Travis J. Hollmann

Genetic Basis for Clinical Response to CTLA-4 Blockade in Melanoma

BACKGROUND Immune checkpoint inhibitors are effective cancer treatments, but molecular determinants of clinical benefit are unknown. Ipilimumab and tremelimumab are antibodies against cytotoxic T-lymphocyte antigen 4 (CTLA-4). Anti-CTLA-4 treatment prolongs overall survival in patients with melanoma. CTLA-4 blockade activates T cells and enables them to destroy tumor cells. METHODS We obtained tumor tissue from patients with melanoma who were treated with ipilimumab or tremelimumab. Whole-exome sequencing was performed on tumors and matched blood samples. Somatic mutations and candidate neoantigens generated from these mutations were characterized. Neoantigen peptides were tested for the ability to activate lymphocytes from ipilimumab-treated patients. RESULTS Malignant melanoma exomes from 64 patients treated with CTLA-4 blockade were characterized with the use of massively parallel sequencing. A discovery set consisted of 11 patients who derived a long-term clinical benefit and 14 patients who derived a minimal benefit or no benefit. Mutational load was associated with the degree of clinical benefit (P=0.01) but alone was not sufficient to predict benefit. Using genomewide somatic neoepitope analysis and patient-specific HLA typing, we identified candidate tumor neoantigens for each patient. We elucidated a neoantigen landscape that is specifically present in tumors with a strong response to CTLA-4 blockade. We validated this signature in a second set of 39 patients with melanoma who were treated with anti-CTLA-4 antibodies. Predicted neoantigens activated T cells from the patients treated with ipilimumab. CONCLUSIONS These findings define a genetic basis for benefit from CTLA-4 blockade in melanoma and provide a rationale for examining exomes of patients for whom anti-CTLA-4 agents are being considered. (Funded by the Frederick Adler Fund and others.).

INI1-deficient tumors: diagnostic features and molecular genetics.

Significant progress has been made in understanding the molecular genetic alterations involved in sarcomagenesis. Cytogenetic and molecular studies have identified nonrandom genetic abnormalities, including tumor suppressor gene inactivation. Mutations, deletions, and other somatic alterations in the tumor suppressor gene INI1 (hSNF5; SMARCB1), which encodes a subunit of the SWI/SNF chromatin remodeling complex, were first described in the malignant rhabdoid tumor of infancy. Since then, INI1 has also been implicated in the pathogenesis of additional tumor types including renal medullary carcinomas and epithelioid sarcomas and a subset of epithelioid malignant peripheral nerve sheath tumors, myoepithelial carcinomas, and extraskeletal myxoid chondrosarcomas. As varied as this group appears, they all show loss of INI1 protein expression, a propensity for rhabdoid cytomorphology, and sometimes other overlapping immunohistochemical and histologic findings. We will review the clinicopathologic features of these tumor types and emphasize the clinical utility of INI1 immunohistochemistry in differential diagnosis.

Alternative transcription initiation leads to expression of a novel ALK isoform in cancer

Activation of oncogenes by mechanisms other than genetic aberrations such as mutations, translocations, or amplifications is largely undefined. Here we report a novel isoform of the anaplastic lymphoma kinase (ALK) that is expressed in ∼11% of melanomas and sporadically in other human cancer types, but not in normal tissues. The novel ALK transcript initiates from a de novo alternative transcription initiation (ATI) site in ALK intron 19, and was termed ALKATI. In ALKATI-expressing tumours, the ATI site is enriched for H3K4me3 and RNA polymerase II, chromatin marks characteristic of active transcription initiation sites. ALKATI is expressed from both ALK alleles, and no recurrent genetic aberrations are found at the ALK locus, indicating that the transcriptional activation is independent of genetic aberrations at the ALK locus. The ALKATI transcript encodes three proteins with molecular weights of 61.1, 60.8 and 58.7 kilodaltons, consisting primarily of the intracellular tyrosine kinase domain. ALKATI stimulates multiple oncogenic signalling pathways, drives growth-factor-independent cell proliferation in vitro, and promotes tumorigenesis in vivo in mouse models. ALK inhibitors can suppress the kinase activity of ALKATI, suggesting that patients with ALKATI-expressing tumours may benefit from ALK inhibitors. Our findings suggest a novel mechanism of oncogene activation in cancer through de novo alternative transcription initiation.

