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Resolution of Squamous-Cell Carcinoma by Restoring T-Cell Receptor Signaling. Cutaneous squamous-cell carcinoma (SCC) is primarily caused by oncogenesis mediated by ultraviolet radiation, and β-human papillomavirus (β-HPV) is believed to be a mere facilitator that is dispensable for the maintenance of cutaneous SCC. Here, we describe a woman with benign and malignant HPV-related diseases that include a recurrent, unresectable, invasive cutaneous SCC with β-HPV19 genomic integration in the context of germline pathogenic mutations in ZAP70, an adapter required for T-cell receptor (TCR) signal transduction. Restoration of the integrity of TCR signaling by allogeneic hematopoietic-cell transplantation led to the resolution of all HPV-related diseases, thereby revealing a direct role of β-HPV in skin carcinogenesis in hosts with defective adaptive T-cell responses. (Funded by the National Institutes of Health.).

Non-melanoma skin cancers, comprising cutaneous squamous cell carcinoma (cSCC) and basal cell carcinoma, remain the most common malignancies in the United States and wordwide1,2, with an increasing incidence in the aging population that approaches the incidence of all other cancers combined3. In general, cSCC has a good prognosis; nonetheless, its high prevalence and occurrence of locally advanced or metastatic disease in 3–5% of cases result in high overall mortality and elevated healthcare costs1,2,4.

ncreasing incidence in the aging population that approaches the incidence of all other cancers combined3. In general, cSCC has a good prognosis; nonetheless, its high prevalence and occurrence of locally advanced or metastatic disease in 3–5% of cases result in high overall mortality and elevated healthcare costs1,2,4. While the causative and prognostic roles of ultraviolet (UV) exposure, Fitzpatrick skin-type, age and sex in cSCC have been well-defined and even incorporated in risk-prediction models4,5, the contribution of immunosurveillance against cutaneous β-human papillomaviruses (β-HPVs) in the pathogenesis of cSCC remains unclear. In fact, while a direct oncogenic role is firmly established for mucosal α-HPVs that are integrated in the host genome in mucosal SCC (i.e., ano-genital, oro-pharyngeal, respiratory tract) and constitutively express the E6/E7 viral oncogenes, cutaneous β-HPVs are believed to have an indirect role as mere facilitators of skin carcinogenesis. In this “hit-and-run” model, β-HPV appear relevant at an early stage of carcinogenesis by enabling the accumulation of UV-induced DNA mutations but are not stably transcribed in established cSCC and, therefore, are considered dispensable to maintain a frankly malignant phenotype6. In addition, although the role of immunosurveillance in skin carcinogenesis is inferred by the high prevalence of cSCC in immunocompromised hosts and by the clinical response to checkpoint inhibitors in cSCC with high mutational burden7, the contribution of immunological escape to viral or UV-mediated neoantigens in skin carcinogenesis remains uncertain.

munosurveillance in skin carcinogenesis is inferred by the high prevalence of cSCC in immunocompromised hosts and by the clinical response to checkpoint inhibitors in cSCC with high mutational burden7, the contribution of immunological escape to viral or UV-mediated neoantigens in skin carcinogenesis remains uncertain. Inborn errors of immunity can help decipher the role of HPVs and specific immunological functions in skin and mucosal carcinogenesis8–11. Herein, we report on the immunopathogenesis and management of recurrent, treatment-refractory invasive cSCC associated with a β-HPV in the context of defective proximal T-cell receptor (TCR) signaling due to germline variants in the ζ-chain of TCR-associated-protein-kinase-70 (ZAP70). The regression of multiple benign and recurrent malignant HPV-related lesions after restoration of HPV-specific T-cell responses following hematopoietic cell transplantation (HCT) illustrates the role of adaptive T-cell functions and the implications of integrated ablative and immunotherapeutic approaches to severe HPV-related diseases.

