Abscopal Effect and Drug-Induced Xenogenization: A Strategic Alliance in Cancer Treatment?

The current state of cancer treatment is still far from being satisfactory considering the strong impairment of patients' quality of life and the high lethality of malignant diseases. Therefore, it is critical for innovative approaches to be tested in the near future. In view of the crucial role that is played by tumor immunity, the present review provides essential information on the immune-mediated effects potentially generated by the interplay between ionizing radiation and cytotoxic antitumor agents when interacting with target malignant cells. Therefore, the radiation-dependent abscopal effect (i.e., a biological effect of ionizing radiation that occurs outside the irradiated field), the influence of cancer chemotherapy on the antigenic pattern of target neoplastic cells, and the immunogenic cell death (ICD) caused by anticancer agents are the main topics of this presentation. It is widely accepted that tumor immunity plays a fundamental role in generating an abscopal effect and that anticancer drugs can profoundly influence not only the host immune responses, but also the immunogenic pattern of malignant cells. Remarkably, several anticancer drugs impact both the abscopal effect and ICD. In addition, certain classes of anticancer agents are able to amplify already expressed tumor-associated antigens (TAA). More importantly, other drugs, especially triazenes, induce the appearance of new tumor neoantigens (TNA), a phenomenon that we termed drug-induced xenogenization (DIX). The adoption of the abscopal effect is proposed as a potential therapeutic modality when properly applied concomitantly with drug-induced increase in tumor cell immunogenicity and ICD. Although little to no preclinical or clinical studies are presently available on this subject, we discuss this issue in terms of potential mechanisms and therapeutic benefits. Upcoming investigations are aimed at evaluating how chemical anticancer drugs, radiation, and immunotherapies are interacting and cooperate in evoking the abscopal effect, tumor xenogenization and ICD, paving the way for new and possibly successful approaches in cancer therapy.

Predicting Ovarian Activity in Women Affected by Early Breast Cancer: A Meta-Analysis-Based Nomogram.

BACKGROUND: The assessment of ovarian reserve in premenopausal women requiring anticancer gonadotoxic therapy can help clinicians address some challenging issues, including the probability of future pregnancies after the end of treatment. Anti-Müllerian hormone (AMH) and age can reliably estimate ovarian reserve. A limited number of studies have evaluated AMH and age as predictors of residual ovarian reserve following cytotoxic chemotherapy in breast cancer patients. MATERIALS AND METHODS: To conduct a meta-analysis of published data on this topic, we searched the medical literature using the key MeSH terms "amenorrhea/chemically induced," "ovarian reserve," "anti-Mullerian hormone/blood," and "breast neoplasms/drug therapy." Preferred Reporting Items for Systematic Reviews and Meta-Analyses statements guided the search strategy. U.K. National Health Service guidelines were used in abstracting data and assessing data quality and validity. Area under the receiver operating characteristic curve (ROC/AUC) analysis was used to evaluate the predictive utility of baseline AMH and age model. RESULTS: The meta-analysis of data pooled from the selected studies showed that both age and serum AMH are reliable predictors of post-treatment ovarian activity in breast cancer patients. Importantly, ROC/AUC analysis indicated AMH was a more reliable predictor of post-treatment ovarian activity in patients aged younger than 40 years (0.753; 95% confidence interval [CI]: 0.602-0.904) compared with those older than 40 years (0.678; 95% CI: 0.491-0.866). We generated a nomogram describing the correlations among age, pretreatment AMH serum levels, and ovarian activity at 1 year from the end of chemotherapy. CONCLUSION: After the ongoing validation process, the proposed nomogram may help clinicians discern premenopausal women requiring cytotoxic chemotherapy who should be considered high priority for fertility preservation counseling and procedures.

Chemotherapy-induced ovarian toxicity in patients affected by endocrine-responsive early breast cancer.

