P16 immunostaining is considered a useful surrogate of transcriptionally active high-risk (hr) HPV infection. Only strong and widespread “block-like” immunoreactivity is considered specific, whereas weak/focal p16 positive immunostaining is considered not specific, and follow-up and HPV molecular detection is not indicated. The aim of the study was to evaluate the presence of HPV DNA and Ki67 immunostaining in 40 cervical atypical biopsies (CALs) with mild and focal histological features suggestive of HPV infection—20 cases with weak/focal p16 positive immunoreactivity and 20 cases negative for p16 expression. In 16/20 weak/focal p16 positive CALs (80%), the INNO-LiPA HPV genotyping detected hrHPV genotypes (HPV 31, 51, 56, 59, 26, 53, 66, 73, and 82). Co-infection of two or more hrHPV genotypes was often evidenced. HPV16 and 18 genotypes were never detected. Ki67 immunostaining was increased in 10/20 cases (50%). In 19/20 p16 negative CALs, hrHPV infection was absent and Ki67 was not increased. These results suggest that weak/focal p16 immunostaining represents the early stage of transcriptionally active infection, strongly related to the presence of less common hrHPV genotypes, probably with a slower transforming power, but with a potential risk of progression if the infection persists. HPV DNA genotyping and follow-up could be useful in these cases to verify if they are able to evolve into overt dysplastic changes and to improve knowledge of less common hrHPV genotypes.

Background: Dumbbell tumors are typically benign schwannomas, neurofibromas, and meningiomas and only rarely there are malignant variants of these lesions or other malignant histotypes. Here, a 34-year-old male presented with a thoracic spinal dumbbell metastatic neuroendocrine carcinoma of unknown primary origin. Case Description: A 34-year-old male presented with 2 months of thoracic pain and progressive mid thoracic sensory loss. A post contrast thoracic MRI showed a dumbbell tumor localized between the T7 and T9 levels with extension laterally into the T7-T8 and T8-T9 foramina. The patient underwent a laminectomy for tumor resection following which his pain and gait improved. Histopathologically, the tumor demonstrated multiple rounded small cells with a Ki67 level around 30%, suggesting a malignant metastatic neuroendocrine tumor of unknown etiology. Conclusion: We successfully treated a 34-year-old male with a T7-T9 malignant spinal dumbbell neuroendocrine tumor of unknown etiology utilizing a decompressive laminectomy.

541 Background: PURE-01 trial enrolled 155 patients who received 3 cycles of pembrolizumab (IO) every 3 weeks before radical cystectomy. We compared tumor-microenvironment immune markers expression in pre- and post-IO TURB specimens in complete/major and non-responders to identify features associated with pathologic response. Methods: We evaluated pre-IO TURB samples of 18 complete responders (CR: ypT0), 6 major responders (MR: ypTa/ypT1) and 19 non-responders (NR: ypT2/ypT3, N0 or N+) and post-IO specimen of 7 CR, 3 MR and 14 NR. Immunohistochemistry analysis for lymphocytes (CD3, CD8, CD20), macrophages (CD68, CD163), immune checkpoints (PD1, PD-L1) and HLA molecule (MHC-I, HLA-DR, B2M) was performed. A semiquantitative count was made on all samples by 3 expert pathologists and a quantitative whole-slide digital evaluation was performed using QuPath (v.0.3) to assess cell density (cells per mm2) and expression of immune checkpoint and HLA molecules. Results: Semiquantitative count showed that seven markers (CD8, CD163, stromal or tumor PD-L1, HLA-DR and B2M on tumor cells) were significantly more expressed on TURB lesions from responders (CR + MR or CR only) compared to NR (N0 or N+) (p-value range: 0.001 to 0.048). In addition to such markers, patients achieving a CR, compared to NR, showed significantly higher expression of CD68 and CD163, of PD-1 on lymphocytes, as well as of tumor MHC-I molecules. Comparison of lesions from patients with CR vs MR revealed significant differences for CD3, CD163, PD-1 and MHC-I, all these markers being more frequently expressed on the former group compared to the latter. Digital pathology analysis confirmed this results on whole-slide evaluation showing higher density of CD8, CD163, PD-L1 and PD-1, and a higher expression B2M, MHC-I and HLA-DR (p-value range: 0.02 to 0.002) in responders. Moreover, N0 patients showed higher expression of PD-L1 and HLA-DR compared to N+ patients. Comparison of pre- and post-IO samples from NR patients showed an increased density of CD8 and CD20 cells (p-value: 0.04 and 0.01), and a decrease of CD163 and HLA-ABC (p-value: 0.003 and 0.02) expressing cells in the post-therapy samples. Conclusions: The tumor immune microenvironment of pre-therapy TURB lesions of patients achieving a complete or major pathologic response after neoadjuvant pembrolizumab shows significant enrichment for T cells and myeloid cells, for stromal or tumor PD-L1 as well as increased MHC-I expression on tumor cells compared to lesions from non responders. Post-IO surgical samples of NR patients showed downregulation of HLA-Class I antigens, in spite of increased CD8 and CD20 density. Clinical trial information: NCT02736266 .