Immunotherapy in HER2-positive breast cancer: state of the art and future perspectives.

Breast cancer (BC) is a complex disease with primary or acquired incurability characteristics in a significant part of patients. Immunotherapeutical agents represent an emerging option for breast cancer treatment, including the human epidermal growth factor 2 positive (HER2+) subtype. The immune system holds the ability to spontaneously implement a defensive response against HER2+ BC cells through complex mechanisms which can be exploited to modulate this response for obtaining a clinical benefit. Initial immune system modulating strategies consisted mostly in vaccine therapies, which are still being investigated and improved. However, the entrance of trastuzumab into the scenery of HER2+ BC treatment was the real game changing event, which embodied a dominant immune-mediated mechanism. More recently, the advent of the immune checkpoint inhibitors has caused a new paradigm shift for immuno-oncology, with promising initial results also for HER2+ BC. Breast cancer has been traditionally considered poorly immunogenic, being characterized by relatively low tumor mutation burden (TMB). Nevertheless, recent evidence has revealed high tumor infiltrating lymphocytes (TILs) and programmed cell death-ligand 1 (PD-L1) expression in a considerable proportion of HER2+ BC patients. This may translate into a higher potential to elicit anti-cancer response and, therefore, wider possibilities for the use and implementation of immunotherapy in this subset of BC patients. We are herein presenting and critically discussing the most representative evidence concerning immunotherapy in HER2+ BC cancer, both singularly and in combination with therapeutic agents acting throughout HER2-block, immune checkpoint inhibition and anti-cancer vaccines. The reader will be also provided with hints concerning potential future projection of the most promising immutherapeutic agents and approaches for the disease of interest.

Targeting immune response with therapeutic vaccines in premalignant lesions and cervical cancer: hope or reality from clinical studies.

Human papillomavirus (HPV) is widely known as a cause of cervical cancer (CC) and cervical intraepithelial neoplasia (CIN). HPVs related to cancer express two main oncogenes, i.e. E6 and E7, considered as tumorigenic genes; their integration into the host genome results in the abnormal regulation of cell cycle control. Due to their peculiarities, these oncogenes represent an excellent target for cancer immunotherapy. In this work the authors highlight the potential use of therapeutic vaccines as safe and effective pharmacological tools in cervical disease, focusing on vaccines that have reached the clinical trial phase. Many therapeutic HPV vaccines have been tested in clinical trials with promising results. Adoptive T-cell therapy showed clinical activity in a phase II trial involving advanced CC patients. A phase II randomized trial showed clinical activity of a nucleic acid-based vaccine in HPV16 or HPV18 positive CIN. Several trials involving peptide-protein-based vaccines and live-vector based vaccines demonstrated that these approaches are effective in CIN as well as in advanced CC patients. HPV therapeutic vaccines must be regarded as a therapeutic option in cervical disease. The synergic combination of HPV therapeutic vaccines with radiotherapy, chemotherapy, immunomodulators or immune checkpoint inhibitors opens a new and interesting scenario in this disease.

Neutrophil-rich gastric carcinomas: light and electron microscopic study of 9 cases with particular reference to neutrophil apoptosis.

The authors report 9 cases of gastric carcinomas characterized by a prominent neutrophilic infiltration of the stroma. These tumors (8 of intestinal type, 1 of diffuse type) showed a pushing growth pattern. Metastatic involvement of regional lymph nodes was seen in 5 cases. The metastatic foci were associated with heavy neutrophilia as well. There was no histologic evidence of Helicobacter pylori infection, whereas various degrees of multifocal intestinal metaplasia were present in the background mucosa. Based on histologic and histochemical results, there were no apparent causes due to other infectious agents responsible for the neutrophil-rich gastric carcinomas. Some of intraepithelial and stromal neutrophils exhibited apoptotic changes, such as chromatin condensation and cell shrinkage, and were TUNEL-positive. Electron microscopy disclosed apoptotic neutrophils in cytoplasmic vacuoles of tumor cells, a finding suggestive of neutrophil-tumor cell phagocytosis (cannibalism). Different stages of neutrophil apoptosis were also shown by electron microscopy and the ultrastructural findings were compared to those described in experimental models, both in vivo and in vitro.

