Dichotomous ALK-IHC Is a Better Predictor for ALK Inhibition Outcome than Traditional ALK-FISH in Advanced Non-Small Cell Lung Cancer.

Purpose: ALK rearrangement detection using FISH is the standard test to identify patients with non-small cell lung carcinoma (NSCLC) eligible for treatment with ALK inhibitors. Recently, ALK protein expression in resectable NSCLC showed predictive value. We evaluated tumor response rate and survival after crizotinib treatment of patients with advanced NSCLC with ALK activation using both dichotomous immunohistochemical (IHC) staining and FISH.Experimental Design: Patients with stage IV NSCLC treated with crizotinib were selected. Tumor response was assessed. ALK rearrangements were detected by FISH (Vysis ALK-break-apart FISH-Probe KIT) and IHC [Ventana ALK (D5F3) CDx assay]. Cohorts of patients with ALK-FISH-positive advanced NSCLC from four other hospitals were used for validation.Results: Twenty-nine consecutive patients with ALK-positive advanced NSCLC diagnosed by FISH and/or IHC on small biopsies or fine-needle aspirations (FNA) were treated with ALK inhibitors. All ALK-IHC-positive patients responded to crizotinib except three with primary resistance. No tumor response was observed in 13 ALK-FISH-positive but ALK-IHC-negative patients. This was confirmed in an external cohort of 16 patients. Receiver operator characteristic (ROC) curves for ALK-IHC and ALK-FISH compared with treatment outcome showed that dichotomous ALK-IHC outperforms ALK-FISH [tumor response area under the curve: (AUC), 0.86 vs. 0.64, P = 0.03; progression-free survival (PFS): AUC 0.86 vs. 0.36, P = 0.005; overall survival (OS): AUC, 0.78 vs. 0.41, P = 0.01, respectively].Conclusions: Dichotomous ALK-IHC is superior to ALK-FISH on small biopsies and FNA to predict tumor response and survival to crizotinib for patients with advanced NSCLC. Our data strongly suggest adapting the guidelines and using dichotomous ALK-IHC as standard companion diagnostic test to select patients with NSCLC who benefit from ALK-targeting therapy. Clin Cancer Res; 23(15); 4251-8. ©2017 AACR.

Bilateral breast cancer, synchronous and metachronous; differences and outcome.

The aims of this study were twofold: to analyze the incidence of patients having synchronous or metachronous bilateral invasive breast cancer (SBBC and MBBC) and to assess the characteristics and outcome compared to those having unilateral breast cancer (UBC). The used data were obtained from our prospective population-based cohort study which had been started in 1983. Bilateral breast cancer (BBC) was categorized as SBBC (≤3 months of the first primary) or MBBC (>3 months after the first primary). The incidence of SBBC was 1% and that of MBBC 7.0 %. Patients with UBC showed more ductal carcinoma compared to patients with BBC. MBBC status was an independent significant predictor of local failure (HR 1.9; 95% CI 1.3-2.7). SBBC status was an independent predictor of distant metastases (HR 2.6; 95% CI 1.4-4.5). Overall survival (OS) was better for MBBC (HR 0.6; 95% CI 0.4-0.8) and worse for SBBC (HR 2.3; 95% CI 1.5-3.6) compared to UBC. We noted: (1) MBBC showed a significant higher local failure compared to UBC, (2) SBBC, compared to MBBC and UBC had a significant higher distant metastases rate, (3) disease-specific survival and OS were significantly worse for SBBC compared to UBC and MBBC, and (4) that the OS for MBBC compared to UBC, was significantly better.

Quality assessment of estrogen receptor and progesterone receptor testing in breast cancer using a tissue microarray-based approach.

Assessing hormone receptor status is an essential part of the breast cancer diagnosis, as this biomarker greatly predicts response to hormonal treatment strategies. As such, hormone receptor testing laboratories are strongly encouraged to participate in external quality control schemes to achieve optimization of their immunohistochemical assays. Nine Dutch pathology departments provided tissue blocks containing invasive breast cancers which were all previously tested for estrogen receptor and/or progesterone receptor expression during routine practice. From these tissue blocks, tissue microarrays were constructed and tested for hormone receptor expression. When a discordant result was found between the local and TMA result, the original testing slide was revised and staining was repeated on a whole-tissue block. Sensitivity and specificity of individual laboratories for testing estrogen receptor expression were high, with an overall sensitivity and specificity [corrected] of 99.7 and 95.4%, respectively. Overall sensitivity and specificity of progesterone receptor testing were 94.8 and 92.6%, respectively. Out of 96 discordant cases, 36 cases would have been concordant if the recommended cut-off value of 1% instead of 10% was followed. Overall sensitivity and specificity of estrogen and progesterone receptor testing were high among participating laboratories. Continued enrollment of laboratories into quality control schemes is essential for achieving and maintaining the highest standard of care for breast cancer patients.

KRAS mutation analysis: a comparison between primary tumours and matched liver metastases in 305 colorectal cancer patients.

