Circulating tumour DNA dynamics predict recurrence in stage III melanoma patients receiving neoadjuvant immunotherapy.

BACKGROUND: Neoadjuvant therapy improves recurrence-free survival (RFS) in resectable stage III cutaneous melanoma. However, accurately predicting individual recurrence risk remains a significant challenge. We investigated circulating tumour DNA (ctDNA) as a biomarker for recurrence in measurable stage IIIB/C melanoma patients undergoing neoadjuvant immunotherapy. METHODS: Plasma samples were collected pre-neoadjuvant treatment, pre-surgery and/or six weeks post-surgery from 40 patients enrolled in the OpACIN-neo and PRADO clinical trials. Patients received two cycles of ipilimumab (anti-CTLA-4) and nivolumab (anti-PD-1) before surgery. Cell free DNA (cfDNA) underwent unbiased pre-amplification followed by tumour-informed mutation detection using droplet digital polymerase chain reaction (ddPCR) with the Bio-Rad QX600 PCR system. RESULTS: Pre-treatment ctDNA was detectable in 19/40 (48%) patients. Among these, 17/19 (89%) zero-converted within six weeks of surgery and none recurred. Positive ctDNA post-surgery (N = 4), irrespective of pre-treatment ctDNA status, was 100% predictive of recurrence (sensitivity 44%, specificity 100%). Furthermore, ctDNA cleared prior to surgery in 7/9 (78%) patients who did not recur, warranting further investigation into ctDNA-guided surgical management. CONCLUSION: Post-surgery ctDNA positivity and zero-conversion are highly predictive of recurrence, offering a window for personalised modification of adjuvant therapy.

A qualitative exploration of the pathway to diagnosis and treatment of cutaneous squamous cell carcinoma of the head and neck with perineural spread.

BACKGROUND: Perineural spread (PNS) is associated with a poor prognosis in cutaneous squamous cell carcinoma of the head and neck (cSCCHN). Hence, investigating facilitators and barriers of early diagnosis and treatment of PNS in cSCCHN may improve outcomes. METHODS: Patients were recruited from an institutional database. Semi-structured interviews were conducted according to the Model of Pathways to Treatment. Thematic analysis was based on the four main intervals in the framework using a data-driven analytical method. RESULTS: Seventeen participants were interviewed. Facilitators included patients' past experiences, symptom progression, trust in healthcare professionals (HCPs), and capacity to leverage relationships. Barriers included difficult diagnoses, limited access to cancer services, lack of care coordination, and lack of awareness of PNS among primary health care providers. CONCLUSION: These findings emphasise the complexity early diagnosis and treatment of PNS. Interventions like clinical practice guidelines, education for HCPs, and telehealth could facilitate timely detection and management.

Single-cell RNA sequencing reveals melanoma cell state-dependent heterogeneity of response to MAPK inhibitors.

BACKGROUND: Melanoma is a heterogeneous cancer influenced by the plasticity of melanoma cells and their dynamic adaptations to microenvironmental cues. Melanoma cells transition between well-defined transcriptional cell states that impact treatment response and resistance. METHODS: In this study, we applied single-cell RNA sequencing to interrogate the molecular features of immunotherapy-naive and immunotherapy-resistant melanoma tumours in response to ex vivo BRAF/MEK inhibitor treatment. FINDINGS: We confirm the presence of four distinct melanoma cell states - melanocytic, transitory, neural-crest like and undifferentiated, and identify enrichment of neural crest-like and undifferentiated melanoma cells in immunotherapy-resistant tumours. Furthermore, we introduce an integrated computational approach to identify subsets of responding and nonresponding melanoma cells within the transcriptional cell states. INTERPRETATION: Nonresponding melanoma cells are identified in all transcriptional cell states and are predisposed to BRAF/MEK inhibitor resistance due to pro-inflammatory IL6 and TNFɑ signalling. Our study provides a framework to study treatment response within distinct melanoma cell states and indicate that tumour-intrinsic pro-inflammatory signalling contributes to BRAF/MEK inhibitor resistance. FUNDING: This work was supported by Macquarie University, Melanoma Institute Australia, and the National Health and Medical Research Council of Australia (NHMRC; grant 2012860, 2028055).

