Brief Communication on Pathologic Assessment of Persistent Stable Metastatic Lesions in Patients Treated With Anti-CTLA-4 or Anti-CTLA-4 + Anti-PD-1 Therapy

Abstract

Despite the wide use of immune checkpoint inhibition for the treatment of melanoma, the mechanisms leading to long-term stable disease are incompletely understood. Patients with metastatic melanoma who had received ipilimumab alone or ipilimumab plus nivolumab 2+years prior and attained at least 6 months of stable disease were identified. Positron emission tomography/computed tomography (PET/CT) was performed. Pretreatment and posttreatment biopsies of areas of stable disease were assessed for tumor, fibrosis, and inflammation. Seven patients underwent PET/CT and tissue biopsy. Fluorodeoxyglucose avid lesions on PET/CT ranged from no activity to an SUV of 22. In 6 patients, the residual stable lesions were composed of necrosis and fibrosis with a prominent pigment containing macrophages and no residual melanoma. In 1 patient, a nodal lesion demonstrated melanoma with active inflammation. In most patients with durable stable disease after treatment with ipilimumab or ipilimumab/nivolumab, residual lesions demonstrated predominantly necrosis and fibrosis consistent with resolving lesions. The presence of melanophages in these samples may suggest ongoing immune surveillance. One patient did demonstrate residual melanoma, indicating the need for ongoing monitoring of this patient population.

BACKGROUND

Until 2011, options for the treatment of melanoma were extremely limited.^1–4 However, immune checkpoint blockade and novel targeted therapies have dramatically advanced our ability to control metastatic melanoma, with patients having longer overall survival and durable responses to therapy.

The pivotal ipilimumab trial randomized untreated melanoma patients to ipilimumab combined with gp100 peptide vaccine, ipilimumab alone, or gp100 alone. An overall survival benefit was seen when the groups receiving ipilimumab were compared with the group receiving gp100 alone.⁵ A phase III trial compared ipilimumab plus dacarbazine with dacarbazine alone in previously untreated patients with metastatic melanoma and confirmed the overall survival benefit of ipilimumab.⁶ Some of these patients did not display an objective response to ipilimumab but remained without long-term progression.^7–9

The combination of ipilimumab and nivolumab has also been used in melanoma with improvement in response and survival. The results of a Phase III trial showed that nivolumab in combination with ipilimumab leads to longer progression free survival and a higher overall response rate than is achieved with ipilimumab alone in treatment-naive patients with advanced melanoma.^10,11 These improvements in ipilimumab monotherapy have led to more patients with long-term benefits when treated with nivolumab or the combination of nivolumab and ipilimumab, many of whom had stable disease or stable disease after an initial response.

It remains unclear if patients with prolonged stable disease after immune checkpoint inhibition have simply formed scars at the site of prior disease or if there are residual tumor cells being controlled by an ongoing immune response. Evaluating tissue from patients with prolonged responses provides a unique opportunity to determine the composition of residual lesions. Correlation with positron emission tomography/computed tomography (PET/CT) helps determine if this is an accurate modality to reflect the presence of residual viable tumor tissue.

METHODS

We identified metastatic melanoma patients that attained long-term stable disease after treatment with ipilimumab, nivolumab, or ipilimumab plus nivolumab at standard dosing. Patients must have received at least 2 doses of ipilimumab, nivolumab, or combination therapy. They were required to have started therapy greater than 2 years before study enrollment and have had stable disease for≥6 months before study enrollment. An initial partial response followed by stabilization of the disease was allowed. Potential patients were identified through physician referrals, clinic visits, medical record review, and review of patient and clinical trial lists. Informed consent was obtained from potentially eligible patients, and these patients were assessed for eligibility criteria. This trial was approved by the Dana-Farber/Harvard Cancer Center Institutional Review Board (IRB).

Patients who fulfilled eligibility criteria were enrolled and underwent PET/CT scans and biopsies of residual areas of stable disease. The PET/CT imaging was compared with standard radiologic imaging to evaluate the activity of the residual disease. H&E-stained pretreatment and posttreatment tissue biopsies underwent pathologic assessment to determine tumor cell content, fibrotic content, and inflammation. Inflammation grading was done as a qualitative assessment of lymphocyte density within the biopsy.

The sample, which contained viable melanoma, was further analyzed by multiplex immunofluorescence staining for immune markers CD8, PD-L1, FOXP3, and PD1 and tumor marker SOX10. Staining was performed on 2 samples (pre-IO and post-IO). Slides were deparaffinized, rehydrated, and serially incubated with primary antibodies followed by a secondary label. Opal Fluorophore Reagents were applied to fluorescently label the antibody complexes. Finally, slides were incubated with Spectral DAPI solution, air dried, and mounted. Image acquisition at 20× resolution was performed, and 2-6 regions of interest (ROIs) were selected that best represent the tissue microenvironment. Mean count density was determined as the average of cell counts obtained among multiple imaging frames. A tumor proportion score (TPS) was calculated and defined as all PD-L1–positive tumor cells each divided by all tumor cells (stained using Sox10). A known inflamed melanoma was used as an internal control to monitor staining accuracy.

