	45%	-
	3. Nab-paclitaxel	39%	-
	4. Liposomal Dox	36%	-
7	1. Liposomal Dox	52%	-	Ovarian cancer tissues from surgery
	2. Apatinib+Etoposide	36%	-
	3. Olaparib	21%	-
	4. Nab-paclitaxel	-2%	-
8	1. Topotecan	70%	-	Malignant ascites
	2. Apatinib+Etoposide	47%	-
	3. Niraparib	20%	-
	4. Liposomal Dox	17%	-
9	1. Topotecan	67%	+	Malignant ascites
	2. Liposomal Dox	42%	-
	3. Lenvatinib	10%	-
	4. Olaparib	-35%	-
10	1. Topotecan	36%	-	Malignant ascites
	2. Liposomal Dox	28%	-
	3. Cyclophosphamide+Bevacizumab	17%	-
	4. Docetaxel+Carboplatin	-9%	+
11	1. Apatinib+Etoposide	53%	-	Malignant ascites
	2. Olaparib	51%	-
	3. Liposomal Dox	46%	-
	4. Irinotecan	32%	-
12	1. Nab-paclitaxel	50%	-	Ovarian cancer tissues from surgery
	2. Chidamide	43%	-
	3. Liposomal Dox	40%	-
	4. Gemcitabine	3%	-

Notes: Inhibition rate was calculated by 1-T/C% (T/C%=treatment group proliferation rate/control group proliferation rate%). Each regimen was used on mini-PDX models with the same doses. Detailed doses for the mini-PDX models: Liposomal doxorubicin (Lipo-Dox): 5 mg/kg, IP (intraperitoneal injection), on Day 1 and Day 5; Irinotecan: 50 mg/kg, IP, on Day 1 and Day 5; Docetaxel: 20 mg/kg, IP, on Day 1 and Day 5; Olaparib: 100 mg/kg, PO (oral administration), Day 1–Day 7; Oxaliplatin: 5 mg/kg, IP, on Day 1 and Day 5; Bevacizumab: 10 mg/kg, IP, on Day 1 and Day 5; Topotecan: 4 mg/kg, IP, Day 1–Day 5; Apatinib: 100 mg/kg, PO, Day 1–Day 7; Nab-paclitaxel (Nab-pac): 20 mg/kg, IV (caudal vein injection), Day 1–Day 5; Gemcitabine+Oxaliplatin (Gem+Oxa): Gemcitabine, 60 mg/kg, IP, Day 1 and Day 5+ Oxaliplatin 5 mg/kg, IP, Day 1; Crizotinib: 50 mg/kg, PO, Day 1–Day 7; Etoposide: 20 mg/kg, IP, Day 1 and Day 5; Apatinib+Etoposide (Apa+Eto): Apatinib, 100 mg/kg, PO, Day 1–Day 7 + Etoposide, 20 mg/kg, IP, Day 1 and Day 5; Docetaxel+Carboplatin: Docetaxel (Doc), 20 mg/kg, IP, Day 1 and Day 5; Carboplatin (Carbo), 50 mg/kg, IP, Day 1; Irinotecan: 50 mg/kg, IP, Day 1 and Day 5; Chidamide: 50 mg/kg, PO, Day 1–7; Gemcitabine (Gemzar): 60 mg/kg, IP, Day 1 and Day 5; Cyclophosphamide (CTX): 100 mg/kg, IP, Day 1 and Day 5; Lenvatinib: 100 mg/kg, PO, Day 1–7; Cyclophosphamide+Bevacizumab (CTX+Bev): Bevacizumab, 10 mg/kg, IP, Day 1 and Day 5+ Cyclophosphamide, 100 mg/kg, IP, Day 1 and Day 5.

Figure 2 Mini-PDX models demonstrated that the expression levels of IHC markers were consistent with epithelial ovarian cancer tissues (pKi67, MUC16, and WT1 positive).

Figure 3 Continued.

Figure 3 Drug-selection test results for the mini-PDX models based on the 12 patients. Tumor cell growth (T/C% = treatment group proliferation rate/control group proliferation rate%) was calculated using the formula: (mean RLU of the treatment group on day 7 - mean RLU on day 0)/(mean RLU of the vehicle group on day 7 - mean RLU on day 0). Abbreviations for chemotherapy: Liposomal doxorubicin (Lipo Dox); Nab-pac (Nab-paclitaxel); Gem+Oxa (Gemcitabine+Oxaliplatin); CTX (Cyclophosphamide); Apa+Eto (Apatinib+Etoposide); Doc+Carbo (Docetaxel+Carboplatin); Bev+CTX (Bevacizumab+Cyclophosphamide); Gemzar (Gemcitabine).