First-line chemoimmunotherapy and immunotherapy in patients with non-small cell lung cancer and brain metastases: a registry study.

Introduction: Brain metastases commonly occur in patients with non-small cell lung cancer (NSCLC). Standard first-line treatment for NSCLC, without an EGFR, ALK or ROS1 mutation, is either chemoimmunotherapy or anti-PD-1 monotherapy. Traditionally, patients with symptomatic or untreated brain metastases were excluded from the pivotal clinical trials that established first-line treatment recommendations. The intracranial effectiveness of these treatment protocols has only recently been elucidated in small-scale prospective trials. Methods: Patients with NSCLC and brain metastases, treated with first-line chemoimmunotherapy or anti-PD-1 monotherapy were selected from the Australian Registry and biObank of thoracic cancers (AURORA) clinical database covering seven institutions. The primary outcome was a composite time-to-event (TTE) outcome, including extracranial and intracranial progression, death, or need for local intracranial therapy, which served as a surrogate for disease progression. The secondary outcome included overall survival (OS), intracranial objective response rate (iORR) and objective response rate (ORR). Results: 116 patients were included. 63% received combination chemoimmunotherapy and 37% received anti-PD-1 monotherapy. 69% of patients received upfront local therapy either with surgery, radiotherapy or both. The median TTE was 7.1 months (95% CI 5 - 9) with extracranial progression being the most common progression event. Neither type of systemic therapy or upfront local therapy were predictive of TTE in a multivariate analysis. The median OS was 17 months (95% CI 13-27). Treatment with chemoimmunotherapy was predictive of longer OS in multivariate analysis (HR 0.35; 95% CI 0.14 - 0.86; p=0.01). The iORR was 46.6%. The iORR was higher in patients treated with chemoimmunotherapy compared to immunotherapy (58% versus 31%, p=0.01). The use of chemoimmunotherapy being predictive of iORR in a multivariate analysis (OR 2.88; 95% CI 1.68 - 9.98; p=0.04). Conclusion: The results of this study of real-world data demonstrate the promising intracranial efficacy of chemoimmunotherapy in the first-line setting, potentially surpassing that of immunotherapy alone. No demonstrable difference in survival or TTE was seen between receipt of upfront local therapy. Prospective studies are required to assist clinical decision making regarding optimal sequencing of local and systemic therapies.

Ex vivo tissue modelling informs drug selection for rare cancers.

The identification and therapeutic targeting of actionable gene mutations across many cancer types has resulted in improved response rates in a minority of patients. The identification of actionable mutations is usually not sufficient to ensure complete nor durable responses, and in rare cancers, where no therapeutic standard of care exists, precision medicine indications are often based on pan-cancer data. The inclusion of functional data, however, can provide evidence of oncogene dependence and guide treatment selection based on tumour genetic data. We applied an ex vivo cancer explant modelling approach, that can be embedded in routine clinical care and allows for pathological review within 10 days of tissue collection. We now report that ex vivo tissue modelling provided accurate longitudinal response data in a patient with BRAFV600E -mutant papillary thyroid tumour with squamous differentiation. The ex vivo model guided treatment selection for this patient and confirmed treatment resistance when the patient's disease progressed after 8 months of treatment.

The molecular and functional landscape of resistance to immune checkpoint blockade in melanoma.

Resistance to immune checkpoint inhibitor therapies in melanoma is common and remains an intractable clinical challenge. In this study, we comprehensively profile immune checkpoint inhibitor resistance mechanisms in short-term tumor cell lines and matched tumor samples from melanoma patients progressing on immune checkpoint inhibitors. Combining genome, transcriptome, and high dimensional flow cytometric profiling with functional analysis, we identify three distinct programs of immunotherapy resistance. Here we show that resistance programs include (1) the loss of wild-type antigen expression, resulting from tumor-intrinsic IFNγ signaling and melanoma de-differentiation, (2) the disruption of antigen presentation via multiple independent mechanisms affecting MHC expression, and (3) immune cell exclusion associated with PTEN loss. The dominant role of compromised antigen production and presentation in melanoma resistance to immune checkpoint inhibition highlights the importance of treatment salvage strategies aimed at the restoration of MHC expression, stimulation of innate immunity, and re-expression of wild-type differentiation antigens.

