Central nervous system acute lymphoblastic leukemia: role of natural killer cells.

Central nervous system acute lymphoblastic leukemia (CNS-ALL) is a major clinical problem. Prophylactic therapy is neurotoxic, and a third of the relapses involve the CNS. Increased expression of interleukin 15 (IL-15) in leukemic blasts is associated with increased risk for CNS-ALL. Using in vivo models for CNS leukemia caused by mouse T-ALL and human xenografts of ALL cells, we demonstrate that expression of IL-15 in leukemic cells is associated with the activation of natural killer (NK) cells. This activation limits the outgrowth of leukemic cells in the periphery, but less in the CNS because NK cells are excluded from the CNS. Depletion of NK cells in NOD/SCID mice enabled combined systemic and CNS leukemia of human pre-B-ALL. The killing of human leukemia lymphoblasts by NK cells depended on the expression of the NKG2D receptor. Analysis of bone marrow (BM) diagnostic samples derived from children with subsequent CNS-ALL revealed a significantly high expression of the NKG2D and NKp44 receptors. We suggest that the CNS may be an immunologic sanctuary protected from NK-cell activity. CNS prophylactic therapy may thus be needed with emerging NK cell-based therapies against hematopoietic malignancies.

Specific Design of Titanium(IV) Phenolato Chelates Yields Stable and Accessible, Effective and Selective Anticancer Agents.

Octahedral titanium(IV) complexes of phenolato hexadentate ligands were developed and showed very high stability for days in water solutions. In vitro cytotoxicity studies showed that, whereas tetrakis(phenolato) systems are generally of low activity presumably due to inaccessibility, smaller bis(phenolato)bis(alkoxo) complexes feature high anticancer activity and accessibility even without formulations, also toward a cisplatin-resistant cell line. An all-aliphatic control complex was unstable and inactive. A leading phenolato complex also revealed: 1) high durability in fully aqueous solutions; accordingly, negligible loss of activity after preincubation for three days in medium or in serum; 2) maximal cellular accumulation and induction of apoptosis following 24-48 h of administration; 3) reduced impact on noncancerous fibroblast cells; 4) in vivo efficacy toward lymphoma cells in murine model; 5) high activity in NCI-60 panel, with average GI50 of 4.6±2 µm. This newly developed family of Ti(IV) complexes is thus of great potential for anticancer therapy.

Highly Effective and Hydrolytically Stable Vanadium(V) Amino Phenolato Antitumor Agents.

Vanadium(V) oxo complexes with no labile ligands, including six octahedral complexes with pentadentate diaminotris(phenolato) ligands and one pentacoordinate complex with a tetradentate aminotris(phenolato) ligand, were synthesized in high yields. All octahedral complexes demonstrated high hydrolytic stability with no signs of decomposition after days in the presence of water, whereas the pentacoordinate complex decomposed within minutes to release the free ligand, demonstrating the marked impact of coordination number and geometry on the complex electrophilicity. All complexes showed marked cytotoxicity toward human colon HT-29 and ovarian OVCAR-3 cells. In particular, the octahedral complexes exhibited especially high activity, higher than that of cisplatin by up to 200-fold. Selected complexes demonstrated similarly high activity also toward the A2780 and the A2780cis cisplatin-resistant line. High cytotoxicity was also recorded after prolonged incubation in a DMSO solution at 4 and 37 °C temperatures and in biological medium. In vivo studies pointed to high efficacy in reducing tumor size, where no clinical signs of toxicity were detected in the treated mice. These results overall indicate high potential of the tested compounds as antitumor agents.

Highly Stable Tetra-Phenolato Titanium(IV) Agent Formulated into Nanoparticles Demonstrates Anti-Tumoral Activity and Selectivity.

Titanium(IV) complexes exhibit high potential as anti-tumor agents, particularly due to their low intrinsic toxicity and cytotoxicity toward cisplatin resistant cells. Nevertheless, Ti(IV) complexes generally undergo rapid hydrolysis that previously hampered their utilization as anticancer drugs. We recently overcame this difficulty by developing a highly stable Ti(IV) complex that is based on tetra-phenolato, hexadentate ligand, formulated into organic nanoparticles. Herein we investigated the activity of this complex in vitro and in vivo. Although inactive when tested directly due to poor solubility, when formulated, this complex displayed (a) high cytotoxicity toward cisplatin resistant human ovarian cells, A2780-cp, with resistance factor of 1.1; (b) additive behavior in combination with cisplatin toward ovarian and colon cancer cells; (c) selectivity toward cancer cells as implied by its mild activity toward non-cancerous, fibroblast lung cells, MRC-5; (d) high stability and durability as manifested by the ability to maintain cytotoxicity, even following one week of incubation in 100% aquatic medium solution; and (e) in vivo efficacy toward solid tumors of human colon cancer cells, HT-29, in nude mice without any clinical signs of toxicity. These features support the formulated phenolato Ti(IV) complex being an effective and selective anti-tumoral agent.