Cytotoxic chemotherapy may variably affect ovarian function depending on age and ovarian reserve at diagnosis, type of chemotherapy and use of tamoxifen. Ascertaining whether a premenopausal patient with endocrine-responsive early breast cancer and chemotherapy-induced amenorrhea has reached menopause is essential not only in order to provide accurate information on residual fertility, but also to appropriately prescribe endocrine therapy. Indeed, aromatase inhibitors are contraindicated in women with residual ovarian reserve. However, the diagnosis of menopause in patients with chemotherapy-induced amenorrhea is challenging, since clinical features, follicle-stimulating hormone and estradiol levels may be inaccurate to this aim. Recent studies demonstrated that the anti-müllerian hormone may improve the assessment of ovarian reserve residual to chemotherapy in women with early breast cancer. Herein, we review the incidence of amenorrhea and menopause induced by cytotoxic chemotherapy in women affected by early breast cancer and the suggested mechanisms that sustain these side-effects. Furthermore, it has been scrutinized the potential of new markers of ovarian reserve that may facilitate the selection of appropriate endocrine treatment for premenopausal women who develop amenorrhea following adjuvant chemotherapy for early breast cancer.

Endocrine side effects induced by immune checkpoint inhibitors.

CONTEXT: In recent years, progress has been made in cancer immunotherapy by the development of drugs acting as modulators of immune checkpoint proteins, such as the cytotoxic T-lymphocyte antigen-4 (CTLA4) and programmed death-1 (PD-1), two co-inhibitory receptors that are expressed on T cells upon activation. These molecules play crucial roles in maintaining immune homeostasis by down-regulating T-cell signaling, thereby preventing unbridled T-cell proliferation while maintaining tolerance to self-antigens, such as tumor-associated antigens. CTLA4 blockade through systemic administration of the CTLA4-blocking antibody ipilimumab was shown to confer significant survival benefit and prolonged stable disease in patients affected by advanced cutaneous melanoma. Other immune checkpoint inhibitors are under clinical evaluation. However, immune checkpoint blockade can lead to the breaking of immune self-tolerance, thereby inducing a novel syndrome of autoimmune/autoinflammatory side effects, designated as "immune-related adverse events," mainly including rash, colitis, hepatitis, and endocrinopathies. DATA ACQUISITION: We searched the medical literature using the words "hypophysitis," "hypopituitarism," "thyroid," "adrenal insufficiency," and "endocrine adverse events" in association with "immune checkpoint inhibitors," "ipilimumab," "tremelimumab," "PD-1," and "PD-1-L." EVIDENCE SYNTHESIS: The spectrum of endocrine disease experienced by patients treated with ipilimumab includes most commonly hypophysitis, more rarely thyroid disease or abnormalities in thyroid function tests, and occasionally primary adrenal insufficiency. Hypophysitis has emerged as a distinctive side effect of CTLA4-blocking antibodies, establishing a new form of autoimmune pituitary disease. This condition, if not promptly recognized, may be life-threatening (due to secondary hypoadrenalism). Hypopituitarism caused by these agents is rarely reversible, and prolonged or lifelong substitutive hormonal treatment is often required. The precise mechanism of injury to the endocrine system triggered by these drugs is yet to be fully elucidated. CONCLUSIONS: Although reports of endocrine side effects caused by cancer immune therapy are abundant, their exact prevalence and mechanism are unclear. Well-designed correlative studies oriented to finding and validating predictive factors of autoimmune toxicity are urgently needed.

Circulating tumor cells in colorectal cancer patients.

The availability of sensitive methods has allowed the detailed study of circulating tumor cells only recently. Evolving evidence support the prognostic and predictive role of these cells in patients affected by several solid tumors, including colorectal cancer. Ongoing studies are aimed at confirming that the molecular characterization of circulating tumor cells in peripheral blood and in bone marrow of patients is a powerful tool to improve the patient risk-stratification, to monitor activity of the drugs, to develop more appropriate targeted therapies and tailored treatments. In parallel, results from these correlative studies promise to gain a better biological understanding of the metastatic process. The clinical utility of the detection of circulating tumor cells in patients affected by colorectal cancer is still hampered by a number of specific hurdles. Improvement in sensitivity and specificity of the available methods of detection, standardization of these methods and functional characterization of circulating tumor cells in well designed and statistically well powered studies are the key steps to reach these ambitious objectives in colorectal cancer patients as well.