IFN-γ Signaling Sensitizes Melanoma Cells to BH3 Mimetics.

Immunotherapy targeting PD-1 and/or CTLA4 leads to durable responses in a proportion of patients with melanoma. However, many patients will not respond to these immune checkpoint inhibitors, and up to 60% of responding patients will develop treatment resistance. We describe a vulnerability in melanoma driven by immune cell activity that provides a pathway towards additional treatment options. This study evaluated short-term melanoma cell lines (referred to as PD1 PROG cells) derived from melanoma metastases that progressed on PD-1 inhibitor-based therapy. We show that the cytokine IFN-γ primes melanoma cells for apoptosis by promoting changes in the accumulation and interactions of apoptotic regulators MCL-1, NOXA, and BAK. The addition of pro-apoptotic BH3 mimetic drugs sensitized PD1 PROG melanoma cells to apoptosis in response to IFN-γ or autologous immune cell activation. These findings provide translatable strategies for combination therapies in melanoma.

High-Dimensional and Spatial Analysis Reveals Immune Landscape-Dependent Progression in Cutaneous Squamous Cell Carcinoma.

PURPOSE: The tumor immune microenvironment impacts the biological behavior of the tumor, but its effect on clinical outcomes in head and neck cutaneous squamous cell carcinomas (HNcSCC) is largely unknown. EXPERIMENTAL DESIGN: We compared the immune milieu of high-risk HNcSCC that never progressed to metastasis with those that metastasized using multiparameter imaging mass cytometry. The cohort included both immunosuppressed patients (IS) and patients with an absence of clinical immune-suppression (ACIS). Spatial analyses were used to identify cellular interactions that were associated with tumor behavior. RESULTS: Nonprogressing primary HNcSCC were characterized by higher CD8+ and CD4+ T-cell responses, including numerically increased regulatory T cells. In contrast, primary lesions from HNcSCC patients who progressed were largely devoid of T cells with lower numbers of innate immune cells and increased expression of checkpoint receptors and in the metastatic lesions were characterized by an accumulation of B cells. Spatial analysis reveals multiple cellular interactions associated with nonprogressing primary tumors that were distinct in primary tumors of disease-progressing patients. Cellular regional analysis of the tumor microenvironment also shows squamous cell-enriched tumor regions associated with primary nonprogressing tumors. CONCLUSIONS: Effective responses from both CD8+ and CD4+ T cells in the tumor microenvironment are essential for immune control of primary HNcSCC. Our findings indicate that the early events that shape the immune responses in primary tumors dictate progression and disease outcomes in HNcSCC.

Survival outcomes of perineural spread in head and neck cutaneous squamous cell carcinoma.

AIM: To present an institution's experience and survival outcomes for patients with head and neck cutaneous squamous cell carcinoma (HNcSCC) and perineural spread (PNS). METHOD: Retrospective study of patients with HNcSCC and PNS treated between January 2010 and August 2020 from the Sydney Head and Neck Cancer Institute database, Sydney, Australia; a high-volume, tertiary, academic head and neck centre. Patient demographics, primary site, involved cranial nerves, treatment modality, loco-regional failure and survival data were obtained. RESULTS: Forty-five patients were identified, of which 32 patients were male (71%). Mean age at diagnosis was 68.7 years (range 43-90). Median follow-up was 16.1 months (range 1-107). The trigeminal nerve was most frequently involved (n = 30, 66.6%) followed by facial nerve (n = 13, 28.9%). Most patients underwent surgery followed by radiotherapy (n = 33, 73%) and eight received definitive radiotherapy. The median overall survival (OS) was 4.5 years (95% CI 3.71-5.38), median disease-specific survival 5.1 years (95% CI 4.21-5.97) and median disease-free survival (DFS) was 1.7 years (95% CI 1.11-2.22). The estimated 5-year OS and DFS were 45% and 25%, respectively. Patients treated with surgery and adjuvant radiotherapy with a clear proximal nerve margin had favourable DFS (P = 0.035) and trended towards better OS (P = 0.134) compared with patients with an involved nerve margin. Patients treated surgically with involved proximal nerve margins had similar outcomes compared with patients with treated definitive radiotherapy (HR 0.80, 95% CI 0.29-2.22, P = 0.664). CONCLUSION: The likelihood of achieving a clear proximal nerve margin should be a strong consideration in the selection of appropriate patients for primary surgery.