BACKGROUND: KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor antibody in metastatic colorectal cancer (CRC). KRAS mutation analysis is usually performed on primary tumour tissue because metastatic tissue is often not available. However, controversial data are available on the concordance of test results between primary tumours and corresponding metastases. We assessed the concordance of KRAS mutation status in a study of 305 primary colorectal tumours and their corresponding liver metastases. METHODS: Patients with histologically confirmed CRC who underwent surgical resection of the primary tumour and biopsy or surgical resection of the corresponding liver metastasis were included. KRAS mutation analysis was performed for codons 12 and 13. RESULTS: KRAS mutation was detected in 108 out of 305 primary tumours (35.4%). In 11 cases (3.6%), we found a discordance between primary tumour and metastasis: 5 primary tumours had a KRAS mutation with a wild-type metastasis, 1 primary tumour was wild type with a KRAS mutation in the metastasis, and in 5 cases the primary tumour and the metastasis had a different KRAS mutation. CONCLUSION: We observed a high concordance of KRAS mutation status of 96.4% (95% CI 93.6-98.2%) between primary colorectal tumours and their corresponding liver metastases. In only six patients (2.0%; 95% CI 0.7-4.2%), the discordance was clinically relevant. In this largest and most homogenous study to date, we conclude that both primary tumours and liver metastases can be used for KRAS mutation analysis.

The impact of margin status in breast-conserving therapy for lobular carcinoma is age related.

PURPOSE: The aim is to look at the impact of margin status and outcome of invasive lobular carcinoma (ILC) treated with breast-conserving therapy (BCT). METHODS: This manuscript describes an analysis on 330 BCT in 318 patients with ILC. RESULTS: The 12-year local relapse free survival (LRFS) is 89%. In multivariate analysis, positive margin status, age>50 years, contra lateral breast cancer, and adjuvant systemic therapy were significant predictors of local relapse free survival. In a separate analysis limited to a positive margin for invasive carcinoma or carcinoma in situ, only a positive margin for invasive carcinoma was a significant predictor of local relapse free survival. This was limited to women

The relationship between HLA class II polymorphisms and somatic deletions in testicular B cell lymphomas of Dutch patients.

Several risk factors including immune deficiencies, infections, and autoimmune diseases have been established for non-Hodgkin's lymphoma (NHL). For diffuse large B cell lymphoma (DLBCL), the most common type of lymphoma, no risk factors have been described, which may be due to the intrinsic heterogeneity of this disorder. Previously we reported that, in contrast to nodal DLBCLs, the majority of testicular DLBCLs manifested complete loss of HLA-DR and -DQ expression associated with homozygous deletions of the corresponding genes. To determine the correlation between HLA class II polymorphisms and these lymphomas, we applied DNA typing for HLA-DRB1 and HLA-DQB1 on 50 Dutch patients with testicular and 48 with nodal DLBCL and compared the frequencies with a cohort of healthy Dutch controls. Both the patients with nodal and those with testicular DLBCL manifested significantly higher frequencies of HLA-DRB1*15 than the controls (p < 0.018, odds ratio 2.09 and p < 0.013, odds ratio 2.12, respectively). Moreover, a positive association was seen with HLA-DRB1*12 (p = 0.043, odds ratio 4.17) in the patients with testicular DLBCL, and a negative association was seen with HLA-DRB1*07 (p = 0.022, odds ratio 0.13) in the patients with nodal DLBCL. Homozygous deletions of the HLA-DR/DQ region, evaluated by interphase fluorescence in situ hybridization were seen in 20 of 48 testicular tumors. No preferential loss or retention of a particular HLA-DR or -DQ allele was seen because all alleles were at least once retained or involved in a homozygous deletion.

Extensive genetic alterations of the HLA region, including homozygous deletions of HLA class II genes in B-cell lymphomas arising in immune-privileged sites.

In B-cell lymphomas, loss of human leukocyte antigen (HLA) class I and II molecules might contribute to immune escape from CD8(+) and CD4(+) cytotoxic T cells, especially because B cells can present their own idiotype. Loss of HLA expression and the possible underlying genomic alterations were studied in 28 testicular, 11 central nervous system, and 21 nodal diffuse large B-cell lymphomas (DLCLs), the first two sites are considered as immune-privileged sites. The analysis included immunohistochemistry, loss of heterozygosity analysis, and fluorescent in situ hybridization (FISH) on interphase cells and isolated DNA fibers. Total loss of HLA-A expression was found in 60% of the extranodal cases and in 10% of the nodal cases (P <.01), whereas loss of HLA-DR expression was found in 56% and 5%, respectively (P <.01). This was accompanied by extensive loss of heterozygosity within the HLA region in the extranodal DLCLs. In 3 cases, retention of heterozygosity for D6S1666 in the class II region suggested a homozygous deletion. This finding was confirmed by interphase FISH that showed homozygous deletions in the class II genes in 11 of the 18 extranodal lymphomas but in none of the 7 nodal DLCLs (P <.001). Mapping by fiber FISH showed variable deletions that always included HLA-DQ and HLA-DR genes. Hemizygous deletions and mitotic recombinations often involving all HLA genes were found in 13 of 18 extranodal and 2 of 7 nodal lymphomas. In conclusion, a structural loss of HLA class I and II expression might help the B-cell lymphoma cells to escape from immune attack.