Digital fibromyxoma (superficial acral fibromyxoma): a detailed characterization of 124 cases.

Digital fibromyxoma (first described by Fetsch and colleagues as superficial acral fibromyxoma) is a distinctive soft tissue tumor with a predilection for the subungual or periungual region of the hands and feet. This report details the histologic, immunophenotypic, and clinical findings in 124 cases of digital fibromyxoma. The study group included 70 male and 54 female patients (1.3:1, M:F), ranging in age from 4 to 86 years (mean, 48 y; median, 49 y). Mean tumor size was 1.7 cm (range, 0.5 to 5 cm; median, 1.5 cm). Nearly half of the patients (41%) presented with a painful mass. Tumors arose on the hands (52%) or feet (45%), with rare tumors arising on the ankle or leg. Most tumors occurred on the digits (94% of hand tumors, 82% of foot tumors), with the majority growing in close proximity to the nail (97% on fingers, 96% on toes). Histologically, 80% of cases were poorly marginated; 70% infiltrated the dermal collagen, 27% infiltrated fat, and 3% invaded bone. In cases in which imaging studies were available, bone involvement by an erosive or lytic lesion was more frequent (9/25, 36%). All tumors were composed of spindle-shaped or stellate-shaped cells with palely eosinophilic cytoplasm and a random or loosely fascicular growth pattern. The tumor cells were separated by dense hyaline collagen alternating with myxoid stroma. Most (86%) of the tumors showed alternating areas of fibrous and myxoid stroma, 11% showed predominantly fibrous stroma, and 3% had predominantly myxoid stroma. Increased mast cells were noted in 88% of tumors. All tumors comprised cells with minimal atypia, occasionally showing scattered larger cells with so-called “degenerative change.” Mitotic figures were infrequent, and all tumors lacked necrosis, pleomorphism, or neural/perineural infiltration. Multinucleate stromal cells were occasionally seen. Tumor cells were reactive for CD34 in 42/61 cases (69%), with rare tumors showing focal reactivity for EMA (3/40, 7.5%), smooth muscle actin (5/42, 12%), and desmin (1/18, 6%). All tumors were negative for S100 (0/66), MUC4 (0/11), GFAP (0/10), AE1/AE3 (0/4), Cam5.2 (0/2), PanK (0/2), Claudin (0/4), and NFP (0/3). Follow-up in 47 cases ranged from 1 to 252 months (mean, 35 mo). Ten tumors (24%) recurred locally (all near the nail unit of the fingers or toes) after a mean interval of 27 months. One tumor recurred twice. All recurrent tumors had positive margins on initial biopsy or subsequent excision and no other clinical or pathologic features correlated with recurrence/persistence. To date, no tumor has metastasized. Finally, sequencing of 8 digital fibromyxomas failed to reveal mutations in exon 8 or 9 of GNAS1, in contrast to intramuscular or cellular myxoma.

Molecular Determinants of Response to Anti-Programmed Cell Death (PD)-1 and Anti-Programmed Death-Ligand 1 (PD-L1) Blockade in Patients With Non-Small-Cell Lung Cancer Profiled With Targeted Next-Generation Sequencing.