Neutrophil-tumor cell phagocytosis (cannibalism) in human tumors: an update and literature review.

The recognition and removal of apoptotic cells by tissue macrophages and nonprofessional phagocytes, in a process called efferocytosis, is critical for development, tissue homeostasis and resolution of inflammation. Apoptotic bodies arising in tumor tissue are ingested by viable neoplastic cells and by resident macrophages. We described tumor cell phagocytosis of apoptotic neutrophils in human gastric carcinomas. This phenomenon is analogous to neutrophil efferocytosis performed by macrophages and by nonprofessional phagocytes during inflammatory reaction but is distinct by other types of cell-in-cell phenomena including emperipolesis and entosis both cytologically and biologically. In this review, we discussed them in their ultrastructural morphology, physiological roles, and clinicopathologic implications. This article is part of a Special Issue entitled "Apoptosis: Four Decades Later".

High risk human papillomavirus genotyping in clinical samples: evaluation of different commercial tests.

The aim of the present study is to compare the performance of several commercial human papillomavirus (HPV) tests in a cohort of 281 women. The hybrid capture II, the PreTect-HPV-Proofer, the linear array, and DR.HPVTMIVD were utilized to detect and type HPV in parallel with in-house PCR tests followed by direct automated sequencing or by sub-cloning and sequencing. The concordance levels along with other tests were evaluated with a Cohen's K value varying between 0.60 to 0.88, indicating good correlation with nearly perfect agreement between hybrid capture II, (HCII) and the linear array test. High sensitivity was recorded by the linear array and HCII with 100% (95% CI, 0.8021 to 1.0000) detection of cervical intraepithelial neoplasia (CIN) III by both methods. Conversely, the PreTect-HPV-Proofer showed high specificity with 12% (95% CI, 0.7966 to 0.9163) positivity on normal samples. The genotyping analysis showed that agreement among tests was only low to moderate with great differences between different HPV types. Multiple infections were detected with poor concordance and sub-cloning assays revealed the presence of a lower number of HPV in comparison to the other methods. In summary, the use of different HPV tests applied to the same group of cervical smears may possibly lead to incongruent results, suggesting the need to standardize type-specific sensitivity of genotyping methods and the need to evaluate their accuracy in detecting multiple HPV infections. This would be a prerequisite for the use of genotyping assays in cervical cancer screening programs.

Predictors of human papilloma virus (HPV) infection in Italian women.

HPV infection is a "necessary cause" of cervical cancer and it is sexually transmitted. Due to upcoming mass vaccination investigation on risk factors for infection is the basis to implement prophylactic strategy even in older women. The aim of the study was to evaluate predictors of high-risk (HR) HPV infection in adult women. Between 2006 and 2008, 100 women aged >18 years, with no previous treatment for cervical lesions, were screened for HR HPV infection in Rome, Italy. Risk factors for HPV infection were investigated through a questionnaire including: ethnicity, religion, education, marital status, sexual behavior, gynecological and obstetrical history, smoking and alcohol intake. Multivariate analysis identified the "never married-separated/divorced" status (OR: 3.38; 95% CI: 1.14-10.12) as predictor of HPV infection, while having a higher age at the first sexual intercourse (FSI) shows a protective effect (OR: 0.84; 95% CI: 0.71-1.00). A trend for the association between the infection and having more than three lifetime partners was also observed (OR: 2.57; 95% CI: 0.86-7.71). No significant association was found for other demographic characteristics investigated. These findings provide a contribution in the knowledge of an adult population defining a "high-risk" sexual behavioral profile and could be helpful to target prophylactic strategies in older woman.

