The human papilloma viruses (HPV) are responsible for ∼30% of viral-induced cancers. There are at least fourteen known oncogenic or high-risk HPV (HPV-HR) types. Prophylactic HPV vaccines have proven effective in preventing benign and malignant manifestations of this virus in both men and women. Unfortunately, these vaccines require at least 10–20 years “lead-in” time before showing an epidemiological-effect in cancer prevention. Despite the proven successes of these vaccines, society has neither embraced the significance nor adequately utilized this medical advancement. In addition, immunotherapies to effectively treat HPV-HR cancers have been elusive. We present two patients with HPV-HR positive gynecological malignancies who recurred following radiation therapy. Both patients were desperate and facing dismal prognoses. We describe the “off-label” use of two FDA-approved immune drugs consisting of intratumoral vaccinations of quadrivalent HPV-L1 vaccine (Gardasil™) and daily application of topical imiquimod resulting complete resolution of radiation refractory disease in both patients.

These are the only two patients treated with this combination of intra-tumoral vaccinations of quadrivalent HPV vaccine and topical imiquimod after presenting with radiation refractory disease. Each female patient had personal and logistical issues limiting travel for inclusion into regional/national research studies. Both patients requested alternative treatment options that could be administered during their current treatment choices. Following Bioethics Committee review and approval, of each patient was consented and received the immune regimen of intratumoral injections of quadrivalent HPV-L1 vaccine (Gardasil™) and topical imiquimod (Table 1). Demographic information for patients A and B is presented in Table 2.

This paper emphasizes the use of sequential, targeted vaccinations of the FDA-approved, quadrivalent, HPV-L1 antigens vaccine injected throughout the previously irradiated tumor. HPV-L1 peptide has been found to produce an effective anti-tumor response [1]. The vaccination technique used for these patients was developed to treat women with HPV-related lower genital tract intra-epithelial neoplasia. Imiquimod, an FDA-approved Toll-like receptor 7 (TLR7) agonist, has been successfully used as a vaccine adjuvant demonstrating anti-tumor immune responses with radiation therapy and vaccines and improves the recognition and uptake of both DNA viruses and radiation induced tumor-associated antigens (neoantigens) [2, 3]. This TLR7-agonist facilitates antigen processing of tumor-specific antigens in regional lymph nodes [4]. This readily available topical medication improves several localized immune processes creating a TH1-dominant microenvironment within the tumor and most importantly, regional lymph nodes [5, 6].

There are several studies describing vaccine-based immunotherapies to treat malignant tumors. A complete review of therapeutic vaccines can be found elsewhere [7], most demonstrate cellular immune responses [3, 7, 8, 9], but few therapeutic vaccines studies report complete, durable responses as described in these two patients with radiation-refractory squamous cell malignancies. This report describes the first account in which the combination of fractionated radiation, sequential intratumoral vaccination and topical imiquimod therapy was used to produced complete, durable responses in two patients with radiation refractory cancers. A recent case report (2018) describes the successful use of systemic and intratumoral vaccinations of the recent, FDA-approved, nonavalent, HPV-L1 vaccine (Gardasil 9 ^TM) treating 90-year-old women with multiple cutaneous, basaloid, squamous cell skin cancers [10]. Though this patient was not treated with nor failed radiation therapy, the authors reported similar tumor changes at 2 weeks post-vaccination as we observed at 2 weeks in our cases. Additionally, no specific side-effects or complications occurred [10].

Radiation therapy is vital in changing the tumor’s well-established, immunosuppressive microenvironment [3]. Fractionated radiation has been reported to produces several tumor-specific biologic and phenotypic cellular changes making tumor cells more sensitive to immunogenic cell death. Some of the important changes include the upregulation of tumor cell’s MHC class 1 and 2 antigens and death receptors such as Fas/CD95 [11]. We postulate that the innate immune system’s nonspecific and rapid response. It may have occurred with the first cycle of intratumoral vaccinations in the form of macrophages, NK and dendritic cells [12, 13]. The cytotoxic Th1 anti-tumor effects are augmented by radiation-induced production of neoantigens and the rapid induction of tumor lysis. This leads to the release, uptake, and processing of neoantigens followed by transport to regional lymph nodes. Importantly, the radiation-induced neoantigens are only present on cancer cells [14, 15]. These tumor-specific antigens allow immune targeting and cross-presentation facilitating the recognition of the radiated tumors heterogeneous mutations producing a sort of personalized “in-situ vaccine.” [3, 12, 14] There is the possibility that the HPV-L1 vaccine which was injected throughout the tumor to account for genetic heterogeneity may have had an effect in attracting local macrophage, NK, and dendritic cells (Figure 2). Moreover, we cannot completely exclude the contribution of the trauma from repeated “needling” of the tumor, which may have added to the inflammatory responses.

Radiation therapy, when use as a single treatment modality, has not consistently produced therapeutic responses outside of the radiated field known as “abscopal effects” without the addition of adjuvant immunotherapies [3, 12]. Unfortunately, patient A refused post-treatment chest imaging which could have objectively documented an “abscopal response” of her pulmonary metastases. The fact she lived 9 months without requiring supplemental oxygen, beginning 2 weeks after her first intratumoral vaccination other, clinical evidence of, at least, a partial-abscopal response.

From a safety standpoint, this immune protocol produced no discernible toxicities in either patient. Our observations suggest that combination of imiquimod and quadrivalent HPV vaccine is likely less toxic and more tolerable than systemic cytotoxic chemotherapies. Most immune therapies do not result in cross-resistance with conventional therapies [16, 17]. Therefore, the possibility of using them in combination or sequentially with other standard therapies could be an option.

We describe two female patients presenting with radiation-refractory, HPV-associated, squamous cell carcinomas of the lower genital tract facing death, patient A, or pelvic exenteration, patient B. Each patient was treated with an experimental therapy consisting of intratumoral injections of the preventative, quadrivalent HPV-L1 vaccine and topical application of imiquimod within 4 weeks following radiation therapy. Each patient demonstrated complete resolution of the treated tumor in addition to patient A’s clinical abscopal response of her pulmonary metastases allowing her to live an additional 9 months 2 weeks after starting this combination immune therapy. Currently, Patient B is alive with no evidence of disease 8 years following the modification of her surgery to a less radical operation due to resolution of her central recurrence 8 days after her second cycle of intratumoral therapy. We believe radiation therapy followed by intratumoral vaccinations and topical TLR-7 agonist resulted in a “prime-boost” response facilitating an appropriate cytotoxic antitumor response. If this combination of cancer therapies can be validated in appropriate clinical trials; this therapy could offer an urgent and unmet need for patients with treatment-refractory, HPV-related malignancies. Additionally, both medications in this report are FDA-approved for other conditions and have established safety profiles. The combination of radiation therapy followed by sequential intratumoral vaccinations and topical imiquimod could conveniently translate to the clinic setting and offers potential cost savings.