The diagnostic process of cervical cancer; areas of good practice, and windows of opportunity.

OBJECTIVE: Despite an extensive screening programme in The Netherlands, some cases of cervical cancer are still diagnosed in late stages of disease. The aim of the present study was to investigate which elements in the diagnostic process of cervical cancer may be improved. METHODS: This is a retrospective study of 120 patients with cervical cancer diagnosed between January 1st 2008 and June 1st 2010 at the University Medical Center Utrecht. Patient charts, referral information, and pathology results were analyzed. RESULTS: 39.1% of cancer cases were screen or interval detected; the other 60.9% of patients had not been screened, either due to non-attendance or because they fell outside the age range for screening. The final diagnosis of cervical cancer was established by biopsy in 77 (64.2%) and by excision of the cervical transformation zone in 35 (29.2%) of the patients. Fifteen (43%) of these excisions could have been avoided if biopsies would have been taken at the first examination, and had shown invasive cancer. CONCLUSIONS: Cervical cancer screening aims at early detection of precursor lesions to decrease the incidence of cancer. This in-depth analysis suggests that improvement of quality of care is to be expected from correct recognition of cervical cancer by physicians and adjustments of the screening programme to reach younger women and non-responders.

Pelvic lymphadenectomy improves survival in patients with cervical cancer with low-volume disease in the sentinel node: a retrospective multicenter cohort study.

OBJECTIVE: In this study, we aimed to describe the value of pelvic lymph node dissection (LND) after sentinel lymph node (SN) biopsy in early-stage cervical cancer. METHODS: We performed a retrospective multicenter cohort study in 8 gynecological oncology departments. In total, 645 women with International Federation of Gynecology and Obstetrics stage IA to IIB cervical cancer of squamous, adeno, or adenosquamous histologic type who underwent SN biopsy followed by pelvic LND were enrolled in this study. Radioisotope tracers and blue dye were used to localize the sentinel node, and pathologic ultrastaging was performed. RESULTS: Among the patients with low-volume disease (micrometastases and isolated tumor cells) in the sentinel node, the overall survival was significantly better (P = 0.046) if more than 16 non-SNs were removed. No such significant difference in survival was detected in patients with negative or macrometastatic sentinel nodes. CONCLUSIONS: Our findings indicate that in patients with negative or macrometastatic disease in the sentinel nodes, an additional LND did not alter survival. Conversely, our data suggest that the survival of patients with low-volume disease is improved when more than 16 additional lymph nodes are removed. If in a prospective trial our data are confirmed, we would suggest a 2-stage operation.

Open versus laparoscopic pelvic lymph node dissection in early stage cervical cancer: no difference in surgical or disease outcome.

OBJECTIVE: This study aimed to investigate in a retrospective study the effect of laparoscopic surgery, introduced in our center in 1994 as part of the standard treatment of early stage cervical cancer, on surgical and disease outcomes. PATIENTS AND METHODS: A total of 169 women with cervical carcinoma stage IB1 (n = 150) or IB2 (n = 19) were included in the study. Seventy-six patients who underwent laparoscopic pelvic lymph node dissection (LPLND), followed either by open radical hysterectomy (n = 63) or, in case of positive lymph nodes, by primary chemoradiation (n = 13), were compared with an historic cohort of 93 patients who underwent a fully open, traditional Wertheim-Meigs procedure (WM). Recorded clinical characteristics of patients included age, International Federation of Gynecology and Obstetrics stage, histologic diagnosis, differentiation grade, tumor diameter, lymph node status, and adjuvant therapy. Operation time; lymph node yield; intraoperative, early, and late complications; site of recurrences; and disease-free and overall survival rates were analyzed and compared between groups. RESULTS: Clinical characteristics did not differ between groups. Duration of total surgery time was longer in patients with LPLND followed by open radical hysterectomy compared with that in the WM group (P < 0.001). In patients with negative lymph nodes (n = 129), the number of resected nodes was higher (P = 0.002) in the LPLND (median, 26 nodes; range, 8-55 nodes) than in the WM group (median, 21 nodes; range, 7-50 nodes). In patients with positive lymph nodes (n = 40), no significant difference in the number of resected lymph nodes between the 2 groups (P = 0.904) was found. Intraoperative, early, and late complications did not differ between the 2 surgical procedures. The number of locoregional recurrences, but not of distant metastases, was significantly higher (P = 0.018) in the WM group compared with the LPLND group. No difference in disease-free or disease-specific survival was found between the LPLND and WM group, neither with nor without adjuvant or primary (chemo)radiation. A benefit in disease-free survival (P = 0.044), but not in disease-specific survival (P = 0.070), was found in the LPLND compared with the WM group in those patients who received adjuvant therapy or primary chemoradiation. CONCLUSIONS: Introduction of a laparoscopic procedure in the surgical staging and treatment of cervical cancer patients did not have a detrimental effect on surgical or disease outcome, and this can be safely applied to the treatment of early stage cervical cancer.