Modifications of nuclear envelope in tumour cells of human gastric carcinomas: an ultrastructural study.

BACKGROUND: Various nuclear envelope derivatives, such as the annulate lamellae, the intranuclear tubules as well as the nuclear projections and pockets may be observed electron microscopically in tumour cells. PATIENTS AND METHODS: In a series of eight gastric adenocarcinomas, ultrastructural features of nuclear envelope changes were analyzed and correlated to the biology of the tumours. RESULTS: Histologically, three tumours were intestinal-type adenocarcinomas and showed annulate lamellae in the cytoplasm of some tumor cells. Five tumors were mixed-type adenocarcinomas, with a solid growth pattern; two of these tumours were characterized by the presence of intranuclear tubules, whereas the remaining three tumours exhibited nuclear pockets and projections. Seven out of eight patients died due to metastatic disease during the follow-up period (median 31 months). CONCLUSION: Ultrastructural evaluation of pleomorphism of the nuclear envelope may be an ancillary method for the pathologist in the study of nuclear grading of gastric carcinomas.

Ultrastructural descriptions of pericyte/endothelium peg-socket interdigitations in the microvasculature of human gastric carcinomas.

BACKGROUND: Angiogenesis requires complex multistep signalling pathways and a high degree of spatial and temporal coordination among endothelial cells and pericytes. The two cell types exhibit numerous contacts in vivo and in vitro, including the occurrence of peg-socket junctions. MATERIALS AND METHODS: Ultrastructural findings in 9 cases of advanced gastric carcinomas were reviewed with special emphasis on endothelium/pericyte peg-socket junctions. RESULTS: The incidence of peg-socket junctions was approximately 8% in 5 out of 9 cases. The remaining 4 cases showed a very low rate, including two cases in whom interactions were totally absent. Peg-socket junctions consisted of cytoplasmic projection from the pericyte protruding into the endothelial indentation. The endothelial cells interacting with pericytes showed ultrastructural signs of partial stabilization such as continuous endothelial lining, regularly constructed interendothelial junctions, more or less integrated pericytes, and multilayered basement membrane. CONCLUSION: Our ultrastructural study confirms previous reports regarding pericyte/endothelial peg-socket interdigitations in murine and human granulation tissues and extends these findings to the microvasculature of human gastric carcinomas.

Mast cell and eosinophil interaction in gastric carcinomas: ultrastructural observations.

BACKGROUND: An increase in the number of mastocytes has been described in some human neoplasms, mainly in gastric and colorectal cancer. MATERIALS AND METHODS: A case of diffuse-type gastric carcinoma, mainly infiltrated by eosinophils and mast cells, was studied using light and electron microscopy. RESULTS: Using light microscopy, cell clusters containing one mast cell and one to three eosinophils were found in the tumour stroma. Electron microscopy of this unusual stromal element revealed that mast cells established areas of junctions with eosinophils. Moreover, focal polarized exocytosis of mast cell granules was found in the areas of junctions with eosinophils. Eosinophils in contact with mast cells showed signs of important in situ activation, such as alterations in the size and number of granules, cytoplasmic vacuoles, and scattered extracellular granules. CONCLUSION: Our ultrastructural study provides morphological evidence of cross-talk between activated mast cells and eosinophils that may play an important role in the enhancement of host immunity against cancer cells.

Gastric hepatoid adenocarcinoma with autophagy-related necrosis-like tumor cell death: report of a case.

A case of hepatoid adenocarcinoma of the stomach is presented. The characteristic features of the tumor are summarized on the basis of the authors' experience and the literature. Ultrastructural examination revealed patchy condensations of chromatin throughout the nucleus suggestive of necrosis-like programmed cell death (PCD). These nuclear alterations were associated with the occurrence of vacuoles and lipofuscins, conferring an autophagic phenotype to this PCD. Thus, the case reported here provides an example of autophagic-related necrosis-like PCD. Alternative PCDs are reviewed and their morphologic distinction is discussed.

