Characterisation of the immune microenvironment of primary breast cancer and brain metastasis reveals depleted T-cell response associated to ARG2 expression.

BACKGROUND: Immune checkpoint inhibition is an established treatment in programmed death-ligand 1 (PD-L1)-positive metastatic triple-negative (TN) breast cancer (BC). However, the immune landscape of breast cancer brain metastasis (BCBM) remains poorly defined. MATERIALS AND METHODS: The tumour-infiltrating lymphocytes (TILs) and the messenger RNA (mRNA) levels of 770 immune-related genes (NanoString™, nCounter™ Immuno-oncology IO360) were assessed in primary BCs and BCBMs. The prognostic role of ARG2 transcripts and protein expression in primary BCs and its association with outcome was determined. RESULTS: There was a significant reduction of TILs in the BCBMs in comparison to primary BCs. 11.5% of BCs presented a high immune infiltrate (hot), 46.2% were altered (immunosuppressed/excluded) and 34.6% were cold (no/low immune infiltrate). 3.8% of BCBMs were hot, 23.1% altered and 73.1% cold. One hundred and twelve immune-related genes including PD-L1 and CTLA4 were decreased in BCBM compared to the primary BCs (false discovery rate <0.01, log2 fold-change >1.5). These genes are involved in matrix remodelling and metastasis, cytokine-chemokine signalling, lymphoid compartment, antigen presentation and immune cell adhesion and migration. Immuno-modulators such as PD-L1 (CD274), CTLA4, TIGIT and CD276 (B7H3) were decreased in BCBMs. However, PD-L1 and CTLA4 expression was significantly higher in TN BCBMs (P = 0.01), with CTLA4 expression also high in human epidermal growth factor receptor 2-positive (P < 0.01) compared to estrogen receptor-positive BCBMs. ARG2 was one of four genes up-regulated in BCBMs. High ARG2 mRNA expression in primary BCs was associated with worse distant metastasis-free survival (P = 0.038), while ARG2 protein expression was associated with worse breast-brain metastasis-free (P = 0.027) and overall survival (P = 0.019). High transcript levels of ARG2 correlated to low levels of cytotoxic and T cells in both BC and BCBM (P < 0.01). CONCLUSION: This study highlights the immunological differences between primary BCs and BCBMs and the potential importance of ARG2 expression in T-cell depletion and clinical outcome.

Dual glutathione-S-transferase-θ1 and -µ1 gene deletions determine imatinib failure in chronic myeloid leukemia.

Approximately 40% of patients with chronic myeloid leukemia (CML) receiving imatinib fail treatment. There is an increased risk of CML in subjects with (i) deletions of genes encoding glutathione-S-transferase (GST)-θ1 (GSTT1) and -µ1, (GSTM1) and (ii) the GST-π1 (GSTP1) single-nucleotide polymorphism (SNP) Ile105Val (GSTP1*B; rs1695); however, their effects on imatinib treatment outcome are not known. Here, we assess the role of these GSTs in relation to imatinib treatment outcome in 193 CML patients. Deletion of GSTT1 alone, or in combination with deletion of the GSTM1 gene, significantly increased the likelihood of imatinib failure (P = 0.021 and P < 0.001, respectively). The GSTP1*B SNP was not associated with time to imatinib failure. Losses of the GSTT1 and GSTM1 genes are therefore important determinants of imatinib failure in CML. Screening for GSTT1 and GSTM1 gene deletions during diagnosis may identify patients who may be better treated using an alternative therapy.

Nilotinib concentration in cell lines and primary CD34(+) chronic myeloid leukemia cells is not mediated by active uptake or efflux by major drug transporters.

