David L. Wachter

Ki67, chemotherapy response, and prognosis in breast cancer patients receiving neoadjuvant treatment

BackgroundThe pathological complete response (pCR) after neoadjuvant chemotherapy is a surrogate marker for a favorable prognosis in breast cancer patients. Factors capable of predicting a pCR, such as the proliferation marker Ki67, may therefore help improve our understanding of the drug response and its effect on the prognosis. This study investigated the predictive and prognostic value of Ki67 in patients with invasive breast cancer receiving neoadjuvant treatment for breast cancer.MethodsKi67 was stained routinely from core biopsies in 552 patients directly after the fixation and embedding process. HER2/neu, estrogen and progesterone receptors, and grading were also assessed before treatment. These data were used to construct univariate and multivariate models for predicting pCR and prognosis. The tumors were also classified by molecular phenotype to identify subgroups in which predicting pCR and prognosis with Ki67 might be feasible.ResultsUsing a cut-off value of > 13% positively stained cancer cells, Ki67 was found to be an independent predictor for pCR (OR 3.5; 95% CI, 1.4, 10.1) and for overall survival (HR 8.1; 95% CI, 3.3 to 20.4) and distant disease-free survival (HR 3.2; 95% CI, 1.8 to 5.9). The mean Ki67 value was 50.6 ± 23.4% in patients with pCR. Patients without a pCR had an average of 26.7 ± 22.9% positively stained cancer cells.ConclusionsKi67 has predictive and prognostic value and is a feasible marker for clinical practice. It independently improved the prediction of treatment response and prognosis in a group of breast cancer patients receiving neoadjuvant treatment. As mean Ki67 values in patients with a pCR were very high, cut-off values in a high range above which the prognosis may be better than in patients with lower Ki67 values may be hypothesized. Larger studies will be needed in order to investigate these findings further.

On a dark-field signal generated by micrometer-sized calcifications in phase-contrast mammography

We show that a distribution of micrometer-sized calcifications in the human breast which are not visible in clinical x-ray mammography at diagnostic dose levels can produce a significant dark-field signal in a grating-based x-ray phase-contrast imaging setup with a tungsten anode x-ray tube operated at 40 kVp. A breast specimen with invasive ductal carcinoma was investigated immediately after surgery by Talbot-Lau x-ray interferometry with a design energy of 25 keV. The sample contained two tumors which were visible in ultrasound and contrast-agent enhanced MRI but invisible in clinical x-ray mammography, in specimen radiography and in the attenuation images obtained with the Talbot-Lau interferometer. One of the tumors produced significant dark-field contrast with an exposure of 0.85 mGy air-kerma. Staining of histological slices revealed sparsely distributed grains of calcium phosphate with sizes varying between 1 and 40 μm in the region of this tumor. By combining the histological investigations with an x-ray wave-field simulation we demonstrate that a corresponding distribution of grains of calcium phosphate in the form of hydroxylapatite has the ability to produce a dark-field signal which would-to a substantial degree-explain the measured dark-field image. Thus we have found the appearance of new information (compared to attenuation and differential phase images) in the dark-field image. The second tumor in the same sample did not contain a significant fraction of these very fine calcification grains and was invisible in the dark-field image. We conclude that some tumors which are invisible in x-ray absorption mammography might be detected in the x-ray dark-field image at tolerable dose levels.

Impaired cell fusion and differentiation in placentae from patients with intrauterine growth restriction correlate with reduced levels of HERV envelope genes

One leading cause of perinatal morbidity and mortality is intrauterine growth restriction (IUGR). Several causes for IUGR have been proposed involving cytotrophoblast dysfunction. Envelope genes of the human endogenous retrovirus (HERV)-W (Syncytin-1), -FRD (Syncytin-2), and -P(b) have fusogenic properties, whereas envelope genes of HERV-R, -V1, and -V2 have putative placental functions. All six HERV envelope genes and three known cellular receptors were analyzed for expression in human control and IUGR placentae (n = 38) and in cultured cytotrophoblasts from control and IUGR (n = 8) placentae. All envelope genes demonstrated downregulation in IUGR compared to control placentae tissues, which were confirmed with cultured cytotrophoblasts. Examination of the Syncytin-1 and Syncytin-2 receptors ASCT-1/-2 and MFSD2 showed that MFSD2 was significantly expressed lower in IUGR than in control placentae and cytotrophoblasts. A reduction of Syncytin-1 protein expression was confirmed for IUGR placentae with immunoblotting and paraffin tissue sections. Embedded placental IUGR tissues showed an overall disorganized syncytiotrophoblast layer with fewer nuclei. Cytotrophoblasts from IUGR placentae demonstrated a lower cell fusion index and nuclei per syncytiotrophoblast in vitro. Fusogenic and non-fusogenic envelope genes are dysregulated in IUGR placentae and may contribute to the etiology of growth restriction in utero.