Life after Cleavage: The Story of a ß-Retroviral (MMTV) Signal Peptide-From Murine Lymphoma to Human Breast Cancer.

An increasing body of evidence in recent years supports an association of the betaretrovirus mouse mammary tumor virus (MMTV) with human breast cancer. This is an issue that still raises heated controversy. We have come to address this association using the signal peptide p14 of the MMTV envelope precursor protein as a key element of our strategy. In addition to its signal peptide function, p14 has some significant post endoplasmic reticulum (ER)-targeting characteristics: (1) it localizes to nucleoli where it binds key proteins (RPL5 and B23) involved (among other activities) in the regulation of nucleolar stress response, ribosome biogenesis and p53 stabilization; (2) p14 is a nuclear export factor; (3) it is expressed on the cell surface of infected cells, and as such, is amenable to, and successfully used, in preventive vaccination against experimental tumors that harbor MMTV; (4) the growth of such tumors is impaired in vivo using a combination of monoclonal anti-p14 antibodies or adoptive T-cell transfer treatments; (5) p14 is a phospho-protein endogenously phosphorylated by two different serine kinases. The phosphorylation status of the two sites determines whether p14 will function in an oncogenic or tumor-suppressing capacity; (6) transcriptional activation of genes (RPL5, ErbB4) correlates with the oncogenic potential of MMTV; (7) finally, polyclonal anti-p14 antibodies have been applied in immune histochemistry analyses of breast cancer cases using formalin fixed paraffin-embedded sections, supporting the associations of MMTV with the disease. Taken together, the above findings constitute a road map towards the diagnosis and possible prevention and treatment of MMTV-associated breast cancer.

Effective Oral Administration of an Antitumorigenic Nanoformulated Titanium Complex.

Orally administered anticancer drugs facilitate treatment, but the acidic conditions in the stomach often challenge their availability. PhenolaTi is a TiIV -based nontoxic anticancer drug with marked in-vivo efficacy. We report that nanoformulation protects phenolaTi from decomposition in stomach-like conditions. This is evidenced by similar NMR characteristics and similar in-vitro cytotoxicity toward murine (CT-26) and human (HT-29) colon cancer cells before and after incubation of nanoformulated phenolaTi (phenolaTi-F) at pH 2, unlike results with the unformulated form of the complex. Furthermore, administration of phenolaTi-F in animal drinking water revealed a notable inhibition of tumor growth in Balb/c and immune-deficient (Nude) mice inoculated with CT-26 and HT-29 cells, respectively. In-vivo efficacy was at least similar to that of the corresponding intraperitoneal treatment with phenolaTi-F and the clinically employed oral drug, capecitabine. No body weight loss or clinical signs of toxicity were evident in the phenolaTi-F-treated animals. These findings demonstrate a new convenient mode of cancer treatment through oral administration by safe titanium-based drugs.

Titanium Tackles the Endoplasmic Reticulum: A First Genomic Study on a Titanium Anticancer Metallodrug.

PhenolaTi is an advanced non-toxic anticancer chemotherapy; this inert bis(phenolato)bis(alkoxo) Ti(IV) complex demonstrates the intriguing combination of high and wide efficacy with no detected toxicity in animals. Here we unravel the cellular pathways involved in its mechanism of action by a first genome study on Ti(IV)-treated cells, using an attuned RNA sequencing-based available technology. First, phenolaTi induced apoptosis and cell-cycle arrest at the G2/M phase in MCF7 cells. Second, the transcriptome of the treated cells was analyzed, identifying alterations in pathways relating to protein translation, DNA damage, and mitochondrial eruption. Unlike for common metallodrugs, electrophoresis assay showed no inhibition of DNA polymerase activity. Reduced in vitro cytotoxicity with added endoplasmic reticulum (ER) stress inhibitor supported the ER as a putative cellular target. Altogether, this paper reveals a distinct ER-related mechanism by the Ti(IV) anticancer coordination complex, paving the way for wider applicability of related techniques in mechanistic analyses of metallodrugs.