A comprehensive description of the experimental methods is provided in Supplementary Appendix along with the clinical protocols approved by the NIH Institutional Review Board. A 34-year-old woman with a history of cryptococcal meningitis with neuro-ophthalmic involvement developed progressively worsening cutaneous and mucosal HPV-related diseases with oral condylomas, diffuse verrucous lesions and multiple recurrent cSCCs in sun-exposed surfaces (43 different biopsy-proven lesions/sites, Figure 1A–C-left-panels, Supplementary Figure 1, 2-upper-panels). The susceptibility to cryptococcosis and recurrent cSCCs were initially attributed to a combined immunodeficiency and radiation-sensitivity blended phenotype due to ZAP70 deficiency and biallelic RNF168 variants12. The presumptive defect in UV-mediated DNA-double-strand breaks (DSBs) repair due to loss of function RNF168 in non-hematopoietic cell-types such as keratinocytes, was believed to significantly increase the risks and reduce the benefits of HCT for ZAP70 deficiency, and thus, conservative management with surgical resections of recurrent cSCCs was pursued (Supplementary Figure 1). The development of a recurrent invasive forehead cSCC refractory to multiple courses of aggressive surgical resections and checkpoint-inhibitor immunotherapy (Figure 1D) prompted referral to NIH/NIAID for further clinical, genetic, and immunological evaluation for advanced immunological therapies. We first pursued a comprehensive histological and molecular characterization of the forehead cSCC. The histological features were consistent with an invasive cSCC at high-risk for recurrence and metastatic spread given its size (5.2 cm), depth of invasion (6 mm), prominent desmoplastic stromal reaction with perivascular and perineural invasion (affecting large caliber nerves measuring ≤3mm) and prevalent moderate differentiation with some areas of poor differentiation13 (Figure 1D–E, Supplementary Figure 6). Despite no previous reports of β-HPVs expression in advanced cSCC14, next-generation RNA sequencing of the tumor revealed high-level of transcription of HPV19, a β1-HPV variant that was similar to others associated to cutaneous malignancies15 (Figure 1F).

of poor differentiation13 (Figure 1D–E, Supplementary Figure 6). Despite no previous reports of β-HPVs expression in advanced cSCC14, next-generation RNA sequencing of the tumor revealed high-level of transcription of HPV19, a β1-HPV variant that was similar to others associated to cutaneous malignancies15 (Figure 1F). Notably, the analysis of junction-spanning reads along with de novo assembly of the viral transcripts revealed two internal junctions within the HPV19 genome with inverse orientation (positive-strand into negative-strand) suggesting that these transcripts originated from recombined viral DNA. Furthermore, the analysis of the assemblies also revealed five host-virus junctions consistent with different host integration events across four chromosomes (Supplementary Figure 3).

with inverse orientation (positive-strand into negative-strand) suggesting that these transcripts originated from recombined viral DNA. Furthermore, the analysis of the assemblies also revealed five host-virus junctions consistent with different host integration events across four chromosomes (Supplementary Figure 3). Next-generation DNA sequencing of the tumor did not identify somatic driver mutations commonly associated with cSCCs (e.g., TP53, NOTCH1/2, CDKN2A)16 nor microsatellite instability, and the tumor mutational burden (TMB) [15.7 mutations-per-megabase (muts/Mb)] was well-below the reported average TMB in cSCCs (50 muts/Mb)16,17. As the somatic genetic variants in cancers represent multiple superimposed mutational signatures causing transitions (A>G/G>A; C>T/T<C) and transversions (A>C/C>A; G>T/T>G) specific for each histotype and oncogenic insult, we analyzed the somatic single-base-substitution (SBS) signatures18 of the patient`s recurrent invasive cSCC to investigate its underlying molecular pathogenesis. Although mutational signatures consistent with UV-exposure [SBS 7a/b (C>T)] were noted, their proportion (26%) was modest compared to what observed in sporadic cSCC (average 77%)16,19 (Figure 1G) and occurred along with signatures of aging, alkylating agents (SBS5 and SBS11, 25%) and a larger proportion of unknown mutational patterns (~50%). In conclusion, the molecular analysis of the recurrent invasive forehead SCC identified high-level E6/E7 oncogene expression from genomically integrated HPV19, modest UV-associated mutational signature enrichment, and absence of any specific driver mutations in cSCC associated tumor-suppressor genes.

