Interobserver variability in the assessment of stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative invasive breast carcinoma influences the association with pathological complete response: the IVITA study.

High stromal tumor-infiltrating lymphocytes (sTILs) in triple-negative breast cancer (TNBC) are associated with pathological complete response (pCR) after neoadjuvant chemotherapy (NAC). Histopathological assessment of sTILs in TNBC biopsies is characterized by substantial interobserver variability, but it is unknown whether this affects its association with pCR. Here, we aimed to investigate the degree of interobserver variability in an international study, and its impact on the relationship between sTILs and pCR. Forty pathologists assessed sTILs as a percentage in digitalized biopsy slides, originating from 41 TNBC patients who were treated with NAC followed by surgery. Pathological response was quantified by the MD Anderson Residual Cancer Burden (RCB) score. Intraclass correlation coefficients (ICCs) were calculated per pathologist duo and Bland-Altman plots were constructed. The relation between sTILs and pCR or RCB class was investigated. The ICCs ranged from -0.376 to 0.947 (mean: 0.659), indicating substantial interobserver variability. Nevertheless, high sTILs scores were significantly associated with pCR for 36 participants (90%), and with RCB class for eight participants (20%). Post hoc sTILs cutoffs at 20% and 40% resulted in variable associations with pCR. The sTILs in TNBC with RCB-II and RCB-III were intermediate to those of RCB-0 and RCB-I, with lowest sTILs observed in RCB-I. However, the limited number of RCB-I cases precludes any definite conclusions due to lack of power, and this observation therefore requires further investigation. In conclusion, sTILs are a robust marker for pCR at the group level. However, if sTILs are to be used to guide the NAC scheme for individual patients, the observed interobserver variability might substantially affect the chance of obtaining a pCR. Future studies should determine the 'ideal' sTILs threshold, and attempt to fine-tune the patient selection for sTILs-based de-escalation of NAC regimens. At present, there is insufficient evidence for robust and reproducible sTILs-guided therapeutic decisions.

Interobserver variability in upfront dichotomous histopathological assessment of ductal carcinoma in situ of the breast: the DCISion study.

Histopathological assessment of ductal carcinoma in situ, a nonobligate precursor of invasive breast cancer, is characterized by considerable interobserver variability. Previously, post hoc dichotomization of multicategorical variables was used to determine the "ideal" cutoffs for dichotomous assessment. The present international multicenter study evaluated interobserver variability among 39 pathologists who performed upfront dichotomous evaluation of 149 consecutive ductal carcinomas in situ. All pathologists independently assessed nuclear atypia, necrosis, solid ductal carcinoma in situ architecture, calcifications, stromal architecture, and lobular cancerization in one digital slide per lesion. Stromal inflammation was assessed semiquantitatively. Tumor-infiltrating lymphocytes were quantified as percentages and dichotomously assessed with a cutoff at 50%. Krippendorff's alpha (KA), Cohen's kappa and intraclass correlation coefficient were calculated for the appropriate variables. Lobular cancerization (KA = 0.396), nuclear atypia (KA = 0.422), and stromal architecture (KA = 0.450) showed the highest interobserver variability. Stromal inflammation (KA = 0.564), dichotomously assessed tumor-infiltrating lymphocytes (KA = 0.520), and comedonecrosis (KA = 0.539) showed slightly lower interobserver disagreement. Solid ductal carcinoma in situ architecture (KA = 0.602) and calcifications (KA = 0.676) presented with the lowest interobserver variability. Semiquantitative assessment of stromal inflammation resulted in a slightly higher interobserver concordance than upfront dichotomous tumor-infiltrating lymphocytes assessment (KA = 0.564 versus KA = 0.520). High stromal inflammation corresponded best with dichotomously assessed tumor-infiltrating lymphocytes when the cutoff was set at 10% (kappa = 0.881). Nevertheless, a post hoc tumor-infiltrating lymphocytes cutoff set at 20% resulted in the highest interobserver agreement (KA = 0.669). Despite upfront dichotomous evaluation, the interobserver variability remains considerable and is at most acceptable, although it varies among the different histopathological features. Future studies should investigate its impact on ductal carcinoma in situ prognostication. Forthcoming machine learning algorithms may be useful to tackle this substantial diagnostic challenge.

Phosphorylated BRCA1 is predominantly located in the nucleus and mitochondria.