Anchored Multiplex PCR Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma mutations using circulating tumor DNA (ctDNA) is a potential alternative to using genomic DNA from invasive tissue biopsies. To date, mutations in the GC-rich TERT promoter region, which is commonly mutated in melanoma, have been technically difficult to detect in ctDNA using next-generation sequencing (NGS) panels. In this study, we developed a custom melanoma NGS panel for detection of ctDNA, which encompasses the top 15 gene mutations in melanoma including the TERT promoter. We analyzed 21 stage III and IV melanoma patient samples who were treatment-naïve or on therapy. The overall detection rate of the custom panel, based on BRAF/NRAS/TERT promoter mutations, was 14/21 (67%) patient samples which included a TERT C250T mutation in one BRAF and NRAS mutation negative sample. A BRAF or NRAS mutation was detected in the ctDNA of 13/21 (62%) patients while TERT promoter mutations were detected in 10/21 (48%) patients. Co-occurrence of TERT promoter mutations with BRAF or NRAS mutations was found in 9/10 (90%) patients. The custom ctDNA panel showed a concordance of 16/21 (76%) with tissue based-detection and included 12 BRAF/NRAS mutation positive and 4 BRAF/NRAS mutation negative patients. The ctDNA mutation detection rate for stage IV was 12/16 (75%) and for stage III was 1/5 (20%). Based on BRAF, NRAS and TERT promoter mutations, the custom melanoma panel displayed a limit of detection of ~0.2% mutant allele frequency and showed significant correlation with droplet digital PCR. For one patient, a novel MAP2K1 H119Y mutation was detected in an NRAS/BRAF/TERT promoter mutation negative background. To increase the detection rate to >90% for stage IV melanoma patients, we plan to expand our custom panel to 50 genes. This study represents one of the first to successfully detect TERT promoter mutations in ctDNA from cutaneous melanoma patients using a targeted NGS panel.

Oral Squamous Cell Carcinoma in Young Patients Show Higher Rates of EGFR Amplification: Implications for Novel Personalized Therapy.

There is an increasing worldwide incidence of patients under 50 years of age presenting with oral squamous cell carcinoma (OSCC). The molecular mechanisms driving disease in this emerging cohort remain unclear, limiting impactful treatment options for these patients. To identify common clinically actionable targets in this cohort, we used whole genome and transcriptomic sequencing of OSCC patient samples from 26 individuals under 50 years of age. These molecular profiles were compared with those of OSCC patients over 50 years of age (n=11) available from TCGA. We show for the first time that a molecular signature comprising of EGFR amplification and increased EGFR RNA abundance is specific to the young subset of OSCC patients. Furthermore, through functional assays using patient tumor-derived cell lines, we reveal that this EGFR amplification results in increased activity of the EGFR pathway. Using a panel of clinically relevant EGFR inhibitors we determine that an EGFR-amplified patient-derived cell line is responsive to EGFR inhibition, suggesting EGFR amplification represents a valid therapeutic target in this subset of OSCC patients. In particular, we demonstrate sensitivity to the second-generation EGFR tyrosine kinase inhibitor afatinib, which offers a new and promising therapeutic avenue versus current EGFR-targeting approaches. We propose that testing for EGFR amplification could easily be integrated into current diagnostic workflows and such measures could lead to more personalized treatment approaches and improved outcomes for this younger cohort of OSCC patients.