Hypophysitis induced by monoclonal antibodies to cytotoxic T lymphocyte antigen 4: challenges from a new cause of a rare disease.

Specific human monoclonal antibodies antagonize cytotoxic T-lymphocyte antigen 4 (anti-CTLA-4 mAbs), a negative regulator of the immune system, inducing unrestrained T-cell activation. In patients with advanced or metastatic melanoma, one of these agents, ipilimumab, produced considerable disease control rates and, for the first time, a clear improvement in overall survival outcomes. However, accumulating clinical experience with anti-CTLA-4 mAbs identified a novel syndrome of autoimmune and autoinflammatory side effects, designated as "immune-related adverse events," including mainly rash, colitis, and hepatitis. Autoimmune hypophysitis has emerged as a distinctive side effect induced by anti-CTLA-4 mAbs. This condition may be life threatening because of adrenal insufficiency if not promptly recognized, but it may easily be diagnosed and treated if clinically suspected. Hypopituitarism caused by these agents is rarely reversible and prolonged or life-long substitutive hormonal treatment is often required. The precise mechanism of injury to the pituitary triggered by anti-CTLA-4 mAbs is yet to be fully elucidated.

Recognizing menopause in women with amenorrhea induced by cytotoxic chemotherapy for endocrine-responsive early breast cancer.

Cytotoxic anticancer treatment may induce amenorrhea or menopause to a variable extent. These side effects may not only impair or impede fertility but also cause sexual dysfunction, bone loss, and menopausal symptoms, with a strikingly negative effect on quality of life in many women. Aromatase inhibitors (AIs) are a recommended adjuvant endocrine treatment option in postmenopausal patients affected by early breast cancer (EBC) but are contraindicated in premenopausal women and in those with residual ovarian function. Women over 40 years of age with chemotherapy-induced amenorrhea (CIA) and routine hormonal levels consistent with menopause may receive an AI as adjuvant endocrine treatment. For these women, the tools available to identify menopause do not appear to be completely reliable. This review focused on the pathophysiology of ovarian toxicity induced by cytotoxic agents and on potentially useful methods to diagnose chemotherapy-induced menopause in patients treated with adjuvant chemotherapy for endocrine-responsive EBC. Moreover, practical approaches are proposed to distinguish true menopausal women, who would benefit from AIs, from those with transient or persistent CIA.

Detection of circulating tumor cells is improved by drug-induced antigen up-regulation: preclinical and clinical studies.

(51)Cr-prelabelled colon cancer cells (simulating 'circulating tumor cells', CTCs) were added to human peripheral blood and exposed to staurosporine (ST) to increase carcinoembryonic antigen (CEA) expression. CTCs were captured with immunomagnetic beads coated with Ber-EP4 monoclonal antibody, recognizing the common epithelial antigen present in the majority of cancer cells of epithelial origin, with capture efficiency of more than 80%. Moreover, ST treatment increased CEA expression without compromising Ber-EP4 capture efficiency. In a pilot clinical study on 37 patients, CTCs were captured using Ber-EP4 beads, and recognized by RT-PCR set for CEA or cytokeratin-19 (CK) mRNA detection. The results showed that: (a) the percentage of CEA-positive CTCs (CTC(CEA), 54.1%) was lower than that of CK-positive CTCs (CTC(CK), 70.3%); (b) in vitro ST treatment converted a significant number of CTC(CEA)-negative into CTC(CEA)-positive cases. Therefore, immunomagnetic capture combined with exposure to ST provides a feasible and sensitive technique for the detection of functionally-active CTCs responsive to ST-mediated CEA up-regulation.