Natural and Induced Humoral Responses to MUC1.

MUC1 is a membrane-tethered mucin expressed on the ductal cell surface of glandular epithelial cells. Loss of polarization, overexpression and aberrant glycosylation of MUC1 in mucosal inflammation and in adenocarcinomas induces humoral immune responses to the mucin. MUC1 IgG responses have been associated with a benefit in survival in patients with breast, lung, pancreatic, ovarian and gastric carcinomas. Antibodies bound to the mucin may curb tumor progression by restoring cell-cell interactions altered by tumor-associated MUC1, thus preventing metastatic dissemination, as well as counteracting the immune suppression exerted by the molecule. Furthermore, anti-MUC1 antibodies are capable of effecting tumor cell killing by antibody-dependent cell-mediated cytotoxicity. Although cytotoxic T cells are indispensable to achieve anti-tumor responses in advanced disease, abs to tumor-associated antigens are ideally suited to address minimal residual disease and may be sufficient to exert adequate immune surveillance in an adjuvant setting, destroying tumor cells as they arise or maintaining occult disease in an equilibrium state. Initial evaluation of MUC1 peptide/glycopeptide mono and polyvalent vaccines has shown them to be immunogenic and safe; anti-tumor responses are scarce. Progress in carbohydrate synthesis has yielded a number of sophisticated substrates that include MUC1 glycopeptide epitopes that are at present in preclinical testing. Adjuvant vaccination with MUC1 glycopeptide polyvalent vaccines that induce strong humoral responses may prevent recurrence of disease in patients with early stage carcinomas. Furthermore, prophylactic immunotherapy targeting MUC1 may be a strategy to strengthen immune surveillance and prevent disease in subjects at hereditary high risk of breast, ovarian and colon cancer.

Vaccine-induced antibody responses in patients with carcinoma.

Cancer vaccines based on defined antigens are capable of inducing antibodies that recognize and kill tumor cells. Antibodies are ideally suited to address minimal residual disease, and vaccination in an adjuvant setting may favorably influence the outcome of a disease. The present article gives a short summary of antibody production by B cells, and the mechanism of action of antibodies, as well as a description of the current methods for measuring antibody responses and for assessing their antitumor efficacy in the context of clinical trials. It concludes with an overview of antibody responses induced by vaccines based on structurally defined tumor-associated antigens tested in patients with carcinomas. Correlation between antibody responses, T-cell responses and clinical outcome has been noted in a few studies, signaling the importance of vaccine design and adjuvants to exploit the interactions of the innate and adaptive immune system. However, humoral responses, which may provide a surrogate marker for T-helper responses and simplify monitoring of large Phase III trials, are still not or incompletely explored in many vaccination trials.

SCC-Ag, lymph node metastases and sentinel node procedure in early stage squamous cell cervical cancer.