Imatinib mesylate and nilotinib are highly effective at eradicating the majority of chronic myeloid leukemia (CML) cells; however, neither agent induces apoptosis of primitive CML CD34(+) cells. One possible explanation is that CD34(+) cells do not accumulate sufficient intracellular drug levels because of either inadequate active uptake or increased efflux. To determine the interaction of nilotinib with major clinically implicated drug transporters, we analyzed their interactions with MDR1 (ABCB1), MRP1 (ABCC1), ABCG2 (BCRP) and human organic cation transporter (hOCT)1 in CML cell lines and primitive (CD34(+)) primary CML cells. Nilotinib is neither dependent on active import by hOCT1, nor effluxed through the ATP-binding cassette transporters analyzed. Indeed, we found nilotinib to be an inhibitor of hOCT1, MDR1 and ABCG2. The efflux transporters MDR1, MRP1 and ABCG2 are expressed on CML CD34(+) cells at 13.5, 108 and 291% of control, respectively, although hOCT1 expression was absent; however, inhibition of efflux transporter activity did not potentiate the effect of nilotinib on apoptosis, Bcr-Abl inhibition or CML CD34(+) cell proliferation. Therefore, we have found no evidence for either active uptake of nilotinib through hOCT1 or efflux through MDR1, MRP1 or ABCG2, and it is therefore unlikely that these transporters will have any effect on the clinical response to this drug.

Expression of the uptake drug transporter hOCT1 is an important clinical determinant of the response to imatinib in chronic myeloid leukemia.

Some chronic myeloid leukemia (CML) patients do not respond to imatinib, whereas others lose an initial response. To identify potential imatinib failures, we investigated the expression of imatinib uptake transporter (human organic cation transporter 1; hOCT1) and efflux transporters (ATP-binding cassette transporters ABCB1, ABCG2, and ABCC1) using real-time quantitative reverse transcription-polymerase chain reaction in 70 CML patients. Patients with high pretreatment hOCT1 expression had superior complete cytogenetic response (CCR) rates (P=0.008), progression-free and overall survival (P=0.01 and 0.004). Pretreatment ABCB1, ABCG2, and ABCC1 levels did not correlate with treatment outcome. Regression analysis demonstrated that pretreatment hOCT1 expression was the most powerful predictor of CCR achievement at 6 months (P=0.002). Imatinib uptake into a CML cell line with high hOCT1 expression was greater than into those with modest or low expression (P=0.002). The expression of hOCT1, but not efflux transporters, is important in determining the clinical response to imatinib.

Gene therapy for prostate cancer: current strategies and new cell-based approaches.

Prostate cancer is the most commonly diagnosed cancer in adult males in the Western world. It accounts for one in ten cancer cases and is the second leading cause of cancer death in men, after lung cancer. A number of curative treatments are available for patients with localized prostate cancer such as radical prostatectomy, radiotherapy, or brachytherapy. However, a proportion of these men will develop progressive disease, and some will present de novo with advanced and metastatic prostate cancer, which is amenable to palliation only with androgen-withdrawal therapy. Most of these patients will eventually develop hormone refractory disease which is incurable, and for whom gene therapy, if feasible may develop as an alternative treatment option. In this review we discuss the gene therapy vectors and strategies that are currently in use, new cell-based approaches, discuss their advantages and disadvantages, and review the potential or proven pre-clinical and clinical efficacy in prostate cancer models/patients.

Human papillomavirus in pterygium.

AIM: To determine the prevalence and significance of human papillomaviral types in conjunctival pterygia. METHODS: Polymerase chain reaction technology was used to identify the presence of human papillomavirus (HPV) in 10 formalin fixed paraffin embedded pterygia samples. 10 conjunctival papillomas were used as positive controls. 20 conjunctival samples, 10 with primary acquired melanosis and 10 with malignant melanoma, were used as negative controls. Sample subgroups were of equal sex, race, and age distribution to eliminate bias. All samples were further analysed (for 21 HPV types) using dot-blot hybridisation techniques. RESULTS: HPV was identified in 90% of the conjunctival papillomas, 50% of the pterygia samples, but no HPV was detected in the negative control group. Two pterygia showed type 6, two type 11, and one type 16. These three HPV types were also detected in papillomas. CONCLUSION: These results suggest that HPV may be involved in the pathogenesis of pterygia and that broadly the same HPV types are found in pterygia and in papillomas. Persistent conjunctival HPV may possibly play a part in the recurrence of pterygia post excision but further larger studies are required to elucidate this hypothesis.

Variation in the E2-binding domain of HPV 16 is associated with high-grade squamous intraepithelial lesions of the cervix.