Combined effects of 5-fluorouracil, folinic acid and oxaliplatin on the expression of carcinoembryonic antigen in human colon cancer cells: pharmacological basis to develop an active antitumor immunochemotherapy.

BACKGROUND: Five-fluorouracil (FU), mainly associated with leucovorin (L), plays an essential role in chemotherapy of colorectal carcinoma. Moreover, FU +/- L has been found to increase the expression of tumor-associated carcinoembryonic antigen (CEA), that may be an important target in therapeutic protocols of active specific immunotherapy. FU + L (FUL) are frequently combined with oxaliplatin (OXA) in advanced colon cancer patients. Thus, we investigated whether FUL in combination with OXA according to 2 different schedules may influence CEA expression in human colon cancer cells in vitro. METHODS: CEA protein expression was evaluated by cytofluorimetric and western blot analysis. Relative quantification of CEA mRNA was assessed by real time RT-PCR analysis. RESULTS: Levels of CEA protein and transcript were found to be higher in FUL-treated cells than in controls. However, when target cells were exposed to OXA before but not after FUL treatment, the up-regulation of CEA was partially inhibited. CONCLUSION: These results suggest that target cells must be exposed to OXA after but not before treatment with the fluoropyrimidine in order to exploit drug-induced up-regulation of CEA. This finding appears to provide useful information to design chemo-immunotherapy protocols based on FUL + OXA, combined with host's immunity against CEA directed cancer vaccines.

Triazene compounds: mechanism of action and related DNA repair systems.

Triazene compounds of clinical interest (i.e. dacarbazine and temozolomide) are a group of alkylating agents with similar chemical, physical, antitumour and mutagenic properties. Their mechanism of action is mainly related to methylation of O(6)-guanine, mediated by methyldiazonium ion, a highly reactive derivative of the two compounds. The cytotoxic/mutagenic effects of these drugs are based on the presence of DNA O(6)-methylguanine adducts that generate base/base mismatches with cytosine and with thymine. These adducts lead to cell death, or if the cell survives, provoke somatic point mutations represented by C:G-->T:A transition in DNA helix. Triazene compounds have excellent pharmacokinetic properties and limited toxicity. Dacarbazine requires hepatic activation whereas temozolomide is spontaneously converted into active metabolite in aqueous solution at physiological pH. Moreover, temozolomide is fully active when administrated orally (100% bioavailability). The biological effects of triazene compounds and cell resistance to them depend on at least three DNA repair systems, (a) O(6)-alkylguanine-DNA-alkyltransferase, called also methyl-guanine methyl-transferase (MGMT); (b) mismatch repair (MMR), and (c) base excision repair (BER). MGMT is a small enzyme-like protein that removes small alkyl adducts from the O(6) position of DNA guanine through a stoichiometric and auto-inactivating reaction. This reaction consists in a covalent transfer of the alkyl group from the alkylated site in DNA to an internal cysteine residue of MGMT protein. High levels of MGMT are responsible for normal and tumour cell resistance to triazenes. Therefore, pre-treatment with MGMT inhibitors - i.e. O(6)-benzylguanine or O(6)-(4-bromotenyl)guanine (Lomeguatrib) - is followed by a great increase in the activity of triazenes against target cells expressing high MGMT levels. MMR is represented by a protein complex dedicated to the repair of biosynthetic errors generated during DNA replication. The MMR system recognizes base mismatches and insertion-deletion loops, cuts the nucleotide sequence containing the lesion, and restores the correct base sequence. Therefore, not only MGMT but also MMR is involved in target cell susceptibility to triazenes. However, the system does not suppress, but instead promotes the cytotoxic effects of triazenes. In fact, MMR is not able to repair the incorrect base pairing determined by treatment with triazenes and, according to a predominant hypothesis, it causes reiterated "futile" attempts of damage repair leading to the activation of cell cycle arrest and apoptosis. BER removes lesions due to cellular metabolism, or to physical or chemical agents. BER is able to repair N(7)-methylguanine and N(3)-methyladenine determined by treatment with triazenes. Therefore, triazene compounds can also kill tumour cells by a N(3)-methyladenine-mediated mechanism if BER activity is inhibited by chemical agents (i.e. PARP inhibitors). In conclusion, in selected cases, triazenes can represent a therapeutic alternative to treatment of neoplastic diseases including haematological malignancies. Moreover, the susceptibility of neoplastic cells to these compounds can be substantially increased through pharmacological modulation of the expression level and functional activity of DNA repair enzymes.