A Key role for cyclic AMP-responsive element binding protein in hypoxia-mediated activation of the angiogenesis factor CCN1 (CYR61) in Tumor cells.

Hypoxia is a prominent feature of solid tumors known to contribute to malignant progression and therapeutic resistance. Cancer cells adapt to hypoxia using various pathways, allowing tumors to thrive in a low oxygen state. Induction of new blood vessel formation via the secretion of proangiogenic factors is one of the main adaptive responses engaged by tumor cells under hypoxic conditions. Hypoxia-inducible factor 1 (HIF-1) is a transcription factor that plays a pivotal role in mediating such responses. In addition, several other transcription factors have also been implicated in hypoxic gene regulation, either independently or in cooperation with HIF-1. In this work, we show that the expression of the angiogenesis-related, immediate early gene CCN1 (formerly known as CYR61), considered to be involved in tumor growth and invasiveness, is enhanced upon hypoxia stress primarily in a protein kinase A and cyclic AMP-responsive element binding protein (CREB) and CRE-dependent manner in various cell lines. The hypoxia-mediated activation of the CCN1 promoter is independent of HIF-1 and HIF-2, as shown by small interfering RNA knockdown. We identify the cis element in the mouse CCN1 promoter responsible for CREB binding to be one of two partial CRE sites present in the promoter. Moreover, we report for the first time that CREB-mediated CCN1 transcription is enhanced in hypoxic regions of tumors in vivo. Identifying and characterizing the molecular mechanisms that govern the response of tumors to hypoxia may be instrumental to identify the tumors that will respond favorably to inhibition of angiogenesis and thus lead to the development of treatments that could complement hypoxia-inducing treatment modalities.

ANKRD44 Gene Silencing: A Putative Role in Trastuzumab Resistance in Her2-Like Breast Cancer.

Trastuzumab is an effective therapeutic treatment for Her2-like breast cancer; despite this most of these tumors develop resistance to therapy due to specific gene mutations or alterations in gene expression. Understanding the mechanisms of resistance to Trastuzumab could be a useful tool in order to identify combinations of drugs that elude resistance and allow a better response for the treated patients. Twelve primary biopsies of Her2+/hormone receptor negative (ER-/PgR-) breast cancer patients were selected based on the specific response to neoadjuvant therapy with Trastuzumab and their whole exome was sequenced leading to the identification of 18 informative gene mutations that discriminate patients selectively based on response to treatment. Among these genes, we focused on the study of the ANKRD44 gene to understand its role in the mechanism of resistance to Trastuzumab. The ANKRD44 gene was silenced in Her2-like breast cancer cell line (BT474), obtaining a partially Trastuzumab-resistant breast cancer cell line that constitutively activates the NF-kb protein via the TAK1/AKT pathway. Following this activation an increase in the level of glycolysis in resistant cells is promoted, also confirmed by the up-regulation of the LDHB protein and by an increased TROP2 protein expression, found generally associated with aggressive tumors. These results allow us to consider the ANKRD44 gene as a potential gene involved in Trastuzumab resistance.

Cranberry juice constituents impair lymphoma growth and augment the generation of antilymphoma antibodies in syngeneic mice.

In addition to its nutritional value, cranberry juice has been effective in treating urinary tract infections. Various reports have also demonstrated its potential for inhibiting in vitro growth of transformed cell lines. Here we show that a fraction [nondialyzable material (NDM) of a molecular weight range 12,000-30,000 (NDM 12-30K)] derived from cranberry juice impairs in vitro growth and invasion through extracellular matrix of Rev-2-T-6 murine lymphoma cells. Furthermore, intraperitoneal injection of this fraction at nontoxic doses both inhibits the growth of Rev-2-T-6 tumors in vivo and enhances the generation of antilymphoma antibodies. These findings demonstrate the in vivo efficacy of cranberry components against malignant lymphoma in immune competent hosts.

In Vivo Anticancer Activity of a Nontoxic Inert Phenolato Titanium Complex: High Efficacy on Solid Tumors Alone and Combined with Platinum Drugs.