Human papillomaviruses (HPV) are strongly associated with cervical intraepithelial neoplasia (CIN) and invasive cancer mainly through the action of the E6 and E7 viral proteins, transcription of which is down-regulated by the E2 protein. To test the hypothesis that HPV 16 E2 variation is important in the development of high-grade squamous neoplasia of the cervix, we carried out a cross-sectional analysis of low-grade and high-grade squamous intraepithelial lesions (SILs) for specific mutations in the HPV 16 E2 gene and for E2 gene disruption in these regions. Isolates were also analysed for the HPV 16 350T-G variant. 22 of 178 low-grade SILs and 43 of 61 high-grade SILs examined, contained HPV 16. No relationship was found between the E6 350T-G variant, or the E2 hinge region 3410C-T variant, and lesion grade. However, disruption of the regions of E2 analysed was significantly more frequent in high-grade lesions, and there was a significant association between the 3684C-A variant in the E2 DNA binding domain and high-grade histology suggesting that this variant may be important in progression to high-grade intraepithelial disease.

Human papillomavirus variants and squamous neoplasia of the cervix.

Human papillomaviruses (HPVs) play a central role in the aetiology of cervical neoplasia. However, only a small proportion of cervical intraepithelial lesions infected with high-risk HPVs will progress to invasive cervical carcinoma, which indicates the involvement of additional factors. An important emerging viral factor is naturally occurring intratypic sequence variation. Such variation has been used to study the geographical spread of HPVs, but there is increasing evidence that it may be important in determining the risk of development of neoplastic disease. The collected data indicate that different HPV variants have altered biochemical and biological properties and represent an additional risk factor in the development of squamous intraepithelial lesions and invasive carcinoma of the cervix. This may be relevant not only to the biology of HPV infection and its association with squamous neoplasia, but also to the use of HPV typing in clinical practice.

Differential expression of p53 and p21 in low grade cervical squamous intraepithelial lesions infected with low, intermediate, and high risk human papillomaviruses.

BACKGROUND: Basal cell tetrasomy in low grade squamous intraepithelial lesions of the cervix is associated with infection by high and intermediate risk but not low risk human papillomaviruses (HPVs). It is known that the viral E6 and E7 proteins interact with p53 and p21, respectively, altering cell cycle control and leading to chromosomal instability. In this study, p53 and p21 expression was analyzed in disomic and tetrasomic low grade squamous intraepithelial lesions infected with a wide range of HPV types. METHODS: HPV identification and typing was performed using both in situ hybridization and the polymerase chain reaction followed by dot blot hybridization with specific HPV probes. Interphase cytogenetic analysis using centromeric chromosomal probes also performed was to identify numeric chromosomal abnormalities. The expression of p53 and p21 was studied by immunohistochemistry using monoclonal antibodies specific for these proteins. RESULTS: Increased expression of p53 and p21 was more widespread in lesions infected with low risk than with intermediate/high risk HPV types (p53, P < 0.001; p21, P < 0.01). p53 status correlated with p21 expression when analyzed according to the distribution of expression by using 3 groups, focal, regional, and diffuse (Pearson coefficient, r = 0.47, P < 0.001). In the lesions infected with intermediate/high risk HPVs, expression of p53 was significantly decreased or completely absent in tetrasomic areas, whereas expression of p21 was similar in both disomic and tetrasomic regions. CONCLUSIONS: The authors' data suggest that low, intermediate, and high risk HPVs have different effects on p53 and p21 protein expression, and that the induction of numeric chromosomal abnormalities by intermediate/high risk HPVs may be related to altered expression of p53.

"High risk" HPV types are frequently detected in potentially malignant and malignant oral lesions, but not in normal oral mucosa.