l patterns (~50%). In conclusion, the molecular analysis of the recurrent invasive forehead SCC identified high-level E6/E7 oncogene expression from genomically integrated HPV19, modest UV-associated mutational signature enrichment, and absence of any specific driver mutations in cSCC associated tumor-suppressor genes. We then characterized the host genetic and immunological context in which the HPV-19-related invasive cSCC developed. Whole-exome-sequencing (WES) performed on peripheral-blood-mononuclear-cells, confirmed a homozygous RNF168 missense variant (p.D103N) which was previously proposed to confer hypersensitivity to ionizing radiation in this patient12. However, this variant had an elevated minor allele frequency for homozygous state (0.14) in the admixed-American population and was predicted to be benign in-silico. We then tested the functional integrity of patient`s RNF168 variant in the DNA DSB-repair response. Upon irradiation of the proband`s lymphocytes, ataxia-telangiectasia-mutated (ATM), structural-maintenance-of-chromosomes-1 (SMC1), and γ-H2AX exhibited normal protein phosphorylation/dephosphorylation kinetics at 1h and 24h respectively (Supplementary Figure 4; Supplementary Table 1–2). These findings were consistent with successful recruitment, retention and function of the pathways involved in repairing radiation-induced DSBs20. Likewise, the levels of DSB-induced apoptosis and cell death after irradiation was within the reference limits derived from healthy controls, further supporting a lack of genetic radiation sensitivity in the proband.

ful recruitment, retention and function of the pathways involved in repairing radiation-induced DSBs20. Likewise, the levels of DSB-induced apoptosis and cell death after irradiation was within the reference limits derived from healthy controls, further supporting a lack of genetic radiation sensitivity in the proband. The proband’s WES also identified the compound ZAP70 heterozygous missense variants (c.733G>A, p.G245R; c.1505C>T, p.P502L) previously reported12. Both ZAP70 variants, which segregated according to the phenotype in the family (Supplementary Figure 5), alter highly conserved residues in the carboxyterminal-SH2 domain (G245R) and kinase domain (P502L) of ZAP70 and were predicted in-silico to be damaging (Figure 2A).

G245R; c.1505C>T, p.P502L) previously reported12. Both ZAP70 variants, which segregated according to the phenotype in the family (Supplementary Figure 5), alter highly conserved residues in the carboxyterminal-SH2 domain (G245R) and kinase domain (P502L) of ZAP70 and were predicted in-silico to be damaging (Figure 2A). Therefore, we pursued a comprehensive functional analysis of the impact of these ZAP70 variants on TCR-signaling to inform management of the invasive treatment-refractory cSCCs. Briefly, upon engagement of the cognate antigen by the TCR, signal transduction occurs through an intracellular phosphokinase signaling circuitry initiated by the CD4/CD8-coreceptor-associated kinase LCK21. The phosphorylation of the TCR-ζ-chain by LCK generates high-affinity binding sites for the SH2-domains, and the cytosolic kinase ZAP70 is then recruited to the TCR-ζ-chain. In turn, the tyrosine phosphorylation of TCR-associated ZAP70 by LCK activates ZAP70`s kinase catalytic activity, resulting in phosphorylation of several downstream signaling proteins (e.g., PLC-γ1, ERK), culminating in the T-cell activation, proliferation, and differentiation (Figure 2B). The proband’s CD4- and CD8 T-cells displayed reduced levels of phosphorylated (p)-ZAP70 upon TCR stimulation and minimal downstream phosphorylated-PLC-γ1 and phosphorylated-ERK compared to a healthy subject (Figure 3A, black vs. red-rectangles). As expected, the ZAP70-associated defective proximal TCR-signaling resulted in a profound impairment of T-cell activation and proliferation in response to anti-CD3/CD28 cross-linking (Figure 3B, black vs red-plots) and β1-HPV19 E6-peptide pools (Figure 3C, black vs red-rectangles).

