Katharina Glatz

Two mitosis-specific antibodies, MPM-2 and phospho-histone H3 (Ser28), allow rapid and precise determination of mitotic activity.

Mitotic figure (MF) counting is important in the evaluation of many tumor types. Inadequate fixation, crush artefacts, the presence of many apoptoses, or the rarity of MFs in a given lesion can make the determination of the mitotic index a very time-consuming or even impossible task, especially for novices. We evaluated the potential of the two commercially available mitotic markers MPM-2 and Phospho-Histone H3 Ser28 (PHH3) for improving mitotic counting. Formalin-fixed tissue of 1 lymphoma, 19 epithelial, 25 mesenchymal, and 10 melanocytic tumors was immunohistochemically stained with both antibodies. Mitotic counts of each tumor sample were determined by a pathologist and three residents in the hematoxylin and eosin and in both immunohistochemical stainings. Because of the higher sensitivity of the immunohistochemical stainings for MFs, average mitotic counts per 10 HPF were higher with MPM-2 (11.0) and PHH3 (10.1) than with hematoxylin and eosin (5.9) staining. The precise distinction of MFs from apoptoses and the visualization of the distribution of MFs uncovering mitotic hotspots, even at low magnification, turned out to be major advantages of both mitotic markers. In addition, the average time needed to establish the mitotic count was reduced by 40.3% with MPM-2 and by 50.4% with PHH3. MPM-2 and PHH3 were subjectively rated by all pathologists involved in this study to be very helpful in mitotic counting, especially in melanocytic and mesenchymal lesions but less so in epithelial tumors. Both markers have hence been successfully introduced in our laboratory for the routine assessment of MFs in melanocytic and mesenchymal tumors.

Trastuzumab emtansine (T-DM1) renders HER2+ breast cancer highly susceptible to CTLA-4/PD-1 blockade

An antibody-drug conjugate overcomes resistance to immune checkpoint blockade in HER2-positive breast cancer. Targeted therapy with more punch Overexpression of the human epidermal growth factor receptor 2 (HER2) is common in breast cancer, and it is associated with poor outcomes despite the availability of trastuzumab, an antibody against HER2, and other HER2-targeted agents. The reason for the poor outcomes is that many patients develop resistance to the targeted drugs. Müller et al. have now shown that this resistance can be overcome with trastuzumab emtansine, an antibody-drug conjugate that combines the HER2-targeting ability of trastuzumab with a cytotoxic drug, which the antibody delivers directly to the tumor. In addition to its cytotoxic effects, treatment with trastuzumab emtansine activated a strong antitumor immune response and effectively combined with immune checkpoint inhibitors, suggesting that it can be used in combination therapy. Targeted drug delivery with antibody-drug conjugates such as the HER2-directed ado-trastuzumab emtansine (T-DM1) has emerged as a powerful strategy for cancer therapy. We show that T-DM1 is particularly effective in eliciting antitumor immunity in patients with early breast cancer (WSG-ADAPT trial) and in a HER2-expressing orthotopic tumor model. In the latter, despite primary resistance to immunotherapy, combined treatment with T-DM1 and anti–CTLA-4/PD-1 (cytotoxic T lymphocyte–associated protein-4/programmed cell death protein-1) was curative because it triggered innate and adaptive immunity. Tumor rejection was accompanied by massive T cell infiltration, TH1 (T helper 1) cell polarization, and, notably, a substantial increase in regulatory T cells. Depletion of regulatory T cells resulted in inflammation and tissue damage, implying their essential role in protecting the host during therapy. This study provides insights into the mechanisms of T-DM1’s therapeutic activity and a rationale for potential therapeutic combination strategies with immunotherapy.

A multinational, internet-based assessment of observer variability in the diagnosis of serrated colorectal polyps.

This Internet-based quiz (http://kathrin.unibas.ch/polyp/) tested the diagnostic variability of 168 pathologists in the diagnosis of 20 colorectal polyps on 3 representative images, including hyperplastic polyps (HPs), traditional serrated adenomas (TSAs), sessile serrated adenomas (SSAs), and tubulovillous adenomas (TVAs). Interobserver variability for each of the 20 lesions was significant and was most pronounced for SSAs. Correct answers were independent of the participants experience with TVAs, HPs, and TSAs. Participants with gastrointestinal subspecialty training and those who had read a reference article on serrated polyps gave a significantly higher percentage of correct answers for SSAs. The nomenclature used for serrated polyps was generally inconsistent. Our results suggest significant shortcomings in the routine H&E diagnosis of serrated colorectal polyps. A diagnostically unifying concept for lesions of the serrated neoplasia pathway, standardization of nomenclature, training of pathologists, and possibly development of ancillary techniques are of paramount importance for accurate patient management.