Studies on the involvement of the human papillomavirus (HPV) in initiation and progression of oral neoplasia have generated conflicting results. The observed discrepancy is attributable mainly to the varying sensitivity of the applied methodologies and to epidemiologic factors of the examined patient groups. To evaluate the role of HPV in oral carcinogenesis, we analyzed 53 potentially neoplastic and neoplastic oral lesions consisting of 29 cases of hyperplasia, 5 cases of dysplasia, and 19 cases of squamous cell carcinomas, as well as 16 oral specimens derived from healthy individuals. A highly sensitive nested polymerase chain reaction (PCR) assay was used, along with type-specific PCR, restriction fragment length polymorphism analysis, dot blotting, and nonisotopic in situ hybridization. Nested PCR revealed the presence of HPV DNA in 48 of the 53 (91%) pathologic samples analyzed, whereas none (0%) of the normal specimens was found to be infected. Positivity for HPV was independent of histology and the smoking habits of the analyzed group of patients. At least one "high risk" type, such as HPV 16, 18, and 33, was detected by type-specific PCR in 47 (98%) infected specimens, whereas only 1 (2%) squamous cell carcinoma was solely infected by a "low risk" type (HPV 6). HPV 16 was the prevailing viral type, being present in 71% of infected cases. Single HPV 16 and HPV 18 infections were confirmed by restriction fragment length polymorphism. HPV 58 was detected by dot blotting in three hyperplastic lesions. HPV positivity and genotyping were further confirmed, and the physical status of this virus was evaluated by nonisotopic in situ hybridization. Diffuse and punctate signals, indicative of the episomal and integrative pattern of HPV infection, were observed for low- and high-risk types, respectively. Our findings are suggestive of an early involvement of high-risk HPV types in oral carcinogenesis.

Basal keratinocyte tetrasomy in low-grade squamous intra-epithelial lesions of the cervix is restricted to high and intermediate risk HPV infection but is not type-specific.

Human papillomavirus (HPV) infection appears to be an early event in cervical carcinogenesis with additional abnormalities being required for biological transformation. We have analysed 179 low-grade cervical squamous intra-epithelial lesions (SILs) and 15 normal cervices for the presence of HPV using both in situ hybridization and polymerase chain reaction (PCR). PCR was performed with GP5+/GP6+ primers followed by hybridization using probes for low (HPV 6, 11, 40, 42, 43, 44), intermediate (HPV 31, 33, 35, 39, 51, 52, 58, 59, 66 and 68) and high-risk HPVs (HPV 16, 18, 45 and 56). Interphase cytogenetic analysis using pericentromeric probes for chromosomes 1, 3, 4, 6, 10, 11, 17, 18 and X was also performed to identify numerical chromosomal abnormalities. Tetrasomy of all nine chromosomes was identified within basal keratinocytes, was restricted to epithelia infected with high risk (17 of 46) or intermediate risk (23 of 83) HPVs but was not HPV type-specific. Tetrasomy was not identified in any of the epithelia infected with low risk HPVs (n = 62). These numbers include multiple infection. These findings indicate that the induction of tetrasomy is a property restricted to high and intermediate-risk HPV types but that it is not type-specific. The factors governing which lesions will develop this abnormality are as yet unclear.

p53 codon 72 ARG/PRO polymorphism is not related to HPV type or lesion grade in low- and high-grade squamous intra-epithelial lesions and invasive squamous carcinoma of the cervix.

A polymorphism at codon 72 of the p53 gene, resulting in either an arginine or a proline residue in the protein, has been reported to affect the susceptibility of p53 to human papillomavirus (HPV) E6-mediated degradation in cultured cells. However, the relevance of this polymorphism to naturally occurring HPV infection is unclear. Therefore, we analysed its relationship to infecting HPV type and lesion grade in a series of low- and high-grade squamous intra-epithelial lesions (SILs) and invasive carcinoma of the cervix. DNA extracted from morphologically characterised, paraffin-embedded tissues (30 normal cervices, 118 low-grade SILs, 118 high-grade SILs and 43 invasive carcinomas) was examined for the presence and type of HPV DNA, and the p53 genotype was identified by both allele-specific PCR and PCR-restriction fragment length polymorphism. There was no significant relationship between the frequency of p53 genotypes and either HPV type or lesion grade. Our data do not support the hypothesis that this p53 polymorphism is involved in the development of high-grade squamous cervical disease in this population.

Detection of Epstein-Barr virus and human papillomavirus in nasopharyngeal carcinoma by the polymerase chain reaction technique.