OBJECTIVES: We analyzed pretreatment SCC-Ag levels, lymph node (LN) status and disease outcome in early stage squamous cell (SCC) cervical cancer. METHODS: Serum SCC-Ag was measured before primary treatment in 91 patients (FIGO stage IB1 72, IB2 10, and IIA 9). Of these, 58 underwent laparoscopic sentinel lymph node (SLN) procedure followed by pelvic lymphadenectomy. RESULTS: No false negative SLN were observed. SCC-Ag levels were higher in patients with positive LN compared to patients with negative LN (p=0.010), but no difference was found in the SLN patients (p=0.344). The accuracy to predict LN metastases of SCC-Ag at ROC established cutoff of 1.65 ng/mL and 5.5 ng/mL was 76% and 78%, respectively, in stage IB1, and 53% and 79%, respectively, in stages IB2+ IIA. Whereas no deaths were observed in patients with negative LN and negative SCC-Ag levels (at previously established cutoff of 1.1 ng/mL), overall survival (OS) for patients with negative LN but elevated SCC-Ag was similar to that of patients with positive LN, irrespective of their marker levels (Kaplan-Meier analysis of all patients and in stage IB1, p=0.002 and p=0.026, respectively). CONCLUSIONS: SCC-Ag (>1.65 ng/mL) can predict LN metastases more accurately in stage IB1 than in stage IB2+ IIA. Since SCC-Ag levels above 1.1 ng/mL are already associated with a poor prognosis, the marker seems to identify a subgroup of LN negative patients with occult disease that may benefit from full lymphadenectomy following a SLN procedure.

Human anti-mouse IgM and IgG responses in ovarian cancer patients after radioimmunotherapy with 90Y-muHMFG1.

BACKGROUND: Human anti-mouse antibody (HAMA)-IgM and IgG in ovarian cancer patients treated with intraperitoneal (i.p.) 90Y-muHMFG1 as consolidating therapy were analyzed for a relationship with outcome of disease. PATIENTS AND METHODS: Serial serum samples from 208 ovarian cancer patients participating in a phase III trial of i.p. 90Y-muHMFG1 and 25 controls were analyzed for HAMA-IgM and HAMA-IgG. Results were correlated with time to, and location of, disease recurrence. RESULTS: Patients receiving i.p. 90Y-muHMFG1 developed a rapid HAMA-IgM peak (week 4 to 8), followed by a HAMA-IgG peak 2-4 weeks later. HAMA levels in the control group remained unchanged. Early maximum HAMA-IgG peaks were associated with early relapse [hazard ratio (HR), 0.975; 95% confidence interval (CI) 0.956 to 0.995; p=0.012]. Patients with a HAMA-IgG maximum before or at 8 weeks were at significantly higher risk for disease recurrence (HR, 1.6; 95% CI 1.1 to 25;p=0.021) as compared to patients with a HAMA-IgG maximum after 8 weeks. CONCLUSION: Besides time point of maximum HAMA-IgG, no evident relation could be found between HAMA-IgM or HAMA-IgG development and time to relapse or location of recurrence.

In vitro expanded human invariant natural killer T-cells promote functional activity of natural killer cells.

Invariant natural killer T (iNKT) cells play a pivotal role in cancer immunity through trans-activation of effector cells via swift cytokine secretion. In mice, iNKT cell activation by alpha-galactosylceramide (alpha-GC) induces potent NK cell-mediated anti-tumour effects. Here we investigated whether human iNKT cells could enhance NK cell functional activity in vitro. iNKT cell activation by alpha-GC treatment of peripheral blood mononuclear cells (PBMC) was not sufficient to enhance NK cell effector functions. However, addition of in vitro expanded iNKT cells to PBMC enhanced NK cell-mediated cytotoxicity in an alpha-GC-dependent manner. NK cell activation by iNKT cells was primarily mediated by soluble factors, and could be enhanced by the NK cell activating cytokine IL-21. These results suggest that adoptive transfer of ex vivo expanded iNKT cells will enhance NK cell function and is expected to enhance the efficacy of cancer immunotherapy, particularly in combination with IL-21 and alpha-GC.