Close association between HER-2 amplification and overexpression in human tumors of non-breast origin

The relationship between HER-2 overexpression and gene amplification is well evaluated in breast cancers but remains unclear or controversial in many other tumor entities. Therefore, we tested the HER-2 status in more than 120 different tumor entities. 5751 tumor samples were analyzed on TMAs by immunohistochemistry (Hercept-Test, DAKO) and fluorescence in situ hybridization (PathVysion, Abbott-Vysis) under highly standardized conditions. HER-2 overexpression (score 2/3+) and amplification occurred most often in breast cancers but was also seen in 18 other tumor entities including cancers of the urinary bladder (amplification in 14.3%, overexpression in 6.7%), stomach (8.3/4.9%), endometrium (6.6/6.8%), lung (2.8/3.1%) and ovary (2.3/1.2%). Remarkably, a strong association between overexpression and amplification was seen in all examined cancer entities. Trastuzumab therapy is highly efficient in HER-2 amplified breast cancer both in metastatic disease and as an adjuvant therapy. A variety of other tumor entities including frequent neoplasms and cancers with often limited therapeutic options have similar patterns of HER-2 alterations as observed in breast cancer (ie high overexpression due to high level gene amplification). Such tumor entities should be carefully evaluated for a possible utility of trastuzumab treatment.

Sequence analysis and high-throughput immunhistochemical profiling of KIT (CD 117) expression in uveal melanoma using tissue microarrays

We aimed to immunohistochemically examine the expression of KIT (CD 117) in human posterior uveal melanoma and to analyze KIT-positive tumors for gene mutations. Brought into a tissue microarray (TMA) format were 101 formalin-fixed, paraffin-embedded posterior uveal melanomas. Immunhistochemistry was performed using the polyclonal anti-CD117 antibody from Dako (A4502). In ten selected KIT-positive tumors, exons 2, 8, 9, 11, 13 and 17 were sequenced. Of the 101 cases, 89 (88%) could be evaluated on the TMAs. Immunohistochemistry for CD 117 was weakly positive in 5 cases (6%), moderately positive in 10 cases (12%) and strongly positive in 57 cases (69%). No KIT mutations were detected in the analyzed exons. In conclusion, human posterior uveal melanoma frequently expresses CD117 at high levels. Although KIT mutations could not be found, it appears justified to investigate the utility of imatinib mesylate in the treatment of these patients.

Frequent Mitotic Activity in Banal Melanocytic Nevi Uncovered by Immunohistochemical Analysis

The presence and distribution of mitotic figures is an important discriminatory parameter in the assessment of melanocytic lesions. We evaluated the number and distribution of mitotic figures in 353 randomly collected melanocytic nevi of various subtypes by hematoxylin and eosin (H&E) staining and immunohistochemically with the 2 mitotic markers Phospho-Histone H3 Ser28 (PHH3) and MPM2. At least 1 mitotic figure was present in 19.5%, 31.3%, and 42.8% of H&E-, PHH3-, and MPM2-stained lesions, respectively. In common compound nevi, the mean number of dermal mitoses amounted to 0.024/mm2 dermal surface area in the H&E staining (PHH3: 0.061; MPM2: 0.087) and to 0.175/mm2 in Spitz nevi (PHH3: 0.325; MPM2: 0.45). Nevi exhibiting mitotic figures were significantly more frequent in the youngest age group (0-20 years) than in patients older than 50 years (P < 0.0001). In the upper half of the dermis, mitotic activity was roughly 3 times as frequent as compared with the lower half. Clusters of mitotic figures within the dermis were not observed. Mitotic activity in obviously benign melanocytic nevi is not rare even in the deep dermal part. More than 2 mitotic figures per lesion can usually be explained either by the nevus subtype, young patient age, traumatization, or inflammation. PHH3 and MPM2 are a valuable diagnostic adjunct in the evaluation of melanocytic tumors allowing more sensitive and faster recognition of mitotic figures and their distribution.

