Mieczyslaw Gajda

Monitoring the Response of Circulating Epithelial Tumor Cells to Adjuvant Chemotherapy in Breast Cancer Allows Detection of Patients at Risk of Early Relapse

PURPOSE To demonstrate that it is possible to monitor the response to adjuvant therapy by repeated analysis of circulating epithelial tumor cells (CETCs) and to detect patients early who are at risk of relapse. PATIENTS AND METHODS In 91 nonmetastatic primary breast cancer patients, CETCs were quantified using laser scanning cytometry of anti-epithelial cell adhesion molecule-stained epithelial cells from whole unseparated blood before and during adjuvant chemotherapy. RESULTS Numbers of CETCs were analyzed before therapy, before each new cycle, and at the end of chemotherapy. The following three typical patterns of response were observed: (1) decrease in cell numbers (> 10-fold); (2) marginal changes in cell numbers (< 10-fold); and (3) an (sometimes saw-toothed) increase or an initial decrease with subsequent reincrease (> 10-fold) in numbers of CETCs. Twenty relapses (22%) were observed within the accrual time of 40 months, including one of 28 patients from response group 1, five of 30 patients from response group 2, and 14 of 33 patients from response group 3. The difference in relapse-free survival was highly significant for CETC (hazard ratio = 4.407; 95% CI, 1.739 to 9.418; P < .001) between patients with decreasing cell numbers and those with marginal changes and between patients with marginal changes and those with an increase of more than 10-fold (linear Cox regression model). CONCLUSION These results show that peripherally circulating tumor cells are influenced by systemic chemotherapy and that an increase (even after initial response to therapy) of 10-fold or more at the end of therapy is a strong predictor of relapse and a surrogate marker for the aggressiveness of the tumor cells.

Exacerbation of antigen-induced arthritis in IFN-γ-deficient mice as a result of unrestricted IL-17 response

Proinflammatory Th1 responses are believed to be involved in the induction and perpetuation of rheumatoid arthritis. However, the role of IFN-γ, the major cytokine produced by Th1 cells, is still incompletely defined. In the present study, we investigated the effects of IFN-γ deficiency (IFN-γ−/−) on the course of experimental murine Ag-induced arthritis (AIA). In the acute stage of disease, IFN-γ−/− AIA mice showed significantly increased inflammatory responses compared with wild-type C57BL/6 AIA mice, i.e., exacerbated joint swelling, increased delayed-type hypersensitivity reaction, and increased histopathological scores of arthritis. Intraarticular administration of exogenous IFN-γ at induction of AIA significantly suppressed these acute aggravation effects. Stimulated cells isolated from lymph nodes and spleen of IFN-γ−/− AIA mice showed increased production of IL-2, IL-4, IL-5, IL-6, but most prominently of IL-17. These elevations were paralleled by decreased humoral immune responses, with low serum levels of total and Ag-specific IgG (IgG1, IgG2ab, IgG2b, IgG3). At immunohistology, the knee joints of IFN-γ−/− AIA mice showed massive neutrophil granulocyte infiltration. Treatment with mAbs neutralizing IL-17 diminished the acute inflammation. In vitro, Th cell expansion and production of IL-17 upon restimulation were effectively and dose dependently inhibited by IFN-γ. These results clearly demonstrate that IFN-γ has anti-inflammatory properties during the initial phase of AIA, and indicate that IFN-γ deficiency exerts disease-promoting effects, preferentially via IL-17-modulated pathways.

The role of regulatory T cells in antigen-induced arthritis: aggravation of arthritis after depletion and amelioration after transfer of CD4+CD25+ T cells

It is now generally accepted that CD4+CD25+ Treg cells play a major role in the prevention of autoimmunity and pathological immune responses. Their involvement in the pathogenesis of chronic arthritis is controversial, however, and so we examined their role in experimental antigen-induced arthritis in mice. Depletion of CD25-expressing cells in immunized animals before arthritis induction led to increased cellular and humoral immune responses to the inducing antigen (methylated bovine serum albumin; mBSA) and autoantigens, and to an exacerbation of arthritis, as indicated by clinical (knee joint swelling) and histological scores. Transfer of CD4+CD25+ cells into immunized mice at the time of induction of antigen-induced arthritis decreased the severity of disease but was not able to cure established arthritis. No significant changes in mBSA-specific immune responses were detected. In vivo migration studies showed a preferential accumulation of CD4+CD25+ cells in the inflamed joint as compared with CD4+CD25- cells. These data imply a significant role for CD4+CD25+ Treg cells in the control of chronic arthritis. However, transferred Treg cells appear to be unable to counteract established acute or chronic inflammation. This is of considerable importance for the timing of Treg cell transfer in potential therapeutic applications.