Purpose Treatment of advanced non-small-cell lung cancer with immune checkpoint inhibitors (ICIs) is characterized by durable responses and improved survival in a subset of patients. Clinically available tools to optimize use of ICIs and understand the molecular determinants of response are needed. Targeted next-generation sequencing (NGS) is increasingly routine, but its role in identifying predictors of response to ICIs is not known. Methods Detailed clinical annotation and response data were collected for patients with advanced non-small-cell lung cancer treated with anti-programmed death-1 or anti-programmed death-ligand 1 [anti-programmed cell death (PD)-1] therapy and profiled by targeted NGS (MSK-IMPACT; n = 240). Efficacy was assessed by Response Evaluation Criteria in Solid Tumors (RECIST) version 1.1, and durable clinical benefit (DCB) was defined as partial response/stable disease that lasted > 6 months. Tumor mutation burden (TMB), fraction of copy number-altered genome, and gene alterations were compared among patients with DCB and no durable benefit (NDB). Whole-exome sequencing (WES) was performed for 49 patients to compare quantification of TMB by targeted NGS versus WES. Results Estimates of TMB by targeted NGS correlated well with WES (ρ = 0.86; P < .001). TMB was greater in patients with DCB than with NDB ( P = .006). DCB was more common, and progression-free survival was longer in patients at increasing thresholds above versus below the 50th percentile of TMB (38.6% v 25.1%; P < .001; hazard ratio, 1.38; P = .024). The fraction of copy number-altered genome was highest in those with NDB. Variants in EGFR and STK11 associated with a lack of benefit. TMB and PD-L1 expression were independent variables, and a composite of TMB plus PD-L1 further enriched for benefit to ICIs. Conclusion Targeted NGS accurately estimates TMB and elevated TMB further improved likelihood of benefit to ICIs. TMB did not correlate with PD-L1 expression; both variables had similar predictive capacity. The incorporation of both TMB and PD-L1 expression into multivariable predictive models should result in greater predictive power.

NF1 Mutations Are Common in Desmoplastic Melanoma.

Desmoplastic melanoma (DM) is a rare variant of melanoma with distinct clinical, histopathologic, and immunohistochemical features. Clinically, DM differs from conventional melanoma by a higher propensity for local recurrence and less frequent metastatic spread to regional lymph nodes. In its pure form, DM has a distinct appearance displaying a low density of fusiform melanocytes in a collagen-rich matrix. Whereas a number of mutations have been identified in primary melanoma, including BRAF, NRAS, GNAQ, GNA11, and KIT, and the occurrence of these mutations has been found to correlate to some extent with the histopathologic features, anatomic site, and/or mode of sun exposure, no distinct set of mutations has so far been reported for DM. To study the potential association of neurofibromin (NF1) mutations with DM, we examined 15 desmoplastic and 20 non-DMs by next-generation sequencing. Mutations of the NF1 gene were found in 14 of 15 (93%) DMs and 4 of 20 (20%) non-DMs. The high frequency of NF1 mutations in DMs suggests an important role for NF1 in the biology of this type of melanoma.

CD25 expression on cutaneous mast cells from adult patients presenting with urticaria pigmentosa is predictive of systemic mastocytosis.

Urticaria pigmentosa (UP) is a clinicopathologic term used to describe reddish-brown cutaneous macules and papules, characterized histologically by mast cell infiltration of the papillary and upper reticular dermis and reactive basal hyperpigmentation of the overlying epidermis. Although typically a benign, self-limited disorder of childhood, a significant proportion (up to 30%) of adolescent and adult-onset UP represents cutaneous involvement by underlying systemic mastocytosis (SM). Predicting the course of cutaneous mast cell disease has been limited by a lack of diagnostic and prognostic markers. In patients with SM, neoplastic bone marrow mast cells show aberrant surface expression of CD25. However, whether CD25 expression on cutaneous mast cells is associated with underlying SM is unknown. In this study, we performed a clinicopathologic analysis of 30 adult patients presenting with UP between 1987 and 2007. Cutaneous mast cell infiltration pattern, cytomorphology, density, and CD25 immunoreactivity were correlated with underlying or subsequent SM. On the basis of clinical and pathologic follow-up, 10 of 30 (33%) patients were diagnosed with SM and 20 of 30 (67%) with limited cutaneous mastocytosis (CM). Although cutaneous mast cell density was slightly higher in patients with SM compared to those with limited CM (P=0.047), neither mast cell cytomorphology nor infiltration pattern correlated with underlying systemic disease. However, cutaneous mast cells from all 10 patients with SM (100%) were immunoreactive for CD25, compared to only 5 of 20 (25%) with limited CM (P<0.001). Our findings suggest that immunoreactivity for CD25 in cutaneous mast cells may be useful for stratifying adult patients presenting with UP for additional clinical evaluation.