Table 5 Treatment After Enrollment, Clinical Response, and Side Effects

Patient Number	Chemotherapy Plan After Drug Selection	Clinical Outcome	Side Effects
1	Liposomal doxorubicin*2 (Day 1: Liposomal doxorubicin 60 mg), Docetaxel+Carboplatin*4 (Day 1: Docetaxel 120 mg, Day 2: Carboplatin 600 mg), Nab-paclitaxel*4 (Day 1: Nab-paclitaxel 260 mg)	SD	Nausea (grade I) Alopecia (grade I)
2	Liposomal doxorubicin+Cisplatin*4 (Day 1: Liposomal doxorubicin 60 mg+Cisplatin 60 mg)	PR	Vomiting (grade I)
3	Nab-paclitaxel+ Bevacizumab*4 (Day 1: Nab-paclitaxel 260 mg+ Bevacizumab 400 mg)	PR	Vomiting (grade I) Paresthesia (grade I)
4	Gemcitabine+Oxaliplatin*2, Pembrolizumab 200 mg once (Day 1: Gemcitabine 1200 mg+Oxaliplatin 100 mg; Day 8: Gemcitabine 1200 mg)	PD	Nausea (grade II) Ventosity (grade II)
5	Liposomal doxorubicin+Topotecan*6 (Day 1: Liposomal doxorubicin: 60 mg Day 1–Day 3: Topotecan 6 mg)	PR	Leukopenia (grade III)
6	Apatinib+Etoposide*4 months (Apatinib: 750 mg qd PO, Etoposide: 75 mg qd PO*3 weeks, following 1 week’s rest)	CR	Vomiting (grade II) Alopecia (grade I)
7	Liposomal dox*4 (Day 1: Liposomal dox 60 mg)	SD	Leukopenia (grade II) Anemia (grade II)
8	Topotecan*2 (Day 1 and Day 8: Topotecan 5 mg)	PD	Leukopenia (grade II) Nausea (grade I)
9	Topotecan+Bevacizumab*4 (Day 1: Bevacizumab 400 mg, Day 1, 8, and 15: Topotecan 4 mg)	PR	Vomiting (grade II) Alopecia (grade I)
10	Bevacizumab+Olaparib (Day 1: Bevacizumab 400 mg, Day 1–21: Olaparib 300 mg bid PO.)	PD	Vomiting (grade I) Alopecia (grade II) Thrombocytopenia (grade II)
11	Apatinib+etoposide*4 months and continuing until end of follow-up (Apatinib: 750 mg qd PO, Etoposide: 75 mg qd PO*3 weeks, following 1 week of rest)	SD	Vomiting (grade I)
12	Liposomal dox*4 (Day 1: Liposomal dox 60 mg)	PR	Vomiting (grade I)

Notes: All drugs were used intravenously if not mentioned otherwise. “*n” refers to the number of cycles of this regimen.The chemotherapy cycle was usually 21 days if not mentioned otherwise. Side effects were graded according to the CTCAEv5.0 standards.

Figure 4 Distribution of treatment outcomes, including the treatment outcomes of targeted lesions (A), non-targeted lesions (B), and overall outcomes (C). The outcomes of treatment were defined according to the RECIST1.1 standard. The overall clinical-benefit rate was 75%.

Abbreviations: CR, complete remission; PR, partial remission; SD, stable disease; PD, progression of disease.

Figure 5 The loss of weight in mice during the 7 days of drug treatment. RCBW% = (BWi-BW0)/BW0*100%; BWi represents the body weight of the mice on day1 while BW0 represents the body weight of mice when the mini-PDX model was first established. Abbreviations for chemotherapy: Liposomal doxorubicin (Lipo Dox); Nab-pac (Nab-paclitaxel); Gem+Oxa (Gemcitabine+Oxaliplatin); CTX (Cyclophosphamide); Apa+Eto (Apatinib+Etoposide); Doc+Carbo (Docetaxel+Carboplatin); Bev+CTX (Bevacizumab+Cyclophosphamide); Gemzar (Gemcitabine).

Figure 6 The rate of CA125 reduction in the 12 patients. The Y axis shows the rate of reduction in CA125, as calculated by the following formula: (CA125 level after all cycles with the observed CA125 level prior to the first cycle of the observed regimen)/CA125 level prior to the first cycle of the observed regimen*100%). This rate was a negative number when the level of CA125 increased after treatment. The X axis represents the 12 patients ranked from the highest to the lowest (negative) rate of CA125 reduction.