Figure 3A, black vs. red-rectangles). As expected, the ZAP70-associated defective proximal TCR-signaling resulted in a profound impairment of T-cell activation and proliferation in response to anti-CD3/CD28 cross-linking (Figure 3B, black vs red-plots) and β1-HPV19 E6-peptide pools (Figure 3C, black vs red-rectangles). In summary, after ruling out an underlying UV hypersensitivity, we attributed a causative role to the ZAP70-mediated TCR defect for recurrent muco-cutaneous HPV-related diseases, including the β1-HPV19-related cSCC. As this invasive unresectable high-risk cSCC with β1-HPV19 genomic integration was refractory to curative treatments in the setting of the underlying TCR-signaling defect, an integrated management plan was designed after ruling out distant metastasis by Positron-Emission-Tomography: a course of cetuximab/5-fluorouracil/cisplatin was followed by cSCC prophylaxis with capecitabine/nicotinamide22, while the patient received an haploidentical HCT for definitive treatment of the underlying immunodeficiency. No clinical complications developed throughout this course, and stable resolution of all HPV-skin related diseases was achieved post-HCT and maintained up to the most recent follow-up (35 months post-HCT, Figure 1A–C right-panels, Supplemental Figure 2 lower-panels). In fact, after engraftment of a ZAP70-wildtype haploidentical donor, the function and integrity of TCR signaling was restored (Figure 3A, blue-rectangle) with normalization of T-cell activation/proliferation (Figure 3B, blue-histograms) and robust expansion of HPV19-specific T-cells (Figure 3C blue-plots). Furthermore, transcriptional analysis of resting and TCR-stimulated CD4 and CD8 T-cells obtained post-HCT reveled a gene expression pattern similar to that observed in TCR-stimulated T-cells from healthy subjects (Figure 3D, E).

A 34-year-old woman with a history of cryptococcal meningitis with neuro-ophthalmic involvement developed progressively worsening cutaneous and mucosal HPV-related diseases with oral condylomas, diffuse verrucous lesions and multiple recurrent cSCCs in sun-exposed surfaces (43 different biopsy-proven lesions/sites, Figure 1A–C-left-panels, Supplementary Figure 1, 2-upper-panels). The susceptibility to cryptococcosis and recurrent cSCCs were initially attributed to a combined immunodeficiency and radiation-sensitivity blended phenotype due to ZAP70 deficiency and biallelic RNF168 variants12. The presumptive defect in UV-mediated DNA-double-strand breaks (DSBs) repair due to loss of function RNF168 in non-hematopoietic cell-types such as keratinocytes, was believed to significantly increase the risks and reduce the benefits of HCT for ZAP70 deficiency, and thus, conservative management with surgical resections of recurrent cSCCs was pursued (Supplementary Figure 1). The development of a recurrent invasive forehead cSCC refractory to multiple courses of aggressive surgical resections and checkpoint-inhibitor immunotherapy (Figure 1D) prompted referral to NIH/NIAID for further clinical, genetic, and immunological evaluation for advanced immunological therapies. We first pursued a comprehensive histological and molecular characterization of the forehead cSCC. The histological features were consistent with an invasive cSCC at high-risk for recurrence and metastatic spread given its size (5.2 cm), depth of invasion (6 mm), prominent desmoplastic stromal reaction with perivascular and perineural invasion (affecting large caliber nerves measuring ≤3mm) and prevalent moderate differentiation with some areas of poor differentiation13 (Figure 1D–E, Supplementary Figure 6). Despite no previous reports of β-HPVs expression in advanced cSCC14, next-generation RNA sequencing of the tumor revealed high-level of transcription of HPV19, a β1-HPV variant that was similar to others associated to cutaneous malignancies15 (Figure 1F).