Antibodies affecting ion channel function in acquired neuromyotonia, in seropositive and seronegative myasthenia gravis, and in antibody-mediated arthrogryposis multiplex congenita.

A new autoimmune disease affecting the neuromuscular junction has been defined. Acquired neuromyotonia is associated with antibodies to voltage-gated potassium channels that act, at least in part, by reducing potassium channel function with resulting neuronal hyperactivity. This condition is quite frequently associated with thymoma and, in many cases, antibodies to acetylcholine receptors are present as well as antibodies to VGKC. Improvements in techniques and the availability of cloned DNA and recombinant forms of the AChR subunits have led to new observations concerning the specificity and roles of antibodies in myasthenia gravis. The transfection of a cell line with the epsilon subunit means that we can now accurately compare antibodies reactive with adult and fetal human AChR. This may help to determine the relationship between AChR subunit expression in different tissues and the induction of antibodies that bind specifically to the two forms, as well as to clarify the role of antibodies to fetal or adult AChR in causing ocular muscle symptoms. Serum antibodies from a few mothers with obstetric histories of recurrent arthrogryposis multiplex congenita in their babies specifically inhibit the function of fetal AChR. These observations not only explain the cause of some cases of arthrogryposis multiplex congenita, but also suggest that other fetal-specific antibodies might be responsible for other fetal or neonatal conditions. An animal model has been established to enable us to investigate the role of maternal serum factors in causing such disorders. Seronegative MG has been the subject of many studies from our laboratory over the last ten years. The transience of the effects of SNMG plasmas on AChR function strongly suggests that the plasma antibodies do not bind directly to the AChR, but inhibit function by some indirect mechanism. They do not appear to act via the cAMP-dependent protein kinase pathway, and studies are in progress to investigate the involvement of other second messenger systems.

Age-related susceptibility to experimental autoimmune myasthenia gravis: immunological and electrophysiological aspects.

Susceptibility to experimental autoimmune myasthenia gravis (EAMG) was found to decrease with aging in both Lewis and Brown Norway (BN) rats. In this study, the difference in susceptibility between young and aged Lewis and BN rats was used to analyze factors determining the clinical severity of EAMG. The incidence and severity of muscular weakness did not correlate with acetylcholine receptor (AChR) loss nor with the ability of antibodies to interfere with AChR function. Aged rats showed significantly lower anti-rat AChR antibody titers than young rats and developed less severe or no clinical signs of disease. In individual young or aged rats, however, no significant correlation was found between the clinical signs of disease and anti-rat AChR titer. Neuromuscular transmission was found to change with aging as measured by single-fiber electromyography (SFEMG). In aged BN rats, increased jitter and blockings were found even before EAMG induction. Despite this disturbed neuromuscular transmission, these aged BN rats were clinically resistant against induction of EAMG. The results of this study indicate that the age-related susceptibility to EAMG is influenced by factors determined by the immune attack as well as mechanisms at the level of the neuromuscular junction.

Association of arthrogryposis multiplex congenita with maternal antibodies inhibiting fetal acetylcholine receptor function.

Arthrogryposis multiplex congenita (AMC), characterized by multiple joint contractures developing in utero, results from lack of fetal movement. Some cases are genetically determined, but AMC occasionally complicates pregnancy in patients with myasthenia gravis (MG) suggesting involvement of circulating maternal antibodies. We previously demonstrated antibodies that inhibited the function of fetal acetylcholine receptor (AChR) in one healthy woman with an obstetric history of recurrent AMC. Here we study sera from this woman, from one other with a similar history, and from three (one asymptomatic) whose babies had neonatal MG and AMC. All five maternal sera had high titers of antibodies that inhibited alpha-Bungarotoxin (alpha-BuTx) binding to fetal AChR, and their sera markedly inhibited fetal AChR function with little effect on adult AChR function. Moreover, in a further survey, 3 of 20 sera from anti-AChR negative AMC mothers inhibited fetal AChR function significantly at 1:100 dilution. These results demonstrate the role of antibodies to fetal AChR and perhaps other muscle antigens in some cases of AMC. More generally, they suggest that placental transfer of antibodies directed at fetal antigens should be considered as a cause of other recurrent fetal or perinatal disorders.

Arthrogryposis multiplex congenita with maternal autoantibodies specific for a fetal antigen.

Fetal arthrogryposis multiplex congenita (AMC) is characterised by non-progressive multiple joint contractures, which may result in fetal death, and is heterogeneous in origin. It can associate with maternal myasthenia gravis and autoantibodies to muscle acetylcholine receptor (AChR). We found maternal antibodies that selectively inhibit the fetal form of the AChR in a mother who herself had no features of myasthenia gravis. Maternal autoantibodies specific for fetal antigens could be an unrecognised cause of other congenital disorders.