Combined effects of protein kinase inhibitors and 5-fluorouracil on CEA expression in human colon cancer cells.

Previous studies showed that 5-fluorouracil (5-FU) and Staurosporine (ST), a protein kinase inhibitor (PKI), were able to increase the expression of carcinoembryonic antigen (CEA) in human colon cancer cells. In the present study, we examined the in vitro effects of five PKIs, i.e. ST, 1-5-isoquinolinyl-sulfonyl-2-methylpiperazine (H-7), bisindolylmaleimide-I (BIS), Genistein (GEN), and Herbimycin A (HERB) alone or in combination with 5-FU on CEA expression. C22-20, a clonal subline, derived from colon cancer HT-29 line, selected for low expression of CEA, was used in our experimental model. Among the PKIs tested, only ST, at non-toxic concentrations of 5 nM, was capable of increasing the level of CEA. The other PKIs did not modify CEA expression when used either alone or in combination with 5-FU. Flow cytometric analysis showed that treatment of cells with 5-FU + ST resulted in a synergistic increase of CEA expression, being higher than that obtainable with both agents alone. Moreover, the increase of CEA expression occurred not only in membrane fractions but also in cytosolic compartments, as indicated by Western blot analysis. The present study suggests that ST-mediated induction of CEA expression in cancer cells is PKC independent and could be of potential clinical interest for the development of new diagnostic and/or immunotherapeutic approaches.

Drug-induced increase of carcinoembryonic antigen expression in cancer cells.

Most of gastrointestinal, breast and lung cancer cells express carcinoembryonic antigen (CEA). Therefore, this protein represents a suitable target for innovative diagnostic and immunotherapeutic strategies of various tumours. Presently CEA can be involved in three main approaches concerning cancer detection and therapy, i.e. (a) detection of tumour cells in the peripheral blood, bone marrow or lymph node using reverse transcriptase-polymerase chain reaction (RT-PCR)-based measurement of CEA mRNA; (b) targeting of anticancer agents or radionuclides by tumour-selective anti-CEA monoclonal antibodies (mAbs); (c) use of antitumour vaccines capable of eliciting major histocompatibility complex (MHC)-restricted immune responses against CEA-derived peptides. Actually, it has been shown that the expression of CEA can be up-regulated by pharmacological agents including, antineoplastic drugs (i.e. 5-fluorouracil), cytokines (i.e. interferons or interleukin-6), differentiating agents (i.e. sodium butyrate) and protein kinase inhibitors (i.e. staurosporine). Therefore, the use of drugs capable of increasing CEA expression, could amplify the sensitivity of diagnostic procedures that rely on CEA determination. Moreover, the same agents could increase the efficacy of vaccines based on immunogenic CEA-derived peptides restricted by the MHC. The purpose of this review is to describe several agents that are able to increase CEA expression and to discuss the rational bases for new strategies in cancer detection and therapy aimed at increasing the expression of tumour-associated antigens.

Treatment of colon and breast carcinoma cells with 5-fluorouracil enhances expression of carcinoembryonic antigen and susceptibility to HLA-A(*)02.01 restricted, CEA-peptide-specific cytotoxic T cells in vitro.