The histopathologic features of the posttreatment biopsy were correlated with PET/CT and standard radiologic imaging to determine if these imaging approaches are capable of informing inflammation, tumor, and fibrotic scar tissue content.

RESULTS

Between April 2017 and January 2020, seven patients were enrolled in the study and underwent PET/CT and tissue biopsy. Three of these patients were treated with ipilimumab alone, and 4 were treated with ipilimumab in combination with nivolumab. Patients ranged in age from 60 to 71, with 3 patients having known cutaneous primaries, 1 patient with an acral primary, and 3 patients with unknown primary lesions. BRAF status was tested on all but 1 of the 7 patients, and 4 had BRAF mutations, 3 of which were V600 mutations (Table 1).

TABLE 1 - Patient Characteristics, PET/CT and Pathology Findings

Pt	Treat-ment	Age at Tx start	BRAF status	Melanoma type	Tx start to biopsy (mo)	Tx stop to biopsy (mo)	Date of SD to biopsy (mo)	PET/CT findings		Pathology findings
	—	—	—	—	—	—	—	SUV	Findings	Tumor, %	Necrosis, %	Fibrosis, %	Inflammation	Comments
1	Ipi	67	Not tested	Cutaneous	46	44	35	22	Intense FDG avid axillary mass	80	5	10	3+inflammation	—
2	Ipi	71	BRAF wt	Acral	33	30	29	0	No FDG avid areas	0	90	5	—	Marked pigmentation
3	Ipi/nivo	60	BRAF V600E	Cutaneous	25	21	16	2.4	Mild FDG uptake in popliteal mass	0	60	40	—	No pigmentation
4	Ipi	60	BRAF wt	Unknown primary	94	91	82	5.5	FDG avid left inguinal LN	0	70	—	2+inflammation	No pigmentation
5	Ipi/nivo	62	BRAF V600E	Unknown primary	43	40	25	4.5-18.1	Several FDG avid LN (LN with FDG 18.1 was biopsied)	0	0	30	3+inflammation,	Scarring with treatment effect
6	Ipi/nivo	69	BRAF L597S	Cutaneous	43	17	32	4.5, 4.6	FDG avid axillary LN, iliac lytic lesion	0	60	20	1+inflammation	Moderate pigmentation
7	Ipi/nivo	69	BRAF V600K	Unknown primary	40	1	18	2.7	Mild FDG uptake in axillary LN	0	90	10	—	Moderate pigmentation

Times from treatment start, stop and date of SD to biopsy in months.

FDG indicates fluorodeoxyglucose; PET/CT, positron emission tomography/computed tomography.

The patients averaged 46 months from the date of treatment start to the biopsy date with a range of 25 to 94 months. Enrolled patients averaged 35 months from treatment stop to biopsy date with a range of 1 to 91 months. The duration of stable disease from the date that the disease stabilized until the biopsy date averaged 34 months with a range of 16 to 82 months (Table 1).

Six patients had fluorodeoxyglucose (FDG) avid lesions on PET/CT, which ranged in intensity from standard uptake values (SUV) 2.4-22. One patient had no FDG avidity in the areas of residual disease observed on CT. The majority of the FDG avid lesions were lymph nodes with other areas, including soft tissue metastasis and bone metastasis.

Biopsies from the residual stable lesions demonstrated predominantly necrosis and fibrosis with a prominent pigment containing macrophages. The samples without tumor consisted of 60% to 90% necrosis, 5% to 40% fibrosis, and some inflammatory cells (Table 1 and Fig. 1).

FIGURE 1: Pathology images from H&E specimens examined for tumor, necrosis, fibrosis, and inflammation. PET/CT images from patients on the study of lesions corresponding to biopsy sites. PET/CT indicates positron emission tomography/computed tomography.

One of the 7 patients underwent resection of an axillary nodal lesion with an SUV of 22; a pathologic review revealed the presence of melanoma with 80% tumor, 3 + inflammation, 5% necrosis, and 10% fibrosis. The pretreatment sample, taken from the primary site, was reviewed and noted to have 10% tumor with no necrosis, 20% fibrosis, and 1 + inflammation. These samples were stained further to evaluate the immune populations observed in the tumor microenvironment and changes between the pretreatment sample, posttreatment sample, and melanoma control. Staining of the posttreatment sample demonstrated clear areas of SOX10 + tumor surrounded by areas of immune activity, which stained for CD8 and PD-L1 (Fig. 2). When compared with the pretreatment sample and melanoma control, it was noted that there was a marked higher density in CD8 + cells in the posttreatment sample while the CD8 + PD1 + cell density was similar. PD-L1 + SOX10 + cells were slightly increased, while PD-L1 + cells that did not stain with SOX10 were very elevated, suggesting a marked increase in PD-L1 + cells in the immune compartment of the posttreatment sample (Fig. 2).