Toll-like receptor agonists and invariant natural killer T-cells enhance antibody-dependent cell-mediated cytotoxicity (ADCC).

huHMFG-1 (AS1402) is a humanised IgG1 against MUC1, which exerts tumour cell killing through antibody-dependent cellular cytotoxicity (ADCC) mediated by natural killer (NK) cells. Here, we explored the capacity of invariant NKT (iNKT) cells, which are known to activate NK cells, and toll-like receptor (TLR) ligands which activate both iNKT and NK cells, to enhance huHMFG-1-ADCC. Addition of iNKT cells, as well as TLR2/6, 7, 8 and 9 agonists to PBMC improved the efficacy of huHMFG-1. These results suggest that transfer of ex vivo expanded iNKT cells or TLR agonist treatment may improve the efficacy of NK cell-mediated antibody-based tumour immunotherapies.

IFN-gamma-producing human invariant NKT cells promote tumor-associated antigen-specific cytotoxic T cell responses.

CD1d-restricted invariant NKT (iNKT) cells can enhance immunity to cancer or prevent autoimmunity, depending on the cytokine profile secreted. Antitumor effects of the iNKT cell ligand alpha-galactosylceramide (alphaGC) and iNKT cell adoptive transfer have been demonstrated in various tumor models. Together with reduced numbers of iNKT cells in cancer patients, which have been linked to poor clinical outcome, these data suggest that cancer patients may benefit from therapy aiming at iNKT cell proliferation and activation. Herein we present results of investigations on the effects of human iNKT cells on Ag-specific CTL responses. iNKT cells were expanded using alphaGC-pulsed allogeneic DC derived from the acute myeloid leukemia cell line MUTZ-3, transduced with CD1d to enhance iNKT cell stimulation, and with IL-12 to stimulate type 1 cytokine production. Enhanced activation and increased IFN-gamma production was observed in iNKT cells, irrespective of CD4 expression, upon stimulation with IL-12-overexpressing dendritic cells. IL-12-stimulated iNKT cells strongly enhanced the MART-1 (melanoma Ag recognized by T cell 1)-specific CD8(+) CTL response, which was dependent on iNKT cell-derived IFN-gamma. Furthermore, autologous IL-12-overexpressing dendritic cells, loaded with Ag as well as alphaGC, was superior in stimulating both iNKT cells and Ag-specific CTL. This study shows that IL-12-overexpressing allogeneic dendritic cells expand IFN-gamma-producing iNKT cells, which may be more effective against tumors in vivo. Furthermore, the efficacy of autologous Ag-loaded DC vaccines may well be enhanced by IL-12 overexpression and loading with alphaGC.

Induction of IgG antibodies to MUC1 and survival in patients with epithelial ovarian cancer.

We investigated whether epithelial ovarian cancer patients participating in a randomized phase III trial comparing single intraperitoneal (IP) administration of yttrium-90-labeled murine HMFG1 ((90)Y-muHMFG1) plus standard treatment (AT) vs. standard treatment (ST) alone developed IgG ab to MUC1 that had an impact on disease outcome. Serial serum samples from 208 patients in the AT and 199 patients in the ST arm were tested for IgG ab to MUC1 (anti-MUC1 IgG). Anti-MUC1 IgG at weeks 4, 8 and 12 ranked higher in the AT than in the ST arm (p < 0.001). The median (range) area under the curve (AUC) of anti-MUC1 IgG for weeks 1 to 12 was 5.53 (1.51-39.51) and 3.92 (1.17-68.74) for the AT and ST arm, respectively (p < 0.001). An anti-MUC1 IgG AUC > 13 was associated with a benefit in overall survival (OS) and disease-free survival (DFS) in the AT arm in univariate (p = 0.043 and 0.036, respectively), but not in multivariate analysis (Cox proportional hazards regression model). Kaplan-Meier analysis showed a benefit in OS and DFS in patients with an anti-MUC1 IgG AUC > 13 in the AT arm (p = 0.043 and 0.036, respectively), but not in the ST arm. A single IP injection with muHMFG1 did not lead to a survival benefit in the randomized trial, but it induced ab to MUC1 that were associated with an improved disease outcome in patients with highest levels of anti-MUC1 IgG. Immunotherapy against MUC1 could be effective in the treatment of epithelial ovarian cancer.