Exploiting the plant secretory pathway to improve the anticancer activity of a plant-derived HPV16 E7 vaccine.

The human papillomavirus 16 (HPV16) E7 oncoprotein can be considered a "tumor-specific antigen", and therefore it represents a promising target for a therapeutic vaccine against HPV-associated tumors. Efficient production of E7 protein with a plant-based transient expression system has been already described and it was demonstrated that E7-containing crude plant extracts confer partial protection against tumor challenge in a mouse model system. Before adopting the plant-based system as a cost-effective method for the production of an E7-based anti-cancer vaccine, some aspects, such as the oncoprotein yield, need further investigation. In the present study, we report the transient expression, mediated by a potato virus X (PVX)-derived vector, of the E7 protein targeted to the secretory system of Nicotiana benthamiana plants by using a plant-derived signal sequence. Targeting the antigen to the secretory pathway enhanced the E7 protein expression levels about five-fold. Mice immunized by s.c. administration with crude foliar extracts containing E7 showed strong stimulation of cell-mediated immune response after five boosters, as detected by ELISPOT. After challenging with the E7-expressing C3 tumor cells, tumor growth was completely inhibited in 80% of the vaccinated animals and a drastic reduction of tumor burden was observed in the remaining tumor-affected mice. These data demonstrate that, by enhancing E7 yield, it is possible to improve the anti-cancer activity of the plant-based experimental vaccine and open the way for a large-scale production of the E7 protein which could be purified or used as in planta formulation, also suitable for oral therapeutic vaccination.

Bovine papillomavirus E5 oncoprotein binds to the activated form of the platelet-derived growth factor beta receptor in naturally occurring bovine urinary bladder tumours.

Studies regarding the functions of the bovine papillomavirus (BPV) E5 oncoprotein in vivo are lacking and no E5-mediated mechanism underlying epithelial carcinogenesis is known. We have shown that BPV-2 DNA is present in the majority of naturally occurring urinary bladder tumours of cattle and that E5 is expressed in the cancer cells. Here we show that the interaction between the platelet-derived growth factor (PDGF) beta receptor and BPV E5, described in vitro in cultured cells, takes place in vivo in bovine urinary bladder cancers. In these cancers, E5 and PDGF beta receptor colocalize, as shown by confocal microscopy, and physically interact, as shown by coimmunoprecipitation. Furthermore, the PDGF beta receptor associated with E5 is highly phosphorylated, suggesting the functional activation of the receptor upon E5 interaction. Our results demonstrate, for the first time, that E5-PDGF beta receptor interaction occurs during the natural history of bovine urinary bladder tumours, suggesting an important role for E5 in carcinogenesis. Finally, the system provides a suitable animal model of papillomavirus-associated cancer to test therapeutic vaccination against E5. Successful bladder tumour regression would provide a valuable model for therapeutic vaccination against papillomavirus-associated tumours.

Spontaneous ocular squamous cell carcinoma in twin goats: pathological and biomolecular studies.

Ocular squamous cell carcinoma (OSCC) is a well-characterized tumour occurring spontaneously in cattle and other mammalian species but not previously reported in the goat. This report describes the histological features of well-differentiated OSCCs in twin goats. Biomolecular investigations led to the identification of Papillomavirus-related DNA sequences within the neoplastic ocular parenchyma of both animals, but immunohistochemical and ultrastructural studies failed to demonstrate viral particles. A putative role of Papillomavirus in the aetiology of OSCC is discussed, together with other possible causative factors.

Presence of HPV in head and neck tumours: high prevalence in tonsillar localization.