Microenvironment-derived factors driving metastatic plasticity in melanoma.

Cellular plasticity is a state in which cancer cells exist along a reversible phenotypic spectrum, and underlies key traits such as drug resistance and metastasis. Melanoma plasticity is linked to phenotype switching, where the microenvironment induces switches between invasive/MITFLO versus proliferative/MITFHI states. Since MITF also induces pigmentation, we hypothesize that macrometastatic success should be favoured by microenvironments that induce a MITFHI/differentiated/proliferative state. Zebrafish imaging demonstrates that after extravasation, melanoma cells become pigmented and enact a gene expression program of melanocyte differentiation. We screened for microenvironmental factors leading to phenotype switching, and find that EDN3 induces a state that is both proliferative and differentiated. CRISPR-mediated inactivation of EDN3, or its synthetic enzyme ECE2, from the microenvironment abrogates phenotype switching and increases animal survival. These results demonstrate that after metastatic dissemination, the microenvironment provides signals to promote phenotype switching and provide proof that targeting tumour cell plasticity is a viable therapeutic opportunity.

Novel High-grade Endometrial Stromal Sarcoma: A Morphologic Mimicker of Myxoid Leiomyosarcoma.

Endometrial stromal sarcomas (ESS) are often underpinned by recurrent chromosomal translocations resulting in the fusion of genes involved in epigenetic regulation. To date, only YWHAE-NUTM2 rearrangements are associated with distinctive high-grade morphology and aggressive clinical behavior. We identified 3 ESS morphologically mimicking myxoid leiomyosarcoma of the uterus and sought to describe their unique histopathologic features and identify genetic alterations using next-generation sequencing. All cases displayed predominantly spindled cells associated with abundant myxoid stroma and brisk mitotic activity. Tumors involved the endometrium and demonstrated tongue-like myometrial infiltration. All 3 were associated with an aggressive clinical course, including multisite bony metastases in 1 patient, progressive peritoneal disease after chemotherapy in another, and metastases to the lung and skin in the last patient. All 3 ESS were found to harbor ZC3H7B-BCOR gene fusions by targeted sequencing and fluorescence in situ hybridization. On the basis of the review of these cases, we find that ESS with ZC3H7B-BCOR fusion constitutes a novel type of high-grade ESS and shares significant morphologic overlap with myxoid leiomyosarcoma.

Primary and Metastatic Cutaneous Melanomas Express ALK Through Alternative Transcriptional Initiation.

A number of common driver mutations have been identified in melanoma, but other genetic or epigenetic aberrations are also likely to play a role in the pathogenesis of melanoma and present potential therapeutic targets. Translocations of the anaplastic lymphoma kinase (ALK), for example, have been reported in spitzoid melanocytic neoplasms leading to kinase-fusion proteins that result in immunohistochemically detectable ALK expression. In this study, we sought to determine whether ALK was also expressed in nonspitzoid primary and metastatic cutaneous melanomas. ALK immunohistochemistry was performed on 603 melanomas (303 primary and 300 metastatic tumors) from 600 patients. ALK immunohistochemistry expression was identified in 7 primary and 9 metastatic tumors. In 5 of 7 primary tumors and in 6 of 9 metastatic lesions, the majority of tumor cells were immunoreactive for ALK. In the other 2 primary and 3 metastatic lesions, positive staining was identified in less than half of the tumor cells. ALK positivity was found in the presence or absence of BRAF or NRAS mutations. In contrast to prior observations with ALK-positive Spitz tumors, none of the ALK-positive melanomas harbored a translocation. Instead, the ALK-positive melanomas predominantly expressed the recently described ALK isoform, ALKATI, which lacks the extracellular and transmembrane domains of wild-type ALK, consists primarily of the intracellular tyrosine kinase domain, and originates from an alternative transcriptional initiation site within the ALK gene. The findings are clinically relevant as patients with metastatic melanoma who have ALK expression may potentially benefit from treatment with ALK kinase inhibitors.