Synergistic antitumor effects of combined cathepsin B and cathepsin Z deficiencies on breast cancer progression and metastasis in mice

The lysosomal cysteine proteases cathepsin B (Ctsb) and cathepsin Z (Ctsz, also called cathepsin X/P) have been implicated in cancer pathogenesis. Compensation of Ctsb by Ctsz in Ctsb −/− mice has been suggested. To further define the functional interplay of these proteases in the context of cancer, we generated Ctsz null mice, crossed them with Ctsb-deficient mice harboring a transgene for the mammary duct–specific expression of polyoma middle T oncogene (PymT), and analyzed the effects of single and combined Ctsb and Ctsz deficiencies on breast cancer progression. Single Ctsb deficiency resulted in delayed detection of first tumors and reduced tumor burden, whereas Ctsz-deficient mice had only a prolonged tumor-free period. However, only a trend toward reduced metastatic burden without statistical significance was detected in both single mutants. Strikingly, combined loss of Ctsb and Ctsz led to additive effects, resulting in significant and prominent delay of early and advanced tumor development, improved histopathologic tumor grading, as well as a 70% reduction in the number of lung metastases and an 80% reduction in the size of these metastases. We conclude that the double deficiency of Ctsb and Ctsz exerts significant synergistic anticancer effects, whereas the single deficiencies demonstrate at least partial reciprocal compensation.

The role of proinflammatory cytokines in the generation and maintenance of joint pain

The proinflammatory cytokines tumor necrosis factor‐α (TNF‐α) and interleukin‐6 (IL‐6) not only promote and maintain inflammation, they also contribute to the generation and maintenance of inflammatory pain by acting at nociceptive nerve cells. A large proportion of dorsal root ganglion (DRG) neurons express TNF receptors and receptor units for stimulation with IL‐6. In the rat model of antigen‐induced arthritis (AIA), neutralization of TNF‐α by etanercept and infliximab reduced inflammation‐evoked mechanical hyperalgesia at the inflamed knee joint. This treatment also attenuated the infiltration of macrophages into the DRGs usually observed during the acute phase of AIA. Intra‐articular application of etanercept reduced the responses of C‐fibers to mechanical stimulation of the inflamed joint but did not influence responses to stimulation of the normal joint. Finally, in cultured DRG neurons TNF‐α increased the proportion of neurons that express the TRPV1 receptor and may thus contribute to the generation of inflammation‐evoked thermal hyperalgesia.

Antinociceptive effects of tumor necrosis factor α neutralization in a rat model of antigen-induced arthritis: Evidence of a neuronal target

OBJECTIVE The reduction of pain in the course of antiinflammatory therapy can result from an attenuation of the inflammatory process and/or from the neutralization of endogenous mediators of inflammation that act directly on nociceptive neurons. The purpose of this study was to investigate whether analgesic effects of the neutralization of tumor necrosis factor alpha (TNFalpha) are due to an attenuation of inflammation or whether direct neuronal effects significantly contribute to pain relief in the course of therapy. METHODS Locomotor and pain-related behavior and histology were assessed in rats with chronic antigen-induced arthritis (AIA) in the knee joint, and the rats were treated with systemic saline, etanercept, or infliximab. The expression of TNF receptors (TNFRs) in dorsal root ganglia was measured using immunohistochemical analysis and polymerase chain reaction. Action potentials were recorded from afferent Adelta fibers and C fibers of the medial knee joint nerve, and etanercept and infliximab were injected intraarticularly into normal or inflamed knee joints (AIA or kaolin/carrageenan-induced inflammation). RESULTS In rats with AIA, both etanercept and infliximab significantly decreased inflammation-induced locomotor and pain-related behavior, while joint swelling was only weakly attenuated and histomorphology still revealed pronounced inflammation. A large proportion of dorsal root ganglion neurons showed TNFRI- and TNFRII-like immunoreactivity. Intraarticular injection of etanercept reduced the responses of joint afferents to mechanical stimulation of the inflamed joint starting 30 minutes after injection, but had no effect on responses to mechanical stimulation of the uninflamed joint. CONCLUSION Overall, these data show the pronounced antinociceptive effects of TNFalpha neutralization, thus suggesting that reduction of the effects of TNFalpha on pain fibers themselves significantly contributes to pain relief.

False-Positive Findings at Contrast-Enhanced Breast MRI: A BI-RADS Descriptor Study

OBJECTIVE Breast MRI has high sensitivity in breast cancer detection, and the BI-RADS MRI lexicon was a step toward standardized description of lesions. However, false-positive findings occur and lead to unnecessary biopsy. The purpose of this investigation was to identify criteria for false-positive findings in clinical practice. MATERIALS AND METHODS Eligible for investigation were all breast MRI examinations from a consecutive 16-month time period that had histopathologic verification and findings classified as BI-RADS category 4-6 in the initial MRI report. Accordingly, 132 patients with 120 malignant and 31 benign lesions were enrolled. Two blinded observers categorized lesions into mass or nonmass and used BI-RADS to identify descriptor distribution differences between the benign and malignant subgroups. RESULTS The ratio of mass to nonmass lesions differed significantly (p < 0.001) between benign (1.2:1) and malignant (7:1) findings. Seventeen mass and 14 nonmass lesions were false-positive, and 105 mass and 15 nonmass lesions were true-positive. Among mass lesions, it was possible to differentiate malignant and benign lesions on the basis of margin (smooth, irregular, or spiculated) and dynamic enhancement features (p < 0.05). Among nonmass lesions, only stippled enhancement had a significant difference between the subgroups (p < 0.05). Tumor diameter had no influence on the correct diagnosis of nonmass lesions (p = 0.301). Conversely, among mass lesions, false-positive lesions were smaller than true-positive lesions (p = 0.01). CONCLUSION Nonmass lesions were the major cause of false-positive breast MRI findings. BI-RADS descriptors are not sufficient for differentiating benign and malignant nonmass lesions.