Human papillomavirus (HPV) seems to be involved in head and neck carcinogenesis. To investigate this association, viral presence and expression were analysed by polymerase chain reaction (PCR)-based methods and correlated to tumour localization, clinical-pathological aspects, and alcohol and tobacco exposure in 65 patients. HPV DNA was found in 16 cases (24.6%); the HPV types detected were: HPV16 (10 cases), HPV 6 (3 cases) HPV 33, 35, and 58 (one case each). The tonsil was the location with the highest HPV positivity (6/8, 75%). This percentage was significantly higher than that found in tumours from any other site (P<0.01). Viral transcripts of early regions were detected in all HPV16 positive tumours. HPV status was not related to age, gender, tumour stage or grade, and use of alcohol and/or tobacco. The results suggest that HPV16 is actively involved in the genesis of a subset of head and neck cancers and that the tonsillar localization may be considered a hot spot for viral transformation.

Bovine papillomavirus type 4 in oesophageal papillomas of cattle from the south of Italy.

Oesophageal papillomas are known to occur in cattle infected with bovine papillomavirus type 4 (BPV-4), and BPV-4 papillomas may undergo malignant progression in cattle that feed on bracken fern. In the south of Italy, where bracken fern is common, examination of 1133 slaughterhouse cattle aged 4-12 years revealed oesophageal lesions (single or multiple peduncuolated proliferations, or mucosal thickening) in 147 (13%). These two types of lesion were consistent with exophytic and inverted papilloma, respectively. BPV-4 was detected by polymerase chain reaction (PCR) analysis in >60% of the samples in which oesophageal papilloma was diagnosed histopathologically. Nucleotide sequencing of the PCR amplicons confirmed the presence of BPV-4 in the papillomas. This is the first report of such infections in a European country other than Britain.

HPV5b variant in a neoplastic lesion of an Italian patient affected by epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) can be defined as a genetic disorder that determines a lifelong infection of the skin by human Papillomaviruses (HPV). The benign lesions contain different HPVs; whereas in the tumors HPV types 5, 8, and, much less frequently, types 14, 17, 20 and 47 can be detected. Variants of HPV5 have been recognized on the basis of the genetic heterogeneity of the E6 open reading frame. We report a typical case of EV in which the presence and expression of the HPV type 5 were clearly detectable. Direct sequence analysis demonstrated a perfect homology with the sequence of the HPV5b variant. This variant was first isolated in a Japanese patient and thereafter in a Polish one. Its presence in the Italian patient indicates that the same variant can be detected in different geographic areas and therefore that some cellular genes may exert a selection pressure so strong as to induce the emergence of this stable HPV5b infectious variant.

[Radioimmuno-guided surgery (RIGS) in breast disease]. / La chirurgia radioimmunoguidata (RIGS) in patologia mammaria.

Radioimmunoguided surgery is a new technology capable of detecting minimal neoplastic lesions using radiocolloids. We used this technique in two fields: to detect sentinel lymph nodes in breast cancer and to remove non-palpable breast lesions. Radioimmunoguided surgery was employed in 135 women; in 32 for sentinel lymph nodes and in 103 for radioguided occult lesion localization using a radioactive tracer (Technetium Tc99m) injected subdermally for sentinel nodes, or near to the non-palpable lesions under US guidance. In our experience these two applications of radioimmunoguided surgery are useful and accurate for determining the nature of lesions and for providing definitive treatment in a single surgical intervention.

Egg yolk antibodies against the E7 oncogenic protein of human papillomavirus type 16.

The E7 oncoprotein is the major transforming protein of Human Papillomavirus type 16 (HPV16) and the most abundant in cervical neoplasia. In this study we report the production of polyclonal antibodies to HPV16 E7 in rabbits and hens. The produced antibodies recognised the denatured and native form of HPV16 E7 protein by Western Blot, and immunoprecipitation. Epitope mapping demonstrated that hen antibodies reacted with a greater number of antigen determinants than the rabbit antibodies. In immunocytochemistry only hen antibodies were able to localize the E7 protein in a HPV positive cell line and in the high-grade squamous intraepithelial lesions, suggesting their possible usefulness in the screening of clinical samples.