Immunoglobulin allotypes influence antibody responses to mucin 1 in patients with gastric cancer.

There are significant interindividual differences in naturally occurring antibody responses to the tumor-associated antigen mucin 1 (MUC1), but the host genetic factors that might contribute to these differences have not been identified. The aim of the present investigation was to determine whether the variation in naturally occurring antibody levels to MUC1 in patients with gastric cancer is associated with GM and KM allotypes, genetic markers of IgG heavy chains and kappa-type light chains, respectively. A total of 169 Caucasian subjects with gastric cancer were allotyped for several GM and KM markers. These subjects were also characterized for IgG and IgM antibodies to MUC1. GM 3 23 5,13 phenotype was highly significantly associated with MUC1 IgG levels; subjects with this phenotype had lower antibody levels compared with those lacking this phenotype (median IgG level 65.5 relative units versus 91.0 relative units, P = 0.0058). In addition, this phenotype had an interactive effect with KM phenotypes on the levels of IgG antibodies to this antigen (P = 0.0081). Levels of MUC1 IgM antibodies were not associated with these genetic markers. These results show, for the first time, that GM and KM allotypes contribute to the interindividual differences in humoral immunity to MUC1.

A combination of serum tumor markers identifies high-risk patients with early-stage squamous cervical cancer.

We aimed to investigate whether pretreatment serum levels of squamous cell carcinoma (SCC) antigen (SCC-Ag), cytokeratin 19 (CYFRA 21-1) and two mucins (CA 15-3 and CA 125) identify patients with occult disease in early-stage SCC of the cervix. Therefore, pretreatment serum samples were obtained from 78 patients with SCC of the cervix (52 IB, 9 IIA and 18 IIB), and tumor markers were measured with commercial immunoassays. SCC-Ag, CYFRA 21-1 and CA 15-3 (analyzed as continuous variables) were significantly associated with overall (OS) and disease-free survival (DFS) in univariate analysis (p < 0.001 in all cases). Multivariate analysis identified lymph node status as the strongest predictor for OS and DFS (p < 0.001 and p = 0.001, respectively), followed by CYFRA 21-1 (p = 0.060 and p = 0.027, respectively) and CA 15-3 (p = 0.082 and p = 0.017, respectively). Clinical cutoff values for each marker were defined by maximizing the log-rank statistics for OS in the total population: 1.1 microg/l for SCC-Ag (n = 47, 60.3%), 1.4 microg/l for CYFRA 21-1 (n = 47, 60.3%), 40 U/ml for CA 15-3 (n = 11, 14.1%) and 30 U/ml for CA 125 (n = 10, 12.8%). Stage IB patients with positive SCC-Ag and CYFRA 21-1 had significantly lower OS (mean 8.3 years, 95% confidence interval, CI, 5.8-10.7 years) and DFS (mean 7.3 years, 95% CI 4.6-10 years) than all other stage IB patients (OS, mean 14.5 years, 95% CI 13.5-15.5 years; DFS, mean 13.9 years, 95% CI 12.5-15.4 years). Stage IB patients with tumors <4 cm or with negative lymph nodes and positive SCC-Ag and CYFRA 21-1 had significantly poorer OS and DFS compared to all other patients in the same group. Elevated levels of both CA 125 and CA 15-3 (3 patients) were associated with an extremely poor prognosis. In conclusion, a combination of SCC-Ag and CYFRA 21-1 may help to identify early-stage cervical cancer patients with occult disease requiring adjuvant therapy.

High level of MUC1 in serum of ovarian and breast cancer patients inhibits huHMFG-1 dependent cell-mediated cytotoxicity (ADCC).