DNA methylation outliers in normal breast tissue identify field defects that are enriched in cancer

Identifying molecular alterations in normal tissue adjacent to cancer is important for understanding cancer aetiology and designing preventive measures. Here we analyse the DNA methylome of 569 breast tissue samples, including 50 from cancer-free women and 84 from matched normal cancer pairs. We use statistical algorithms for dissecting intra- and inter-sample cellular heterogeneity and demonstrate that normal tissue adjacent to breast cancer is characterized by tens to thousands of epigenetic alterations. We show that their genomic distribution is non-random, being strongly enriched for binding sites of transcription factors specifying chromatin architecture. We validate the field defects in an independent cohort and demonstrate that over 30% of the alterations exhibit increased enrichment within matched cancer samples. Breast cancers highly enriched for epigenetic field defects, exhibit adverse clinical outcome. Our data support a model where clonal epigenetic reprogramming towards reduced differentiation in normal tissue is an important step in breast carcinogenesis.

SMARCB1(INI1)-deficient Sinonasal Basaloid Carcinoma A Novel Member of the Expanding Family of SMARCB1-deficient Neoplasms

Poorly differentiated sinonasal carcinomas are a heterogenous group of aggressive neoplasms that encompasses squamous cell carcinoma including basaloid variant, lymphoepithelial carcinoma, sinonasal undifferentiated carcinoma, and neuroendocrine-type small cell carcinoma. We herein describe 3 cases of a hitherto unreported variant combining features of basaloid carcinoma with variable intermingled rhabdoid cells. Patients were 2 women (aged 28 and 35) and a man (52 y) who presented with sinonasal masses. All had advanced local disease with bone involvement (pT4). None had a history of irradiation or a family history of rhabdoid tumors. Treatment was surgery and adjuvant chemoradiation. One patient developed liver, lung, pleural, and pericardial metastases (63 mo) and is currently (70 mo) alive under palliative treatment. Another developed recurrent cervical lymph node metastases and died of disease 8.5 years later. The youngest patient was disease-free at last follow-up 7 years later. Histologic features were very similar in all 3 cases and showed intimate admixture of compact basaloid cell nests with peripheral palisading, perivascular pseudorosettes, and a few scattered rhabdoid cells. Rhabdoid cells were more extensive in the metastasis in 1 case but formed a minor inconspicuous component in the primary tumors in all cases. Striking features common to all cases were (1) basaloid “blue” appearance at low power, (2) papilloma-like exophytic component, (3) extensive pagetoid surface growth with prominent denuding features, and (4) replacement of underlying mucous glands mimicking an inverted papilloma. Clear-cut origin from benign papilloma and overt squamous differentiation were lacking. Diffuse (2) or partial (1) p16 expression was noted, but all cases lacked human papillomavirus DNA by molecular tests. In situ hybridization was negative for Epstein-Barr virus. Immunohistochemistry showed diffuse expression of pancytokeratin. CK5 and vimentin showed intermingling of CK5+/vimentin− basaloid and CK5−/vimentin+ rhabdoid cells. Complete loss of nuclear SMARCB1 expression was seen in all cases including also the denuding carcinoma in situ–like surface lesions. To our knowledge, this variant of sinonasal carcinoma has not been reported before. The identical features in all 3 cases suggest a specific disease rather than a nonspecific dedifferentiated phenotype. Awareness of this rare variant and thus reporting of additional cases is necessary for defining its full morphologic and biological spectrum.

Retransplantation of Cryopreserved Ovarian Tissue: the First Live Birth in Germany

BACKGROUND Cryopreserved ovarian tissue can be retransplanted to restore fertility after radiation or chemotherapy. To date, 15 live births after retransplantation have been reported worldwide. We report the first pregnancy and the first live birth after retransplantation in Germany. CASE REPORT A 25-year-old female patient received initial chemotherapy and radiation of the mediastinum for Hodgkins lymphoma in 2003 and suffered a relapse two years later. Ovarian tissue was laparoscopically removed and cryopreserved, and she was then treated with high-dose chemotherapy and stem cell transplantation. She remained in remission for 5 years and she could not conceive during this time. The cryopreserved ovarian tissue was thawed and laparoscopically retransplanted into a peritoneal pouch in the ovarian fossa of the right pelvic wall. Three months later, her menopausal symptoms resolved, and she had her first spontaneous menstruation. Six months after retransplantation, after two normal menstrual cycles, low-dose follicle stimulating hormone (FSH) treatment induced the appearance of a dominant follicle in the tissue graft. Ovulation was then induced with human chorionic gonadotropin (HCG), whereupon the patient conceived naturally. After an uncomplicated pregnancy, she bore a healthy child by Caesarean section on 10 October 2011. Histological examination of biopsy specimens revealed that the ovarian tissue of the graft contained follicles in various stages of development, while the original ovaries contained only structures without any reproductive potential. CONCLUSION This was the first live birth after retransplantation of cryopreserved ovarian tissue in Germany and also the first case with histological confirmation that the oocyte from which the patient conceived could only have come from the retransplanted tissue. In general, young women who will be undergoing chemotherapy and/or radiotherapy for cancer must be informed and counseled about the available options for fertility preservation.

