Cutaneous squamous-cell carcinoma of the head-neck area refractory to chemo-radiotherapy: benefit from anti-PD-1 immunotherapy.

OBJECTIVE: Radiotherapy provides excellent results in locally advanced cutaneous squamous-cell carcinoma of the head and neck area (cSCC-HN), with a 2-year local progression-free interval obtained for about 80% of patients. Overexpression of immune checkpoint co-inhibitory molecules, like PD-L1 (programmed death ligand 1), by cancer cells may define local immunosuppression, tumour escape from immune surveillance and reduced radiotherapy efficacy. METHODS: A 65-year-old female, with a large exophytic cSCC-HN invading adjacent soft tissues, was treated with hypofractionated accelerated chemo-radiotherapy. The patient received four bi-weekly cycles of chemotherapy concurrently with eight fractions of 5.5 Gy (two fractions per week). Two months after the end of chemo-radiotherapy, the tumour was stable in dimensions, without any signs of symptomatic relief. The patient was, after that, treated with anti-PD-1 immunotherapy (nivolumab). The tumour gradually regressed, reaching partial response after four cycles and complete response after 16 cycles of nivolumab. No side-effects related to immunotherapy were recorded. The patient is alive and without evidence of disease 28 months after radiotherapy. CONCLUSIONS: Treatment of patients with chemo- and radio-resistant cSCC-HN with immunotherapy may optimize the efficacy of radiotherapy by stimulating immunological tumour rejection mechanisms. cSCC-HN patients who fail to respond to chemo-radiotherapy completely are expected to benefit the most from immunotherapy because of the radio-vaccination effect expected from the preceded radiotherapy.

Patterns of LC3A Autophagy Protein Expression in Keratoacanthomas.

To investigate the expression patterns of autophagy marker light chain protein 3 (LC3A) in keratoacanthoma (KA). KAs are generally regarded as benign but malignant behavior, including rare metastases, may occur. 85 KAs were assessed for the LC3A autophagic protein by immunohistochemistry. Diffuse cytoplasmic staining and a "stone-like structure" (SLS) characterized positive expression. Thirty-four out of 85 KAs (40%) had diffuse cytoplasmic LC3A immunostaining (percentage of positive cells ranging from 5 to 60%). In contrast, only 4 of the 85 KAs (4.7%) expressed SLSs. Only one SLS was detected per histologic section of each tumor. The p53 oncoprotein was encountered in all cases with expression ranging from 1 to 90% of cells (median 30%). The Ki-67 index was expressed in 63 cases (74% of cases; range 1-50% of cells; median value 5%). Neither of these two parameters nor diffuse cytoplasmic LC3A staining was significantly correlated with SLS expression or lack thereof. Expression of SLSs, a hallmark of malignancy, was found in 4.7% of KAs. Further study is necessary to determine whether this fraction represents the exceptional cases that harbor latent malignant potential.

Beclin-1 and LC3A expression in cutaneous malignant melanomas: a biphasic survival pattern for beclin-1.

Autophagy is an intracellular pathway for the degradation of long-lived proteins and damaged organelles. It is, in essence, a recycling process allowing cells to survive oxygen and nutrient depletion. The expression of two autophagy-related proteins, beclin 1 and light chain 3A (LC3A) was investigated in 79 nodular cutaneous melanomas. The results were correlated with histopathological factors, vascular density, and hypoxia-related proteins [hypoxia-inducible factors (HIF1α and HIF2α) and lactate dehydrogenase 5]. The reactivity of both autophagy-related proteins was uniformly cytoplasmically diffused. High beclin 1 and LC3A reactivity was related to tumor hypoxia, as this was inferred from the intense expression of HIF1α and lactate dehydrogenase 5, whereas low beclin 1 and LC3A expression was linked with an increased vascular density. In addition, beclin 1 was related to disease-specific survival which, however, exposed a biphasic pattern. A strong beclin 1 expression extending over a tumor area of more than 50% (high) was associated with an increased rate of early deaths, whereas a similarly strong, but less-extensive cytoplasmic reactivity (<10% tumor area; low) defined a sharp fall in the survival 5 years after surgery. Furthermore, the low beclin 1 expression was associated with high Breslow's depth, high Clark's level, and ulceration. Low LC3A expression was also related to ulceration, but not to other histopathological features nor prognosis. In multivariate analysis, beclin 1 was an independent prognostic variable. It is concluded that extensive autophagic activity is generated by tumor hypoxia and anaerobic glycolysis, whereas angiogenesis maintains low autophagic activity. Atg6/beclin 1 was proved to be capable of deciphering the prognosis in cutaneous malignant melanoma, but the matter requires further investigation.

LC3A-positive "stone-like" structures in cutaneous squamous cell carcinomas.