The huHMFG-1 (AS1402) antibody is a humanised IgG1 directed against MUC1 and is currently in clinical trials for the treatment of breast carcinoma. Adenocarcinomas over-express and shed MUC1, and high MUC1 serum levels are associated with progressive disease. Here, we have investigated the effects of MUC1 present in sera from breast and ovarian cancer patients and that of NK cells on in vitro huHMFG-1-mediated ADCC, performed with and without the addition of various cytokines. Screening for patients with high levels of NK cells bearing the FcgammaRIIIa-158V polymorphism, adjusting the dosage to circulating levels of MUC1 and co-administration of NK cell activating cytokines may increase the efficacy of huHMFG-1 treatment.

Serum CA-125 in relation to adnexal dysplasia and cancer in women at hereditary high risk of ovarian cancer.

PURPOSE: Serum CA-125 level is commonly used as indicator for ovarian cancer recurrence. However, its value for the prediction of neoplastic lesions is unknown. The aim of this study was to investigate whether CA-125 concentrations are indicative of adnexal dysplasia and cancer in women at hereditary high risk of ovarian/tubal cancer. PATIENTS AND METHODS: CA-125 was obtained from 424 women at hereditary high risk of ovarian/tubal cancer attending the VU University Medical Center (Amsterdam, the Netherlands) between 1993 and 2005. Serum samples obtained at the second-to-last (n = 64) and last (n = 98) visit before surgery were tested in women who underwent adnexal surgery for diagnostic (n = 9) or prophylactic (n = 89) reasons. Serum samples obtained from 370 age-matched healthy women were used as controls. RESULTS: Both the absolute value (P < .0001) and the serial change (P < .0001) of CA-125 were predictive for ovarian cancer (n = 8). For adnexal dysplasia (n = 23), the absolute value of CA-125 (P = .003) was predictive, but the serial change in CA-125 was not (P = .32). The odds ratio for adnexal dysplasia versus nondysplasia in the highest tertile (CA-125 levels 14 U/mL) compared with the lowest tertile (CA-125 < 10 U/mL) was 6 (95% CI, 1.32 to 36.66). CONCLUSION: In patients at hereditary high risk for adnexal cancer, both the absolute value of serum CA-125 and the change in serial CA-125 are predictors for ovarian cancer. Remarkably, the absolute value of CA-125 is also predictive for adnexal dysplasia. CA-125 values should, therefore, be taken into account in the decision toward prophylactic bilateral salpingo-oophorectomy.

Cancer specific Mucin-1 glycoforms are expressed on multiple myeloma.

Present therapies cannot cure the large majority of patients with multiple myeloma (MM) and therefore new treatment strategies are imperative. This study analysed the different glycosylation profiles of Mucin-1 (MUC1) on MM and acute myeloid leukaemia (AML) cells using a series of anti-MUC1 antibodies. Seventy-three per cent of the MM patients had plasma cells that expressed the fully glycosylated forms of MUC1. In contrast to controls, normal bone marrow cells and AML cells, the differentiation-dependent and cancer-associated glycoforms of MUC1 were present on 59% and 36% MM tumour cells respectively. This indicated that aberrantly glycosylated MUC1 is a potential immunotherapeutic target in MM patients.

Molecular basis of incomplete O-glycan synthesis in MCF-7 breast cancer cells: putative role of MUC6 in Tn antigen expression.

An incomplete elongation of O-glycan saccharide chains in mucins have been found in epithelial cancers, leading to the expression of shorter carbohydrate structures, such as the Tn antigen (GalNAc-O-Ser/Thr). This antigen is one of the most specific human cancer-associated structures and is capable of inducing effective immune responses against cancer cells. We aimed to investigate the causes of the expression of Tn antigen in the Tn-rich MCF-7 breast cancer cell line focusing on the first step of the O-glycosylation process. Interestingly, amino acid sequences derived from "non-mammary" apomucins (MUC5B and MUC6) were very good acceptor substrates for ppGalNAc-Ts, which are the enzymes catalyzing the Tn antigen synthesis. MUC6 peptide glycosylation with MCF-7 microsome extracts as source of ppGalNAc-T activity yielded 95% conversion of the peptide into MUC6-Tn. In addition, the MUC6-Tn glycopeptide was a poor acceptor substrate for core 1 beta3Gal-T, the next enzyme involved in the saccharide chain biosynthesis, yielding only 5% conversion of MUC6-Tn into MUC6-TF. These results indicate that non-mammary apomucin expression could be responsible, at least in part, for Tn antigen expression in MCF-7 breast cancer cells due to a combined action on glycosyltransferases: an increase of ppGalNAc-T activity and a decrease of core 1 beta3Gal-T activity. Our hypothesis is supported by experiments in vivo showing that (a) native MUC6 glycoproteins express the Tn antigen in MCF-7 cells and (b) Tn antigen expression is increased after transfection with a construct encoding for a MUC6 recombinant protein into the low Tn-expressing breast cancer cell T47D. These results open new horizons in breast cancer glycoimmunology, stressing the potential role of non-mammary apomucins.