Due to the toxicity of platinum compounds used in the clinic as anticancer chemotherapies, titanium serves as a safe and attractive alternative. Lately, we introduced a new family of Ti complexes based on readily available phenolato ligands, demonstrating incredibly high hydrolytic stability, with the lead compound phenolaTi demonstrating wide cytotoxic activity toward the NCI-60 panel of human cancer cell lines, with an average GI50 value of 4.7±2 µm. Herein, we evaluated in vivo: a) the safety, and b) the growth inhibitory capacity (efficacy) of this compound. PhenolaTi was found to be effective in vivo against colon (CT-26) and lung (LLC-1) murine cell lines in syngeneic hosts and toward a human colon cancer (HT-29) cell line in immune-deficient (Nude) mice, with an efficacy similar to that of known chemotherapy. Notably, no clinical signs of toxicity were observed in the treated mice, namely, no effect on body weight, spleen weight or kidney function, unlike the effects observed with the positive control Pt drugs. Studies of combinations of phenolaTi and Pt drugs provided evidence that similar efficacy with decreased toxicity may be achieved, which is highly valuable for medicinal applications.

Association of Mouse Mammary Tumor Virus With Human Breast Cancer: Histology, Immunohistochemistry and Polymerase Chain Reaction Analyses.

PURPOSE: The purpose of this study is to determine whether mouse mammary tumor virus (MMTV)-associated human breast cancer has the same or similar histology to MMTV-associated mouse mammary tumors. Such associations may indicate a role for MMTV in human breast cancer. METHODS: Immunohistochemical techniques (using antibodies directed against the signal peptide p14 of the envelope precursor protein of MMTV) and polymerase chain reaction (PCR) analyses were used to identify MMTV proteins and MMTV-like envelope gene sequences in a series of breast cancers from Australian women. The histological characteristics of these human breast cancer specimens were compared with MMTV positive mouse mammary tumors. The same methods were used to study benign breast tissues which 1-11 years later developed into breast cancer. RESULTS: MMTV p14 proteins were identified in 27 (54%) of 50 human breast cancers. MMTV env gene sequences were identified by PCR in 12 (27%) of 45 human breast cancers. There was a significant correlation between the presence of MMTV (identified by p14 immunohistochemistry) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.001). There was a non-significant correlation between the presence of MMTV env gene sequences (identified by PCR) in human breast cancers and histological characteristics similar to MMTV positive mouse mammary tumors (p = 0.290). MMTV p14 proteins were identified in 7 (54%) of 13 benign breast specimens that later developed into human breast cancers. MMTV by PCR was identified in two benign specimens one of whom later developed MMTV positive breast cancer. DISCUSSION: These observations offer evidence that MMTV may be associated with characteristic human breast cancer histology. p14-based immunohistochemistry appears to be a more reliable technique than PCR for the identification of MMTV in human breast cancer. Identification of MMTV-associated p14 proteins in benign breast tissues confirms prior PCR-based studies that MMTV infection occurs before the development of MMTV positive breast cancer. CONCLUSION: Many MMTV positive human breast cancers have similar histology to MMTV positive mouse mammary tumors. MMTV infection identified in benign breast tissues precedes development of MMTV positive human breast cancer. When considered in the context of prior studies, these observations indicate a likely role for MMTV in human breast cancer.

Mouse mammary tumor virus Env-derived peptide associates with nucleolar targets in lymphoma, mammary carcinoma, and human breast cancer.

We have previously shown that the leader peptide (p14) of the Env-precursor of mouse mammary tumor virus is translocated into the nucleoli of murine T cell lymphomas that harbor this virus. Using a polyclonal antibody against recombinant p14, we show here that p14 is also localized to the nucleoli of murine mammary carcinomas and some human breast cancer samples. Affinity purification studies define a number of proteins, mostly nucleolar, that bind p14. Taken together, these findings point towards a more general involvement of p14 in lymphomagenesis and mammary carcinogenesis.

International Central Nervous System and Ocular Lymphoma Workshop: recommendations for the future.