Grating-based darkfield imaging of human breast tissue

Mastectomy specimens were investigated using a Talbot-Lau X-ray imaging set-up. Significant structures in the darkfield were observed, which revealed considerably higher contrast than those observed in digital mammography. Comparison with the histomorphometric image proofs that the darkfield signal correlates with a tumor region containing small calcification grains of 3 to 30μm size.

Diagnostic Value of Immunohistochemical IMP3 Expression in Core Needle Biopsies of Pancreatic Ductal Adenocarcinoma

The oncofetal protein, insulin-like growth factor II messenger ribonucleic acid-binding protein 3 (IMP3), has been analyzed in many different tumors. Various studies have found that IMP3 is a marker for malignancy and is correlated with increased tumor aggressiveness and reduced overall survival. The diagnosis of pancreatic ductal adenocarcinoma (PDAC) in core needle biopsies can be challenging, and immunohistochemical markers are needed. We studied IMP3 expression in 177 core needle biopsies of the pancreas, including 112 PDACs, 55 cases with chronic sclerosing pancreatitis, and 10 biopsies with tumor-free pancreatic tissue without inflammation. An additional 18 biopsies of PDAC metastases (16 liver biopsies and 2 lymph node biopsies) were analyzed. To study IMP3 expression in large tissue sections, 45 pancreatic resection specimens (26 with PDAC and 19 with chronic sclerosing pancreatitis) were investigated. In contrast to normal or inflamed pancreatic tissue, which was negative in 47 of 65 (72.3%) cases and weakly positive in 15 of 65 (23.1%) cases, strong IMP3 expression was found in 99 of 112 (88.4%) PDACs. Therefore, sensitivity and specificity of IMP3 expression in the differential diagnosis of PDAC and chronic sclerosing pancreatitis using core needle biopsies were found to be 88.4% and 94.6%, respectively. These results were confirmed in the pancreas resection specimens. Furthermore, strong IMP3 expression was found in 17 of 18 (94.4%) of the PDAC metastases that were analyzed. Our study shows that IMP3 is an easy to use and potentially new immunohistochemical marker for the diagnosis of PDAC in core needle biopsies.

Xenotransplantation of cryopreserved ovarian tissue from patients with ovarian tumors into SCID mice—no evidence of malignant cell contamination

This study examined the possible presence of malignant cells in ovarian cortex from patients with ovarian tumors after xenografting of the ovarian tissue into severe combined immunodeficiency mice. None of the mice presented symptoms of reintroduced malignancy nor did microscopic and immunohistochemical evaluation of the grafts raise any suspicion of residual malignant disease.

Akt and p53 are potential mediators of reduced mammary tumor growth by Chloroquine and the mTOR inhibitor RAD001

PI3K/Akt/mTOR and p53 signaling pathways are frequently deregulated in tumors. The anticancer drug RAD001 (everolimus) is a known mTOR-inhibitor, but mTOR-inhibition leads to phosphorylation of Akt inducing resistance against RAD001 treatment. There is growing evidence that conflicting signals transduced by the oncogene Akt and the tumorsuppressor p53 are integrated via negative feedback between the two pathways. We previously showed that the anti-malarial Chloroquine, a 4-alkylamino substituted quinoline, is a p53 activator and reduced the incidence of breast tumors in animal models. Additionally, Chloroquine is an effective chemosensitizer when used in combination with PI3K/Akt inhibitors but the mechanism is unknown. Therefore, our aim was to test, if Chloroquine could inhibit tumor growth and prevent RAD001-induced Akt activation. Chloroquine and RAD001 caused G1 cell cycle arrest in luminal MCF7 but not in mesenchymal MDA-MB-231 breast cancer cells, they significantly reduced MCF7 cell proliferation on a collagen matrix and mammospheroid formation. In a murine MCF7 xenograft model, combined treatment of Chloroquine and RAD001 significantly reduced mammary tumor growth by 4.6-fold (p = 0.0002) compared to controls. Chloroquine and RAD001 inhibited phosphorylation of mTOR and its downstream target, S6K1. Furthermore, Chloroquine was able to block the RAD001-induced phosphorylation of Akt serine 473. The Chloroquine effect of overcoming the RAD001-induced activation of the oncogene Akt, as well as the promising antitumor activity in our mammary tumor animal model present Chloroquine as an interesting combination partner for the mTOR-inhibitor RAD001.