We used the PCR technique to detect the Epstein-Barr virus (EBV) and human papillomavirus (HPV) DNA in paraffin-embedded tissues from Greek patients with nasopharyngeal carcinoma (NPC). The oligonucleotide primers used for the detection of EBV amplify a 375-bp long sequence from the EcoRI B fragment of the viral genome, whereas for HPV the primers amplify a 151-bp long sequence of the viral genome. The PCR products were analysed by agarose gel electrophoresis and visualised by UV illumination after staining with ethidium bromide. Sixty-three specimens were examined. EBV specific sequence was amplified in 20 (32%) and HPV in 12 (19%) out of the 63 samples. There was no co-infection with EBV and HPV. Although there is a high correlation of EBV infection with poorly differentiated NPC in patients from Southern China and South-East Asia, the restricted distribution suggests genetic or environmental cofactors in the development of the neoplasm. Our results confirm this suggestion since there was only a 32% correlation of EBV with NPC in Greece. HPV may also be involved in the carcinogenesis of EBV-negative squamous cell nasopharyngeal carcinomas.

The presence of herpesviruses in pterygium.

Pterygium is a chronic disease of unknown origin and pathogenesis. It is a vision threatening disease where surgical excision is effective. We examined surgically excised symptomatic pterygia for the presence of herpesviruses such as cytomegalovirus (CMV), herpes simplex virus (HSV) and Epstein-Barr virus (EBV) DNA using the polymerase chain reaction (PCR) technique. Samples of normal conjuctival tissue from limpus at 12 or 6 hours were excised in some of the eyes treated; they were used as controls. HSV DNA was detected in 9 and CMV DNA in 8 out of the 20 examined samples. In 3 out of the 20 examined samples both HSV and CMV DNA were detected whereas EBV DNA was not found in any of the examined samples. These results suggest that HSV and CMV may contribute to the pathogenesis of pterygium.

Detection of the epstein-barr-virus by the polymerase chain-reaction in immunosuppressed and immunocompromised patients.

DNA extracted from the blood of immunosuppressed and immunocompromised individuals and from patients with infectious mononucleosis, leukaemias and lymphomas were studied using the Polymerase Chain Reaction (PCR) technique. The oligonucleotide primers used for the detection of the Epstein-Barr virus (EBV) amplify a 375bp sequence from the EcoRI B fragment of the viral genome. EBV specific sequences were amplified from the blood samples of 18 out of 65 patients, most of which were transplant patients (9 out of 31). The results confirmed the association of EBV with clinical disorders in immunodeficient and immunocompromised patients and the importance of PCR method in routine diagnosis.

Detection of human cytomegalovirus and epstein-barr-virus by the polymerase chain-reaction in patients with Beta-thalassemia.

Infections caused by Human Cytomegalovirus (HCMV) and Epstein-Barr virus (EBV) are common in multiple transfused patients, such as patients with beta-thalassaemia. The ability of the Polymerase Chain Reaction (PCR) to amplify HCMV and EBV DNA from blood and other samples makes this technique a valuable diagnostic tool for the detection of both viruses in the early stages of the infection. PCR was used for the amplification of a 435 bp region of the immediate early-1 (IE-1) gene of HCMV and a 375 bp sequence from the EcoRI B fragment of EBV genome. Blood samples from 80 patients with beta-thalassaemia were examined. HCMV was found in 14 and EBV in 12 patients. The results obtained confirm the implications of HCMV and EBV in the diagnosis of viral infections in multiple transfused patients as well as the importance of PCR technique as a valuable diagnostic tool.

Detection of hsv, CMV and ebv by the polymerase chain-reaction technique in patients with inflammatory eye-diseases.

Herpes simplex virus (HSV), cytomegalovirus (CMV) and Epstein-Barr virus (EBV) have been recognized as pathogenic agents of intraocular inflammatory conditions. The ability of the Polymerase Chain Reaction (PCR) technique to amplify HSV, CMV and EBV DNA from aqueous specimens makes this technique a valuable diagnostic tool for the detection of these viral pathogens in patients with ophthalmic lesions. We used PCR for the amplification of a 476 bp long sequence from the pol I gene of HSV genome, a 435 bp region of the immediate early-1 (IE-1) gene of CMV and a 375 bp sequence from the EcoRI B fragment of EBV genome. We examined 22 aqueous humour specimens from patients with uveitis and retinitis, inflammatory eye diseases, diagnosed clinically. We found HSV in 4 (18.2%), CMV in 6 (27.3%) and EBV in 1 (4.5%) out of the 22 examined patients. None of the 22 examined samples was found to be infected with more than one of the examined viral pathogens. These data confirm the implications of the members of Herpesvirus family in inflammatory inner eye diseases and the importance of PCR technique as a diagnostic tool in clinical virology.