PURPOSE: To bring together multidisciplinary experts to discuss primary central nervous system lymphoma (PCNSL) and primary intraocular lymphoma (PIOL). METHODS: NIH campus workshop discussion focusing on future work in both clinical and basic lymphoma research. RESULTS: The discussion lead to recommendations on elucidating disease pathobiology, improving diagnostic accuracy and sensitivity, and novel therapeutic strategies. CONCLUSIONS: Approaches which have been successfully applied to other neoplasms, such as microarray, may be applied to improve diagnostic accuracy and sensitivity of PCNSL and PIOL and should be systematically incorporated into clinical trials of both. Development of animal models of PCNSL and PIOL may be useful in understanding the unique ocular and CNS milieu. Disease detection by radiological, nuclear medicine, molecular and flow cytometric approaches should be systematically studied to improve early diagnosis, accurate staging, and response evaluation. Improved therapy remains the ultimate goal. Efforts in these arenas should be coordinated on a national and international level.

Insights into molecular mechanism of action of salan titanium(IV) complex with in vitro and in vivo anticancer activity.

Titanium compounds, in particular, Ti(IV) based diaminobis(phenolato) "salan" complexes demonstrate high cytotoxicity towards a wide range of cancer cell lines in vitro, and still, very little is known on their mode of action. A representative salan Ti(IV) complex was tested both in vitro and in vivo on human HT-29 colorectal adenocarcinoma and A2780 ovarian carcinoma cells. Both cell lines were sensitive in vitro with A2780 demonstrating an enhanced rate of uptake and intracellular accumulation and thus an earlier response to the drug. HT-29 cells responded in vivo by impaired tumor development in nude mice. Both cell lines responded in vitro (but to a different extent) by upregulation of p53 with no apparent effect on p21 followed by cell cycle arrest, apoptosis and necrosis as demonstrated by sub-G1 cell accumulation and staining by Annexin-V and propidium iodide. Furthermore, time dependent activation of cysteine-aspartic proteases9 (caspase9) as well as some minor activation of cysteine-aspartic proteases3 (caspase3) support a direct effect on the apoptotic pathway. The differential response of the two cell lines to the salan titanium(IV) complex suggests that more than one pathway is involved in their growth regulation and thus could inhibit development of drug resistant variants.

A new immunization and treatment strategy for mouse mammary tumor virus (MMTV) associated cancers.

Mouse Mammary Tumor Virus (MMTV) causes mammary carcinoma or lymphoma in mice. An increasing body of evidence in recent years supports its involvement also in human sporadic breast cancer. It is thus of importance to develop new strategies to impair the development, growth and metastasis of MMTV-associated cancers. The signal peptide of the envelope precursor protein of this virus: MMTV-p14 (p14) is an excellent target for such strategies, due to unique characteristics distinct from its regular endoplasmic reticulum targeting function. These include cell surface expression in: murine cancer cells that harbor the virus, human breast cancer (MCF-7) cells that ectopically express p14, as well as cultured human cells derived from an invasive ductal breast carcinoma positive for MMTV sequences. These findings support its use in signal peptide-based immune targeting. Indeed, priming and boosting mice with p14 elicits a specific anti-signal peptide immune response sufficient for protective vaccination against MMTV-associated tumors. Furthermore, passive immunization using a combination of anti-p14 monoclonal antibodies or the transfer of T-cells from immunized mice (Adoptive Cell Transfer) is also therapeutically effective. With reports demonstrating involvement of MMTV in human breast cancer, we propose the immune-mediated targeting of p14 as a strategy for prevention, treatment and diagnosis of MMTV-associated cancers.

Summary statement on primary central nervous system lymphomas from the Eighth International Conference on Malignant Lymphoma, Lugano, Switzerland, June 12 to 15, 2002.

Under the sponsorship of the International Extranodal Lymphoma Study Group, a Multidisciplinary Workshop on primary CNS lymphoma (PCNSL) with over 50 participants from Europe, North America, Israel, and Australia was held as part of the Eighth International Conference on Malignant Lymphoma in Lugano, Switzerland (June 12 to 15, 2002). The main purposes of the Workshop were to exchange the latest scientific information, to analyze methodologic issues in the design of clinical trials, to reach a consensus on treatment recommendations and prognostic factors, to discuss clinical and molecular targets for future studies, and to establish an international collaborative group to conduct laboratory and clinical investigations in PCNSL. This article summarizes the contents of the Workshop, analyzes the current knowledge on the most relevant biologic and clinical issues in PCNSL, and focuses on fundamental challenges to be addressed in future studies.

Murine model of primary intraocular lymphoma.