FIGURE 2: Additional multiplex immunofluorescence testing on post sample from patient 1. (A) Whole slide scan (0.3× zoom), (B) Whole slide scan (2.4× zoom), (C) Zoomed ROI on Akoya Inform [DAPI (blue), CD8 (white), PD-L1 (green), FoxP3 (yellow), PD1 (orange), SOX10 (magenta)] (D) Count density (cells/mm²) of immune markers in the tumor resection for patient 1 comparing melanoma control, pretreatment specimen, and posttreatment specimen. CD8 + (white), CD8 + PD1 + (tan), PD1 + + (orange), FOXP3 + (yellow) (E) PD-L1 quantification. PD-L1 Tumor Proportion Score (TPS) measures the mean percent of PD-L1 + SOX10 + tumor cells (light green). The mean percent of PD-L1 + in inflammatory cells is quantified for cells that were phenotyped as CD8 + or Other.

The patient whose biopsy revealed residual melanoma had not had any changes in this area noted on imaging over the 35 months between developing SD and the biopsy; this patient received no additional therapy and continues to remain without evidence of melanoma. All patients who were enrolled in the study remain alive and disease free at the time of this manuscript publication.

DISCUSSION

The goal of this study was to evaluate the composition of persistent but stable lesions in patients with prolonged clinical benefit following checkpoint blockade. Most residual lesions demonstrated necrosis and fibrosis, suggesting that long-term stable disease represents predominantly resolving scar without viable malignancy. The presence of melanophages in these lesions indicates ongoing immune surveillance.

One of the 7 patients did have residual melanoma despite having stable disease for 35 months and being off therapy for 44 months. Staining of this sample suggested that the tumor that was present was surrounded by CD8 + immune cells. Overall, the tumor would have been considered negative for PD-L1 staining with a relatively low TPS score. However, there was a high rate of PD-L1 + in stromal inflammatory cells in this sample, which suggests a large population of macrophages. This finding suggests that there is an active immune reaction consisting of CD8 + T-cells and macrophages, which is continually working to contain this tumor.

Correlation with PET/CT was performed to help guide the use of PET/CT to detect residual viable tumor tissue. In our study, increased FDG uptake on PET/CT alone was not a consistent marker of residual disease, although the level of uptake may be helpful as the patient with the residual disease did have the highest FDG uptake. Further exploration of the use of PET/CT to detect melanoma response to immunotherapy is underway (PET-Stop trial NCT04462406).

Our study is limited by the relatively small number of patients. It proved very difficult to find patients that met the study criteria who also had lesions that would be safe to biopsy. Interestingly, despite including patients who received nivolumab alone in the eligibility and in the lists created, none were identified that fit the study criteria. This suggests that there may be slightly different patterns observed in patients who receive ipilimumab versus those who receive nivolumab alone.

Patients with durable, stable disease after treatment with ipilimumab or ipilimumab and nivolumab combination therapy represent a growing population of melanoma patients treated with immune checkpoint inhibition. An examination of the residual lesions observed in these patients demonstrated predominantly necrosis and fibrosis consistent with resolving lesions. The presence of melanophages in these samples may suggest some ongoing immune surveillance. One patient did demonstrate residual melanoma, indicating the need for ongoing monitoring of this patient population. This study demonstrated the need for novel and combination approaches to evaluate residual disease. Efforts are ongoing to explore alternate methods to detect residual disease through blood tests such as ctDNA and other methods. These novel methods will hopefully help better inform patient testing and follow-up.

CONFLICTS OF INTEREST/FINANCIAL DISCLOSURES

E.B. has served on advisory boards for Nektar, Novartis, Apexigen, Shionogi, and BMS and received research funding from Lilly, Novartis, Partners Therapeutics, Genentech, BMS, and BVD. P.O. receives research funding from and serves advisory roles for Neon Therapeutics, Bristol-Myers Squibb, Merck, CytomX, Pfizer, Novartis, Celldex, Amgen, Array, AstraZeneca/MedImmune, Armo BioSciences, Xencor, Oncorus, Evaxion, Immunetune, and Roche/Genentech. S.R. receives research support from Bristol-Myers Squibb and KITE/Gilead. S.R. is on the SAB of Immunitas Therapeutics. F.S.H. serves as an advisor or consultant for BMS, Merck, EMD Serono, Novartis, Surface oncology, Compass Therapeutics, Apricity, Sanofi, Pionyr, Bicara, Checkpoint, Genentech/Roche, Bioentre, Gossamer, Iovance, Trillium, Catalym, Immunocore, Amgen, Kairos, Eisai, Rheos, Zumutor, and Corner Therapeutics. The remaining authors e authors report no conflicts of interest.