What makes MUC1 a tumor antigen?

The epithelial mucin 1 (MUC1) is an accepted serum tumor marker and cellular tumor antigen. We discuss recent views on the difference(s) between normal and tumor MUC1, and its implication for the development of cancer vaccines and antibody therapies, with special emphasis on the role of glycosylation.

Lobulitis is a frequent finding in prophylactically removed breast tissue from women at hereditary high risk of breast cancer.

The aim of this study was to investigate closely the nature of premalignant lesions that occur in prophylactically removed breast tissue from patients at hereditary high risk of breast cancer. Breast tissues obtained from 41 patients who underwent prophylactic mastectomy (pM) because of a hereditary high risk of breast cancer and control tissues from 82 age-matched healthy controls who underwent breast reduction surgery were screened for premalignant lesions. Premalignant and malignant lesions were more frequent (p = 0.0016) in pM samples (5/41) than in controls (1/82). Interestingly, lobulitis, defined as more than 100 lymphocytes and/or plasma cells per lobule in more than one section in morphologically normal lobules, was encountered in 21 of 41 (51%) pM patients, in contrast to only 8 of 82 (10%) controls (p < 0.0001). Preliminary observations indicate a predominance of T-cells in these infiltrates, in agreement with the already known frequent presence of lymphocytic infiltration in hereditary ductal in situ and infiltrating ductal/medullary carcinomas. This novel finding implies an immune reaction to an as yet unidentified antigen frequently present in women at hereditary high risk of breast cancer, possibly as part of an early carcinogenic event.

Sequence-variant repeats of MUC1 show higher conformational flexibility, are less densely O-glycosylated and induce differential B lymphocyte responses.

The human epithelial cancer mucin MUC1 is able to break tolerance and to induce humoral immune responses in healthy subjects and in cancer patients. We recently showed that clusters of sequence-variant repeats are interspersed in the repeat domain of MUC1 at high frequency, which should contribute to the structural and immunological features of the mucin. Here we elucidated the potential effects exerted by sequence-variant repeats on their O-glycosylation. Evidence from in vitro glycosylation with polypeptide N-acetylgalactosaminyltransferases GalNAc-T1 and GalNAc-T2 in concert with mass spectrometric analyses of in vivo glycosylated MUC1 probes from transiently transfected HEK293 cells indicated reduced glycosylation densities of repeats with three concerted replacements: AHGVTSAPESRPAPGSTAPA. The Pro to Ala replacement in STAPA exerts not only proximal effects on the ppGalNAc-T2 preferred site at -3 and -4, but also more distant effects on the ppGalNAc-T1 preferred site at -15 (TSAPESRPAPGSTAPA). We also examined the conformational changes of MUC1 glycopeptides induced by the concerted DT to ES replacements and revealed a higher conformational flexibility of ES/P peptides compared to DT/P peptides. Differences in conformational flexibilities and in O-glycosylation densities could underlie the observed differential humoral responses in humans. We were able to show that the natural immunoglobulin G (IgG) responses to the repeat domain of MUC1 in sera from nonmalignant control subjects are preferentially directed to variant repeat clusters. In contrast, the IgG response in patients with adenocarcinoma shifted to higher frequencies of preferential DTR peptide binding.