Immune Checkpoint Inhibitor Induced Pericarditis and Encephalitis in a Patient Treated With Ipilimumab and Nivolumab for Metastatic Melanoma: A Case Report and Review of the Literature.

Immune checkpoint inhibitors (ICIs) have dramatically improved outcomes in melanoma. Common ICI toxicities have become familiar to clinicians; however, rare delayed toxicities remain challenging given the paucity of data with such presentations. We present the unique case of a 61-year-old with metastatic melanoma with two rare, delayed ICI-induced toxicities. After resection of a large symptomatic parietal metastases, this patient received two doses of combination ipilimumab and nivolumab. Five weeks following his second dose, he developed ICI-induced pericarditis with associated pericardial effusion and early signs of tamponade. Corticosteroids were not administered due to a concurrent cerebral abscess. Administration of colchicine, ibuprofen, judicious monitoring, and cessation of immunotherapy led to the complete resolution of the effusion over several weeks. Seven months following his last dose of immunotherapy, the patient developed ICI-associated grade four autoimmune encephalitis, presenting as status epilepticus. High-dose steroid initiation led to rapid clinical improvement. The patient remains in near-complete response on imaging with no recurrence of pericardial effusion and partial resolution of neurological symptoms. ICI-induced pericardial disease and encephalitis carry substantial mortality rates and prompt diagnosis and management is critical. Clinicians must therefore remain vigilant for these rare toxicities regardless of duration of drug exposure or time since cessation of therapy.

Efficacy of immunotherapy in KRAS-mutant non-small-cell lung cancer with comutations.

KRAS-mutant non-small-cell lung cancer is the most common molecular driver of lung adenocarcinoma in western populations. No KRAS specific therapy has been approved by the US FDA until 2021. Despite significant heterogeneity in comutations, patients typically receive single-agent immunotherapy or chemoimmunotherapy as standard first-line therapy. It is unclear whether KRAS mutations predict outcomes with immunotherapy; however, there is emerging data suggesting improved outcomes in patients with a TP53 comutation and worse outcomes in patients with a STK11/LKB1 or KEAP1 comutation.

Circulating tumour DNA and melanoma survival: A systematic literature review and meta-analysis.

We reviewed and meta-analysed the available evidence (until December 2019) about circulating tumour DNA (ctDNA) levels and melanoma patients survival. We included twenty-six studies (>2000 patients overall), which included mostly stage III-IV cutaneous melanoma patients and differed widely in terms of systemic therapy received and somatic mutations that were searched. Patients with detectable ctDNA before treatment had worse progression-free survival (PFS) (summary hazard ratio (SHR) 2.47, 95 % confidence intervals (CI) 1.85-3.29) and overall survival (OS) (SHR 2.98, 95 % CI 2.26-3.92), with no difference by tumour stage. ctDNA detectability during follow-up was associated with poorer PFS (SHR 4.27, 95 %CI 2.75-6.63) and OS (SHR 3.91, 95 %CI 1.97-7.78); in the latter case, the association was stronger (p = 0.01) for stage IV vs. III melanomas. Between-estimates heterogeneity was low for all pooled estimates. ctDNA is a strong prognostic biomarker for advanced-stage melanoma patients, robust across tumour (e.g. genomic profile) and patients (e.g. systemic therapy) characteristics.

Dosing of BRAK and MEK Inhibitors in Melanoma: No Point in Taking a Break.

Resistance to BRAF/MEK inhibitor treatment occurs in most patients with advanced BRAFV600-mutant melanoma. Intermittent drug dosing had been proposed as a strategy to circumvent resistance. In a clinical trial published in Nature Medicine, continuous dosing of BRAF/MEK inhibitors produced superior progression-free survival compared to intermittent dosing in BRAFV600-mutant melanoma patients.

Tumor MHC Expression Guides First-Line Immunotherapy Selection in Melanoma.