Multiple copies of the mitochondrial genome in eukaryotic cells are organized into protein-DNA complexes called nucleoids. Mitochondrial genome repair mechanisms have been reported, but they are less well characterized than their nuclear counterparts. To expand our knowledge of mitochondrial genome maintenance, we have studied the localization of the BRCA1 protein, known to be involved in nuclear repair pathways. Our confocal and immunoelectron microscopy results show that BRCA1 is present in mitochondria of several human cancer cell lines and in primary breast and nasal epithelial cells. BRCA1 localization in mitochondria frequently overlapped that of nucleoids. Small interfering RNA-mediated knockdown of BRCA1 in human cancer cells (confirmed by Western blot) results in decreased nuclear, cytoplasmic, and mitochondrial staining after immunofluorescence microscopy, establishing the specificity of the BRCA1 immunolabeling. Furthermore, using cell fractionation, dephosphorylation, and enzyme protection experiments, we show that a 220-kDa phosphorylated isoform of BRCA1 is enriched in mitochondrial and nuclear fractions but reduced in cytoplasmic subcellular fractions. Submitochondrial fractionation confirmed the presence of BRCA1 protein in isolated mitoplasts. Because phosphorylation of BRCA1 and subsequent changes in subcellular localization are known to follow DNA damage, our data support a universal role for BRCA1 in the maintenance of genome integrity in both mitochondria and nucleus.

The masquerading presentation of a systemic anaplastic large cell lymphoma, ALK positive: a case report and review of the literature.

We report an unusual case of systemic anaplastic large cell lymphoma (ALCL), ALK positive with leukemic involvement in a 57-year-old woman. The patient presented with a fulminant respiratory infection unresponsive to treatment requiring intensive care and ventilatory support. The CT scan demonstrated mediastinal and bilateral lymphadenopathy. On peripheral smear a few atypical lymphocytes were visualized. Based on the increasing number of atypical lymphocytes in the daily peripheral bloodsmears, the diagnosis ALCL was suggested. Definitive diagnosis was made on a bone marrow biopsy, with lymphocytes being immunoreactive for CD30, EMA, and ALK. Leukemic peripheral blood involvement in ALCL is an uncommon clinicopathologic entity with unfavorable prognosis. The case we present is perhaps unusual in that a complete respons was achieved, highlighting the importance of prompt diagnosis and judicious management.

The role of biological markers as predictors of response to preoperative chemotherapy in large primary breast cancer.

The aim of this prospective study was to evaluate biological markers, their correlation with response and outcome, and the change in these markers under the influence of preoperative chemotherapy (PCT) in patients with a large primary breast cancer. One hundred and thirty-five women were treated with PCT, followed by locoregional therapy and adjuvant treatment. Estrogen receptor (ER), progesterone receptor (PgR), HER-2, p53, and cathepsin D were determined by immunohistochemistry (IHC) before and after PCT. The overall response (OR) was 70% and the pathologic complete response (pCR) was 13%. Forty-four percent of the patients could be offered breast-conserving surgery (BCS). At a median follow-up of 50 mo the overall survival is 82% and the disease-free survival is 70%. No local recurrence (LR) has developed following BCS. Invasive ductal carcinoma (IDC) was more frequently ER-negative and HER-2-positive than invasive lobular carcinoma (ILC). P53-negative and ER-negative patients seemed to be more chemosensitive compared to p53-positive patients (74% vs 53%) and ER-positive patients (75% vs 65%), but this difference did not reach statistical significance. A trend toward higher complete pathologic remission rate was seen for ER-negative patients (p = 0.0609). PgR, HER-2, and cathepsin D were not related to response. The pattern of biological markers did not change with PCT, making repeated determination useless.

The role of heregulin-alpha as a motility factor and amphiregulin as a growth factor in wound healing.

Wound healing is a complex process of which growth and motility are essential features. The aim of this study was to search for keratinocyte-derived secreted factors that may play a role in these mechanisms, and their corresponding receptors. Growth and motility factors were purified from conditioned medium from cultured primary keratinocytes. Receptor and growth factor expression profiles were investigated by immunohistochemical, western blotting, and in situ hybridization analysis on cultured keratinocytes and tissue sections derived from chronic wounds. The most potent autocrine growth factor for keratinocytes, which it was possible to purify and sequence from keratinocyte-conditioned medium, is amphiregulin. Its receptor HER-1 is up-regulated on the membranes of keratinocytes lining the edge of the wound. From the same keratinocyte-conditioned medium, heregulin-alpha was purified as a potent motility factor for keratinocytes. Its receptor is HER-3, which is up-regulated on the membranes of keratinocytes lining the edge of the wound and on keratinocytes that had migrated towards the centre of the wound. HER-4 - another receptor for heregulin-alpha - is weakly present in occasional cells near the edge of the wound. The co-receptor for HER-3 and HER-4 is HER-2/neu, which is also present in epidermal cells but not overexpressed. This study shows that heregulin-alpha is a potent motility factor for normal epithelial cells and that it plays a central role in the process of wound healing of stratified epithelia. Heregulin-alpha has already been shown to be the motility factor leading to migration of HER-2/neu-overexpressing breast cancer cells. The role of amphiregulin as a growth factor and of heregulin-alpha as a motility factor for keratinocytes in epidermal and mucosal wound healing parallels their motility and growth induction in carcinogenesis.