cSCC, a common cutaneous malignancy, is primarily caused by UV-mediated somatic DNA mutations resulting in oncogene activation and tumor suppressor genes inactivation in keratinocytes1,16. However, the increased risk of cSCC in solid organ transplantation, chronic lymphocytic leukemia and other immunocompromising conditions1 along with the robust clinical response to immune-checkpoint inhibitors, indicate that impaired adaptive T-cell responses are associated with both cSCC development and progression. Nonetheless, it remains unclear to what extent skin commensal β-HPVs, somatic neoantigens or both are involved in generating and maintaining T-cell responses that can prevent or control skin carcinogenesis. Current evidence and a well-established model of skin carcinogenesis indicate that β-HPVs are not transcriptionally active in established cSCC, but instead act just as early facilitators of the accumulation of UV-induced mutations in driver host oncogenes6. In contrast to this model, we found that a recurrent, invasive, treatment-refractory cSCC had high expression of β1-HPV19 viral oncogenes in the setting of host genome integration. This tumor lacked typical cSCC-associated somatic driver mutations and had only modest UV-mediated and overall mutational burden. This patient’s tumor showed β-HPV integration in cSCC, and we attribute the patient’s benign and malignant HPV-related diseases to the specific immunological context determined by a ZAP-70-mediated impairment in HPVs-specific T-cell responses. While biallelic loss-of-function ZAP70 mutations result in early-onset combined immunodeficiency, ZAP70 hypomorphic variants, like the ones identified in this patient, can be partially permissive for T-cell development but still impair TCR-mediated signal transduction. The precise genotype-phenotype correlation identified in this case allowed us to decipher the long-term clinical effect of altered TCR-signaling on adaptive immunity against commensal HPV and consequent skin carcinogenesis.

e partially permissive for T-cell development but still impair TCR-mediated signal transduction. The precise genotype-phenotype correlation identified in this case allowed us to decipher the long-term clinical effect of altered TCR-signaling on adaptive immunity against commensal HPV and consequent skin carcinogenesis. As the intrinsic oncogenic potential of commensal β-HPVs (e.g., β1-HPV5 and HPV8) is described in the context of epidermodysplasia verruciformis23, and the recruitment of β-HPV-specific adaptive immune responses can protect from the development of SCC in animal models24,25, our findings indicate that impaired HPV-specific T-cell responses and uncontrolled expression of β1-HPV viral oncogenes can promote and sustain the development of cSCC with aggressive clinical and histological features in some immunocompromised hosts.

onses can protect from the development of SCC in animal models24,25, our findings indicate that impaired HPV-specific T-cell responses and uncontrolled expression of β1-HPV viral oncogenes can promote and sustain the development of cSCC with aggressive clinical and histological features in some immunocompromised hosts. Furthermore, our case study suggests an alternate role for β1-HPV in skin carcinogenesis in the context of defective adaptive T-cell function: In addition to possibly promoting conventional UV-mediated cSCC oncogenesis, individuals with impaired T-cell responses may also be susceptible to direct β-HPV-mediated oncogenesis. These findings have significant clinical implications: In our specific case, despite initial anchoring to a hypothesis of UV-radiosensitivity-associated susceptibility to cSCC, a more precise immunological and molecular study of the underlying immunological defect informed the decision to proceed with HCT to restore ZAP70 function and TCR-signaling integrity. The restoration of T-cell function and consequent development of robust HPV-specific T-cell responses led to the stable resolution of all HPV-related skin and mucosal diseases, including the aggressive and treatment-refractory cSCC. The complete reversion of the phenotype post-HCT further emphasizes that neither RNF168 variants nor UV-radiosensitivity contributed to the pathogenesis of HPV-related diseases in this patient.

to the stable resolution of all HPV-related skin and mucosal diseases, including the aggressive and treatment-refractory cSCC. The complete reversion of the phenotype post-HCT further emphasizes that neither RNF168 variants nor UV-radiosensitivity contributed to the pathogenesis of HPV-related diseases in this patient. Similarly, the characterization of T-cell functions and β-HPVs transcriptional activity in patients with recurrence of aggressive cSCC may be valuable in other clinical contexts in which boosting T-cell-based immunity against β-HPVs (e.g., by therapeutic immunization or immune checkpoint inhibition) can be developed or further optimized to improve the prevention and management of cSCC25.