Immunotherapy targeting T-cell inhibitory receptors, namely programmed cell death-1 (PD-1) and/or cytotoxic T-lymphocyte associated protein-4 (CTLA-4), leads to durable responses in a proportion of patients with advanced metastatic melanoma. Combination immunotherapy results in higher rates of response compared to anti-PD-1 monotherapy, at the expense of higher toxicity. Currently, there are no robust molecular biomarkers for the selection of first-line immunotherapy. We used flow cytometry to profile pretreatment tumor biopsies from 36 melanoma patients treated with anti-PD-1 or combination (anti-PD-1 plus anti-CTLA-4) immunotherapy. A novel quantitative score was developed to determine the tumor cell expression of antigen-presenting MHC class I (MHC-I) molecules, and to correlate expression data with treatment response. Melanoma MHC-I expression was intact in all tumors derived from patients who demonstrated durable response to anti-PD-1 monotherapy. In contrast, melanoma MHC-I expression was low in 67% of tumors derived from patients with durable response to combination immunotherapy. Compared to MHC-I high tumors, MHC-I low tumors displayed reduced T-cell infiltration and a myeloid cell-enriched microenvironment. Our data emphasize the importance of robust MHC-I expression for anti-PD-1 monotherapy response and provide a rationale for the selection of combination immunotherapy as the first-line treatment in MHC-I low melanoma.

Mismatch repair protein loss in cutaneous head and neck squamous cell carcinoma.

BACKGROUND: The treatment of advanced cutaneous head and neck cutaneous squamous cell carcinomas (HNcSCC) results in significant morbidity. Recently, immune checkpoint inhibitor treatment has been approved for DNA mismatch repair (MMR) deficient patients in a histology-agnostic manner. This study aims to evaluate the incidence of MMR deficiency in advanced HNcSCC and its association with clinicopathologic factors. METHODS: The cohort included 176 consecutive HNcSCC cases treated with curative intent. Immunohistochemistry for MMR proteins (hMLH1, hMSH2, hMSH6, and hPMS2) was performed. Clinicopathological and survival data was collected prospectively. RESULTS: The incidence of MMR protein deficiency was 9.1%. There was no association between age, incidence of metachronous malignancies, clinicopathological factors, or survival outcomes. CONCLUSION: A higher incidence of MMR deficiency was observed in this cohort of advanced HNcSCC. The lack of association with young age at onset or increased incidence of metachronous malignancies suggests that MMR deficiency is likely to be sporadic in HNcSCC.

Design and Testing of a Custom Melanoma Next Generation Sequencing Panel for Analysis of Circulating Tumor DNA.

Detection of melanoma-associated mutations using circulating tumor DNA (ctDNA) from plasma is a potential alternative to using genomic DNA from invasive tissue biopsies. In this study, we developed a custom melanoma next-generation sequencing (NGS) panel which includes 123 amplicons in 30 genes covering driver and targetable mutations and alterations associated with treatment resistance. Analysis of a cohort of 74 stage III and IV treatment-naïve melanoma patients revealed that sensitivity of ctDNA detection was influenced by the amount of circulating-free DNA (cfDNA) input and stage of melanoma. At the recommended cfDNA input quantity of 20 ng (available in 28/74 patients), at least one cancer-associated mutation was detected in the ctDNA of 84% of stage IV patients and 47% of stage III patients with a limit of detection for mutant allele frequency (MAF) of 0.2%. This custom melanoma panel showed significant correlation with droplet digital PCR (ddPCR) and provided a more comprehensive melanoma mutation profile. Our custom panel could be further optimized by replacing amplicons spanning the TERT promoter, which did not perform well due to the high GC content. To increase the detection rate to 90% of stage IV melanoma and decrease the sensitivity to 0.1% MAF, we recommend increasing the volume of plasma to 8 mL to achieve minimal recommended cfDNA input and the refinement of poorly performing amplicons. Our panel can also be expanded to include new targetable and treatment resistance mutations to improve the tracking of treatment response and resistance in melanoma patients treated with systemic drug therapies.