This study was set to investigate the relation between autophagic activity and the aggressiveness of cutaneous squamous cell carcinomas (SCC), as indicated by tumor thickness and proliferative activity. The anti-LC3A antibody, recognizing both the soluble and the autophagosome-bound forms of the protein, and a standard immunohistochemical technique were applied to 75 cutaneous SCC of variable tumor thickness. The study was complemented by staining for MIB1. Three patterns of LC3A reactivity were recognized: diffuse cytoplasmic, cytoplasmic/perinuclear, and "stone-like" structures (SLS), that is, large, rounded, densely stained amorphous material, 5 µm on average, enclosed within cytoplasmic vacuoles. Higher numbers of SLS were counted in >6-mm-thick SCC compared with the intermediate-thickness tumors (2.1-6 mm) and the <2-mm-thick tumors; the mean recorded values, being 8.8, 4.55, and 1.55, respectively, were statistically significant. The diffuse cytoplasmic staining showed a nearly inverse trend, whereas the perinuclear pattern, expressed in <10% of the total, was not evaluated. With regard to MIB1 proliferation index, this increased with tumor thickness and, in linear regression analysis, was directly linked with SLS counts and inversely with the cytoplasmic pattern. These data suggest that autophagic activity in SCC, when expressed as high LC3A/SLS counts, can be regarded as an indicator of tumor aggressiveness.

Radiochemotherapy with cetuximab, cisplatin, and amifostine for locally advanced head and neck cancer: a feasibility study.

PURPOSE: Radiotherapy (RT) combined with cisplatin or cetuximab is the standard of care for patients with locally advanced head/neck cancer (LA-HNC). The feasibility of radiochemotherapy with cisplatin and cetuximab, supported with amifostine, was herein investigated. METHODS AND MATERIALS: Forty-three patients with LA-HNC were recruited. Conformal hypofractionated/accelerated RT with amifostine cytoprotection (2.7 Gy/fraction, 21 fractions in 4 weeks) was combined with cisplatin (30 mg/m(2)/week) and cetuximab (standard weekly regimen) therapy. The dose of amifostine was individualized according to tolerance. RESULTS: A high daily amifostine dose (750-1,000 mg) was tolerated by 41.8% of patients, and a standard dose (500 mg) was tolerated by 34.9% of patients. A high amifostine dose was linked to reduced RT delays (p = 0.0003). Grade 3 to 4 (3-4) mucositis occurred in 7/43 (16.2%) patients, and fungal infections occurred in 18/43 (41.8%) patients. Radiation dermatitis was not aggravated. Interruption of cetuximab due to acneiform rash was necessary in 23.3% of patients, while amifostine-related fever and rash were not observed. Severe late radiation sequelae consisted of laryngeal edema (9% laryngeal cases) and cervical strictures (33% of hypopharyngeal cases). Good salivary function was preserved in 6/11 (54.5%) nasopharyngeal cancer patients. The complete response rate was 68.5%, reaching 77.2% in patients with minor radiotherapy delays. The 24-month local control and survival rates were 72.3% and 91%, respectively (median follow-up was 13 months.). CONCLUSIONS: In this feasibility study, weekly administration of cisplatin and cetuximab was safely combined with accelerated RT, supported with amifostine, at the cost of a high incidence of acneiform rash but a reduced incidence of amifostine-related fever/rash. A high daily dose of amifostine allows completion of therapy with minor delays.

CD30 (Ki-1) molecule expression in human embryonal epithelial cells of the basal layer of the developing epidermis and epidermal buds and its potential significance for embryogenesis.

OBJECTIVE: CD30 antigen has long been considered to be restricted to tumour cells of Hodgkin's disease, of anaplastic large cell lymphoma and T and B activated lymphocytes. Expression of CD30 antigen has been reported in the decidual stroma, cultivated macrophages, lipoblasts, myoepithelial cells, reactive and neoplastic vascular lesions, mesotheliomas, embryonal carcinoma and seminoma cells. The fact that the CD30 molecule can mediate signals for cell proliferation or apoptosis prompted us to perform a systematic investigation of CD30 antigen expression in embryonal tissues during the proliferation and differentiation stages. We first targeted foetal human intestinal cryptae cells with positive results. The epidermis is a dynamic epithelium that is constantly renewed throughout life. Its turnover is estimated at about 7 days in mice and about 60 days in humans. This rapid replacement demands, as with other epithelial tissues, that an adult has stem cells capable of supplying differentiated cells throughout life. The most basic and widely accepted criteria for these stem cells are that they have a high capacity for self-renewal and the ability to generate daughter cells that undergo terminal differentiation. Not all of the proliferative cells in the basal layer are stem cells and we were intrigued to find out if stem or other cells in the basal layer can express the CD30 antigen. MATERIALS AND METHODS: We investigated the immunohistochemical expression of CD30 antigen in 15 paraffin-embedded tissue samples representing epidermis and epidermal buds from foetuses after spontaneous abortion in 8th, 10th, and 12th week of gestation, respectively, using the monoclonal antibody Ki-1. RESULTS: The results showed that the epithelial cells of the epidermis in the developing skin express the CD30 antigen and CD30 expression in these epithelial cells is higher in cases of hormonal administration than in normal gestation. A similar positive reaction was observed in the epidermal buds associated with the development of the skin appendages.