Integration of the HPV16 genome does not invariably result in high levels of viral oncogene transcripts

Virus integration into the host genome is a characteristic step during cervical carcinogenesis. Experimental data provide evidence that integration could result in increased levels of oncogene (E6/E7) transcripts. This is the first study in which the level of viral transcripts is correlated to the physical state of the viral genome in cervical intraepithelial neoplasia (CIN) and cervical carcinomas (CxCa). Using the APOT-assay integrate-derived transcripts only were detected in 3/28 (11%) CIN and in 28/55 (51%) carcinomas, respectively. The remaining biopsies contained either episome-derived transcripts only or both mRNA species. SybrGreen real time reverse transcriptase–PCR assays were used to quantify viral gene expression for (i) all transcripts initiated from p97, (ii) full-length E6, (iii) E6*I and (iv) E5 transcripts. E6/E7 transcript levels showed a broad distribution but similar median values irrespective of histopathological grading and physical state of the viral genome. Biopsies with integrate-derived transcripts only generally lacked E5-specific mRNA. Our data do not support the hypothesis that HPV integration invariably results in high levels of oncogene transcripts. Instead, constitutive expression of oncogene transcripts rather than the level of expression appears to be decisive for transformation and the maintenance of the malignant phenotype.

Reduced tumour cell proliferation and delayed development of high-grade mammary carcinomas in cathepsin B-deficient mice

Expression levels of the papain-like cysteine protease cathepsin B (Ctsb) have been positively correlated with mammary tumour progression and metastasis; however, its roles in the hallmark processes of malignant growth remain poorly defined. Using Ctsb-deficient mice we investigated tumour cell differentiation, proliferation and apoptosis in the Tg(MMTV-PyMT) mouse mammary cancer model. Absence of Ctsb significantly impaired development of high-grade invasive ductal carcinomas and reduced the metastatic burden in the lungs. Mice lacking Ctsb exhibited reduced cell proliferation in mammary carcinomas and their lung metastases. Notably, intravenous injection of primarily isolated, Ctsb-expressing tumour cells into congenic Ctsb-deficient mice revealed impaired cell proliferation in the resulting experimental lung metastases, providing evidence for the involvement of Ctsb in paracrine regulation of cancer cell proliferation. No Ctsb genotype-dependent difference in tumour cell death was observed in vivo or by treatment of isolated PyMT cancer cells with tumour necrosis factor-α. However, cancer cells lacking Ctsb exhibited significantly higher resistance to apoptosis induction by the lysosomotropic agent Leu-Leu-OMe. Thus, our results indicate an in vivo role for Ctsb in promoting cellular anaplasia in mammary cancers and proliferation in lung metastases.

Sensitivity and specificity of unenhanced MR mammography (DWI combined with T2-weighted TSE imaging, ueMRM) for the differentiation of mass lesions

ObjectiveThis study was performed to assess the sensitivity and specificity for malignant and benign mass lesions of a diagnostic approach combining DWI with T2-weighted images (unenhanced MR mammography, ueMRM) and compare the results with contrast-enhanced MR mammography (ceMRM).Materials and methodsConsecutive patients undergoing histopathological verification of mass lesions after MR mammography without prior breast interventions (contrast-enhanced T1-weighted, T2-weighted and DWI sequences) were eligible for this retrospective investigation. Two blinded observers first rated ueMRM and then ceMRM according to the BIRADS scale. Lesion size, ADC values and T2-weighted TSE descriptors were assessed.ResultsThis study examined 81 lesions (27 benign, 54 malignant). Sensitivity of ueMRM was 93% (observer 1) and 86% (observer 2), respectively. Sensitivity of ceMRM was 96.5% (observer 1) and 98.3% (observer 2). Specificity was 85.2% (ueMRM) and 92.6% (ceMRM) for both observers. The differences between both methods and observers were not significant (P ≥ 0.09). Lesion size measurements did not differ significantly among all sequences analyzed. Tumor visibility was worse using ueMRM for both benign (P < 0.001) and malignant lesions (P = 0.004).ConclusionSensitivity and specificity of ueMRM in mass lesions equal that of ceMRM. However, a reduced lesion visibility in ueMRM may lead to more false-negative findings.