PURPOSE: Primary intraocular lymphoma (PIOL) is a subtype of central nervous system lymphoma. Although this lymphoma is rare, its incidence has tripled in the past 15 years. Currently, the only available model is a murine metastatic malignant lymphoma that occurs after intraperitoneal inoculation of Rev-2-T-6 lymphoma cells into newborn syngeneic mice. The current study was conducted to develop a new experimental model for PIOL. METHODS: Rev-2-T-6 cells (0.5 x 10(5) or 1.0 x 10(5)) were inoculated into the vitreous of adult BALB/c mice. Mice were monitored clinically every other day and under fundoscopic examination weekly. They were euthanatized on weeks 3, 5, 6, 7, or 8, after inoculation. All eyes were processed for histology. Immunohistochemistry was performed with an antibody (p14) specific for Rev-2-T-6 cells. Cytokine mRNA expression (IL-2, -4, -6, -10, and IFN-gamma and CC chemokine receptor-1 [CCR1]) was assayed in the lymphoma cells by microdissection and RT-PCR. IL-10 and -6 levels in the vitreous were measured by ELISA. RESULTS: Within 2 to 4 weeks, tumor cells from the vitreous migrate through the retina and gather between the RPE cell and retina. Rarely (>2 months after inoculation), Rev-2-T-6 cells may break through the RPE and infiltrate the choroid and sclera. Tumor localization was confirmed by immunohistochemistry. The intraocular lymphoma cells produce high levels of IL-10, IFN-gamma, and CCR1 transcripts. A high level of IL-10 was detected in the vitreous inoculated with Rev-2-T-6 cells. CONCLUSIONS: The data suggest that RPE cells constitute a barrier to the spread of intraocular lymphoma. Intravitreal injection of Rev-2-T-6 cells is a novel model of PIOL in immune-competent hosts that will aid in understanding the molecular mechanisms of the disease.

Intraocular Lymphoma Models.

Primary vitreoretinal lymphoma (PVRL) is a subtype of primary central nervous system lymphoma (PCNSL), a high-grade, extranodal, non-Hodgkin's lymphoma, predominantly of B-cell origin. PVRL is an aggressive disease with a poor prognosis. Human studies are not ideally suited for the study of intraocular lymphoma pathogenesis or treatment strategies due to the rare nature of the disease, its variable presentation, limited volume of available ocular fluids, and fragility of sampled lymphoma cells. Animal models have been critical in making progress in understanding intraocular lymphoma pathogenesis and investigating potential therapeutic strategies. Early murine models for intraocular lymphoma used intraperitoneal injection of mouse T-cell lymphomas. This was followed by intravitreal T-cell murine models. More recent murine models have used B-cell lymphomas to more closely mimic human disease. The most current B-cell lymphoma models employ a combined approach of inoculating both the mouse vitreous cavity and brain. The challenge in murine models for intraocular lymphoma lies in recreating the clinical features, disease behavior, molecular profile, systemic immunity, and the microenvironment observed in human disease. In the future, animal models will continue to be central to furthering our understanding of the disease and in the investigation of potential treatment targets.

Human saliva as route of inter-human infection for mouse mammary tumor virus.

Etiology of human breast cancer is unknown, whereas the Mouse Mammary Tumor Virus (MMTV) is recognized as the etiologic agent of mouse mammary carcinoma. Moreover, this experimental model contributed substantially to our understanding of many biological aspects of the human disease. Several data strongly suggest a causative role of MMTV in humans, such as the presence of viral sequences in a high percentage of infiltrating breast carcinoma and in its preinvasive lesions, the production of viral particles in primary cultures of breast cancer, the ability of the virus to infect cells in culture. This paper demonstrates that MMTV is present in human saliva and salivary glands. MMTV presence was investigated by fluorescent PCR, RT-PCR, FISH, immunohistochemistry, and whole transcriptome analysis. Saliva was obtained from newborns, children, adults, and breast cancer patients. The saliva of newborns is MMTV-free, whereas MMTV is present in saliva of children (26.66%), healthy adults (10.60%), and breast cancer patients (57.14% as DNA and 33.9% as RNA). MMTV is also present in 8.10% of salivary glands. RNA-seq analysis performed on saliva of a breast cancer patient demonstrates a high expression of MMTV RNA in comparison to negative controls. The possibility of a contamination by murine DNA was excluded by murine mtDNA and IAP LTR PCR. These findings confirm the presence of MMTV in humans, strongly suggest saliva as route in inter-human infection, and support the hypothesis of a viral origin for human breast carcinoma.