Interobserver agreement on what constitutes visceral pleural invasion by non-small cell lung carcinoma : An internet-based assessment of international current practices

Visceral pleural invasion (VPI) increases the T category of non-small cell lung carcinomas (NSCLCs) that otherwise only meet T1 criteria to T2. The American Joint Committee on Cancer provides no guidelines on what constitutes VPI. Penetration beyond the visceral pleural elastic layer (VPEL) has been proposed as the minimum criterion but is not internationally accepted. The purposes of this study were to elucidate current international practices regarding assessment of VPI and identify histologic features pathologists consider necessary for VPI. We examined responses to an online quiz consisting of 15 NSCLCs adjacent to or involving the visceral pleura. Of 103 participants from 22 countries, 84.5% were in academic practice; 42.7% had a subspecialty interest in pulmonary pathology. Interobserver percentage agreement about whether VPI was present, absent, or indeterminate ranged from 36.9% to 93.2% (mean, 73.0%). The K for participants for all quiz cases was 0.35. There was considerable diagnostic variability in cases with extensive pleural elastosis and when tumor cells were intermingled with the VPEL. It seems that the majority of participants consider penetration of the VPEL necessary and also sufficient to categorize VPI as present. However, the formation of internationally recognized guidelines for assessing VPI by NSCLC is likely to improve diagnostic consensus.

An online quiz uncovers limitations of morphology in equivocal lung cytology

Equivocal atypia in respiratory cytology can be a diagnostic challenge. In such cases fluorescence in situ hybridization (FISH) may be used for the analysis of chromosomal aberrations and often allows a reliable distinction of benign and malignant cells.

Expression and amplification of therapeutic target genes in retinoblastoma

PurposeWe set out to evaluate alterations of the therapeutic target genes KIT (CD 117), EGFR, and HER-2 in human retinoblastoma.MethodsNinety-five formalin-fixed, paraffin-embedded retinoblastomas were brought into a tissue microarray (TMA) format. Immunohistochemistry was performed to analyze the expression of CD117, EGFR, and HER-2. Fluorescence in situ hybridization (FISH) was utilized for detection of EGFR amplifications. Three tumors with strong CD117 positivity were sequenced for KIT exon 11 mutations.ResultsDetectable CD117 expression was seen in 19% of all interpretable cases. Sequence analysis of the three tumors with the strongest CD117 expression revealed no mutations. EGFR was positive in 14% of all cases. No EGFR amplification was observed by FISH, however. All tumors were negative for HER-2 expression.ConclusionsOur data suggest that selected cases of retinoblastoma may be candidates for anti-EGFR and imatinib mesylate (STI571) therapy.

Noninvasive Contrast-Enhanced Ultrasound Molecular Imaging Detects Myocardial Inflammatory Response in Autoimmune Myocarditis

Background—Cardiac tests for diagnosing myocarditis lack sensitivity or specificity. We hypothesized that contrast-enhanced ultrasound molecular imaging could detect myocardial inflammation and the recruitment of specific cellular subsets of the inflammatory response in murine myocarditis. Methods and Results—Microbubbles (MB) bearing antibodies targeting lymphocyte CD4 (MBCD4), endothelial P-selectin (MBPSel), or isotype control antibody (MBIso) and MB with a negative electric charge for targeting of leukocytes (MBLc) were prepared. Attachment of MBCD4 was validated in vitro using murine spleen CD4+ T cells. Twenty-eight mice were studied after the induction of autoimmune myocarditis by immunization with &agr;-myosin-peptide; 20 mice served as controls. Contrast-enhanced ultrasound molecular imaging of the heart was performed. Left ventricular function was assessed by conventional and deformation echocardiography, and myocarditis severity graded on histology. Animals were grouped into no myocarditis, moderate myocarditis, and severe myocarditis. In vitro, attachment of MBCD4 to CD4+ T cells was significantly greater than of MBIso. Of the left ventricular ejection fraction or strain and strain rate readouts, only longitudinal strain was significantly different from control animals in severe myocarditis. In contrast, contrast-enhanced ultrasound molecular imaging showed increased signals for all targeted MB versus MBIso both in moderate and severe myocarditis, and MBCD4 signal correlated with CD4+ T-lymphocyte infiltration in the myocardium. Conclusions—Contrast-enhanced ultrasound molecular imaging can detect endothelial inflammation and leukocyte infiltration in myocarditis in the absence of a detectable decline in left ventricular performance by functional imaging. In particular, imaging of CD4+ T cells involved in autoimmune responses could be helpful in diagnosing myocarditis.