Melanoma PDX established by the Wistar/MD Anderson/Penn PDTC

Meenhard Herlyn and Michael Davies, MPI

We have banked 457 PDX of melanomas (Krepler et al. Cell Reports 2017, Garman et al. Cell Reports 2017) We observed in melanoma an overall take rate of 87% and tumor growth was achieved with as few as of 100 cells. The ~450 PDX are from patients representing all clinical and histological groups. The high success rate in generating PDX from patients’ tissues was observed for all biologic, genetic and clinical types  of the disease except early primary melanomas and primary ocular melanomas. The majority (~180) of PDX are from treatment-naïve patients prior to therapy (Figure 1). On the other hand, we have specimens procured from patients whose tumors progressed on treatment with BRAF inhibitors, BRAF inhibitors plus MEK inhibitors, BRAF inhibitors plus anti-CTLA-4, BRAF inhibitors plus anti-PD-1, BRAF inhibitors plus anti-CTLA-4 plus anti-PD-1, and BRAF inhibitors plus MEK inhibitors plus combinations among checkpoint inhibitors such as anti-CTLA-4 & anti-PD-1. Several specimens were procured when patients responded well to therapy, but this represents the most difficult subgroup for tissue collection. For specimens from patients whose tumors progressed on BRAF inhibitors or BRAF inhibitors plus MEK inhibitors, the animals bearing those tumors are fed with diet containing the inhibitors reaching similar concentrations in blood as in patients. This continued treatment assures that molecular mechanisms underlying therapy resistance seen in patients’ tumors are well maintained in PDX.

  F igure 1. Patients’ treatment history prior to surgery for PDX generation.    While the majority of patients were treatment-naïve, up to 50 patients per category who received prior therapy relapsed after initial responses. Specimens derived from patients who relapsed on BRAF or BRAF/MEK inhibitor therapy were grown as PDX in the presence of the respective inhibitors. Targeted therapy (TT;) immune checkpoint inhibitors (IT;) sequential therapies (Seq); combination therapies (Combo).

Figure 1. Patients’ treatment history prior to surgery for PDX generation. While the majority of patients were treatment-naïve, up to 50 patients per category who received prior therapy relapsed after initial responses. Specimens derived from patients who relapsed on BRAF or BRAF/MEK inhibitor therapy were grown as PDX in the presence of the respective inhibitors. Targeted therapy (TT;) immune checkpoint inhibitors (IT;) sequential therapies (Seq); combination therapies (Combo).

We have 15 specimens from patients without prior treatment, from which we established both permanent cell lines and PDX. 

Using a targeted capture approach of 108 genes previously implicated in melanomagenesis, massively parallel sequencing was performed on 488 melanoma patient-derived xenografts, tumor biopsies and cell lines. All previously reported cutaneous melanoma subtypes were recapitulated: BRAF, NRAS, NF1, KIT and WT/WT/WT/WT.  We found that melanomas with non-V600 region BRAF, non-Q61 NRAS, and NF1 mutations, required additional activating mutations in the MAPK signaling pathway.  Multiple combinations were observed together, such as mutations in the BRAF loop domain and RAS 12/13, NF1 and KIT, and NF1 and MAP3K5/MAP3K9. We have then detected hotspot mutations in KIT, RAC1, mutations and amplifications of RTKs, and amplifications of MDM2 and have confirmed published mutations in the TERT promoter. The genetic profiles of PDX reflect those of patients with differences only in more frequent deletion/inactivation of CDKN2A and p53. Figure 6A also summarizes prior therapy and concomitant data on metastasis formation (approximately 40%) and the status of protein expression profiles. Figure 6B provides information of genetic alterations and protein expression of samples from patients who are resistant to either BRAF inhibitor alone or the combination of BRAF inhibitors plus MEK inhibitors. Those signaling inhibitor-resistant PDX are of critical importance for this application.

 

Krepler C, Sproesser K, Brafford P, Beqiri M, Xiao M, Shannan B, Watters A, Perego M, Zhang G, Vultur A, Yin X, Liu Q, Garman B, Anastopoulos IN, Wubbenhorst B, Wilson M, Xu W, Karakousis G, Feldman M, Xu X, Amaravadi RK, Gangadhar TC, Elder DE, Haydu L, Wargo JA, Davies MA, Lu Y, Mills GB, Frederick DT, Barzily-Rokni M, Flaherty KT, Hoon DS, Guarino M, Bennett J, Ryan RW, Petrelli NJ, Shields CL, Terai M, Sato T, Aplin AE, Roesch A, Darr D, Angus S, Kumar R, Halilovic E, Caponigro G, Jeay S, Wuerthner J, Walter A, Ocker M, Boxer MB, Schuchter LM, Nathanson KL, Herlyn M. A comprehensive melanoma patient-derived xenograft and liver tumor collection representing the clinical, genetic, and biologic heterogeneity of the disease as models for experimental therapy. Cell Rep. 2017 Nov 14;21(7):1953-1967. doi: 10.1016/j.celrep.2017.10.021. PMID: 29141225

Garman C, Anastopoulos IN, Krepler C, Brafford P, Sproesser K, Jiang Y, Wubbenhorst B, Amaravadi RK, Bennett J, Beqiri M, Elder M, Flaherty KT, Frederick D, Gangadhar TC, Guarino M, Hoon D, Karakousis GC, Liu Q, Mitra N, Petrelli NJ, Schuchter LM, Shannan B, Shields C, Wargo J, Wenz B, Wilson MA, Xiao M, Xu W, Xu X, Yin X, Zhang NR, Davies M, Herlyn M, Nathanson KL. Targeted, massively parallel sequencing identifies novel genetic subsets of cutaneous melanoma. Cell Rep. 2017 Nov 14;21(7):1936-1952. doi: 10.1016/j.celrep.2017.10.052. PMID: 29141224

 

FUNDING

This project has been funded in whole or in part with Federal funds from the National Cancer Institute, National Institutes of Health, Department of Health and Human Services, under Contract No. 1U54CA224070-01.