Cancer vaccines directed against tumor associate antigen (TAA) have produced encouraging results in preclinical models but not in cancer patients. A major limitation of this strategy is the relative degree of tolerance to these antigens and the low and heterogeneous tumor cell expression of TAA and major histocompatibility complex (MHC). Previous studies have shown that 5-fluorouracil (5-FU) can upregulate the expression of membrane-associated carcino-embryonic antigen (CEA), and MHC molecules in colon and breast carcinoma cell lines. We have investigated whether this drug can also enhance their sensitivity to the lytic effects of CEA-peptide specific Cytotoxic T cell lymphocytes (CTL). The CEA peptide-specific CTLs generated in our laboratory from normal HLA-A(*)02.01(+) donor PBMCs, were able to kill HLA-A(*)02.01(+)/CEA(+) breast (MCF-7-T103) and colon (HLA-A(*)02.01 gene-transfected HT-29 and C22.20) carcinoma cells in HLA-A(*)02.01 restricted manner. The treatment of target cells with 5-FU, enhanced their CEA expression and susceptibility to CTL-mediated lysis. Cold competition assays confirmed these results, thus supporting the hypothesis that immune target cell lysis and 5-FU mediated enhancement were dependent on CEA peptide presentation by cancer cells. 5-FU treatment of functionally "mature" CTL after in vitro expansion, did not reduce their cytolytic activity against MT-2 target cells but, when the anti-metabolite was added during the immune-sensitization phase, CTL generation was significantly inhibited. These results provide a rationale for investigating a possible new role of 5-FU as an immuno targeting amplifier agent in breast and colorectal cancer patients immunized with CEA-directed cancer vaccines.

Residual telomerase activity: a marker of cell survival after exposure to gamma radiation in vitro.

BACKGROUND: Residual telomerase activity (TA) could be used as a marker of malignant or normal cell survival after exposure to cytotoxic agents. Therefore TA after treatment with ionizing radiation was used in a radiosensitivity assay. MATERIALS AND METHODS: Radiosensitive MOLT-4 and relatively radioresistant Jurkat leukemia cells or normal peripheral blood mononuclear cells (MNC) were irradiated with 5-160 Gy. Cell count, MTT assay and telomerase activity were evaluated on day 3 and clonogenic assay on day 10. RESULTS: In Jurkat cells, irradiation inhibited tumor growth and total TA per culture (TA/culture), but up-regulated TA per viable cell (TA/cell). TA/culture and TA/cell were profoundly depressed by irradiation of MOLT-4 or phytohemagglutinin-activated MNC. CONCLUSION: Susceptibility of normal or neoplastic cells to high-dose radiation can be monitored by evaluating residual TA/culture. In some cases, however, difficulties in the interpretation of the results could stem from radiation-induced increase of TA/cell, as observed for the Jurkat cell line.

Pharmacological modulation of carcinoembryonic antigen in human cancer cells: studies with staurosporine.

Preliminary studies, performed in our laboratory, showed that staurosporine (ST), a protein-kinase (PK) inhibitor, increases the expression of the carcinoembryonic antigen (CEA) in a human colon cancer cell line. The present study explores the cellular and molecular effects of ST on the CEA expression in breast cancer MCF-7 line and in a number of colon cancer cell lines characterized by the different basal levels of the antigen, including two cloned sublines (i.e. C22.20 and C6.6, expressing low and high CEA levels, respectively). In all cases, increase of the CEA expression was observed at drug concentrations devoid of marked cytostatic effects (e.g. 5 nM) and was accompanied by the enhanced CEA shedding in the supernatant. Moreover, the increase of the CEA levels both occurred in the cell membranes and in the cytosolic compartments and appeared to be the result of the enhanced CEA gene transcription. Similar results have been previously obtained with interferon-gamma. However, ST treatment, different from interferon-gamma, did not up-regulate the level of the HLA class I molecules. A preliminary investigation also showed that other PKC inhibitors did not substantially modulate the CEA expression. Therefore, the biochemical mechanism underlying the effect of ST should not be correlated with that involved in the PKC inhibition. The present study suggests that ST and, presumably, its analogs used in the cancer treatment could enhance the CEA expression on neoplastic cells in patients affected by the CEA-positive malignancies. This appears to be of potential clinical interest for the development of new immunotherapeutic or diagnostic approaches based on the pharmacological modulation of this antigenic marker.