Susumu Wakai

ROS1-rearranged lung cancer: a clinicopathologic and molecular study of 15 surgical cases.

Recent discovery of ROS1 gene fusion in a subset of lung cancers has raised clinical interest, because ROS1 fusion–positive cancers are reportedly sensitive to kinase inhibitors. To better understand these tumors, we examined 799 surgically resected non–small cell lung cancers by reverse transcriptase polymerase chain reaction and identified 15 tumors harboring ROS1 fusion transcripts (2.5% of adenocarcinomas). The most frequent fusion partner was CD74 followed by EZR. The affected patients were often younger nonsmoking female individuals, and they had overall survival rates similar to those of the ROS1 fusion–negative cancer patients. All the ROS1 fusion–positive tumors were adenocarcinomas except 1, which was an adenosquamous carcinoma. Histologic examination identified an at least focal presence of either solid growth with signet-ring cells or cribriform architecture with abundant extracellular mucus in 53% of the cases. These 2 patterns are reportedly also characteristic of anaplastic lymphoma kinase (ALK)-rearranged lung cancers, and our data suggest a phenotypic resemblance between the ROS1-rearranged and ALK-rearranged tumors. All tumors except 1 were immunoreactive to thyroid transcription factor-1. Fluorescence in situ hybridization using ROS1 break-apart probes revealed positive rearrangement signals in 23% to 93% of the tumor cells in ROS1 fusion–positive cancers, which were readily distinguished using a 15% cutoff value from 50 ROS1 fusion–negative tumors tested, which showed 0% to 6% rearrangement signals. However, this perfect test performance was achieved only when isolated 3′ signals were included along with classic split signals in the definition of rearrangement positivity. Fluorescence in situ hybridization signal patterns were unrelated to 5′ fusion partner genes. All ROS1 fusion–positive tumors lacked alteration of EGFR, KRAS, HER2, ALK, and RET genes.

Immunohistochemical detection of ROS1 is useful for identifying ROS1 rearrangements in lung cancers

The recent discovery and characterization of an oncogenic ROS1 gene fusion in a subset of lung cancers has raised significant clinical interest because small molecule inhibitors may be effective to these tumors. As lung cancers with ROS1 rearrangements comprise only 1–3% of lung adenocarcinomas, patients with such tumors must be identified to gain optimal benefit from molecular therapy. Recently, immunohistochemical analyses using a novel anti-ROS1 rabbit monoclonal antibody (D4D6) have shown promise for accurate identification of ROS1-rearranged cancers. To validate this finding, we compared the immunostaining results of tissue microarrays (TMAs) containing 17 ROS1-rearranged and 253 ROS1-non-rearranged lung carcinomas. All 17 ROS1-rearranged cancers showed ROS1 immunoreactivity mostly in a diffuse and moderate-to-strong manner with an H-score range of 5–300 (median, 260). In contrast, 69% of ROS1-non-rearranged cancers lacked detectable immunoreactivity, whereas the remaining 31% showed reactivity mainly in a weak or focal manner. The H-score for the entire ROS1-non-rearranged group ranged from 0 to 240 (median, 0). The difference in H-score between the two cohorts was statistically significant, and the H-score cutoff (≥150) allowed optimal discrimination (94% sensitivity and 98% specificity). Similar but slightly less-specific performance was achieved using the extent of diffuse (≥75%) staining or ≥2+ staining intensity as cutoffs. CD74-ROS1 and EZR-ROS1 fusions were significantly associated with at least focal globular immunoreactivity and plasma membranous accentuation, respectively, and these patterns were specific to ROS1-rearranged cases. Although full-length ROS1 is expressed in some ROS1-non-rearranged cases, we showed that establishment of an optimal set of interpretative criteria makes ROS1 immunohistochemistry a valuable method to rapidly and accurately screen lung cancer patients for appropriate molecular therapy.

Anaplastic lymphoma kinase status in rhabdomyosarcomas

Rhabdomyosarcoma is a rare soft tissue sarcoma that typically affects children, adolescents, and young adults. Despite treatment via a multidisciplinary approach, the prognosis of advance-stage rhabdomyosarcomas remains poor, and a new treatment strategy is needed. Anaplastic lymphoma kinase (ALK) is a receptor tyrosine kinase that is a potential target for specific inhibitors. In this study, we investigated 116 rhabdomyosarcomas using a polymer-based ALK immunostaining method and correlated the results with clinicopathological parameters. In addition, we examined ALK status using dual-color fluorescence in situ hybridization, PCR, and sequencing. In immunohistochemical analysis, ALK was detected in 2 (6%) of 33 embryonal rhabdomyosarcomas, 42 (69%) of 61 alveolar rhabdomyosarcomas, and 0 (0%) of 22 other subtypes, including pleomorphic, adult-spindle-cell/sclerosing, and epithelioid variants. Compared with ALK-negative alveolar rhabdomyosarcomas, ALK-positive ones are presented with metastatic spread more frequently and showed a greater extent of myogenin reactivity. Overall survival was not associated with ALK expression. FOXO1 rearrangement was significantly associated with ALK immunoreactivity. The median ALK copy number was greater in ALK-positive tumors than in ALK-negative tumors. Most (93%) cases tested showed no selective increase in the ALK gene dosage. ALK selective amplification and low-level selective gain were noted in one and three cases, respectively. Further, a high-polysomy pattern (≥4 ALK copies in ≥40% of cells) was observed in seven cases. A significant increase in the ALK copy number was exclusive to the ALK-immunopositive cohort, but it was uncommon, accounting for only 30% of the 37 ALK-positive rhabdomyosarcomas. ALK gene rearrangement was not observed in either cohort, while an ALK somatic mutation (I1277T) was found in one ALK-negative embryonal case. Although it remains controversial whether ALK expression without gene rearrangement is therapeutically relevant, this comprehensive analysis may help future studies on the utility of ALK-targeted therapy for patients with rhabdomyosarcoma.

Aberrant anaplastic lymphoma kinase expression in high-grade pulmonary neuroendocrine carcinoma

Aims In lung cancer with anaplastic lymphoma kinase (ALK) gene rearrangement, accurate diagnosis is essential to identify patients who can be treated with a specific kinase inhibitor. Sensitive ALK immunostaining can provide nearly complete concordance with gene rearrangement status and is expected to serve as a surrogate biomarker for tailored treatment with an ALK inhibitor. In the present report, we describe aberrant ALK expression in a small proportion of pulmonary neuroendocrine carcinomas (NECs) that do not have ALK gene alteration. Methods A total of 227 pulmonary NECs were assembled on tissue microarrays and were subjected to highly sensitive ALK staining methods. Results We observed focal positivity with heterogeneous intensity in 2 (2.9%) of 69 small-cell carcinomas and 1 (0.9%) of 106 large-cell NECs. In contrast, 52 carcinoid tumours were all negative for ALK expression. Neither ALK rearrangement nor amplification was observed using fluorescence in situ hybridisation, and no somatic mutation was detected in three ALK positive NECs. Conclusions Thus, this aberrant expression is probably of a wild-type ALK and a potential pitfall when implementing sensitive ALK immunohistochemistry in the molecular diagnosis of lung cancer.

Primary Retroperitoneal Myxoid Liposarcomas.

Myxoid liposarcomas (MLSs) are genetically defined by the presence of DDIT3 gene fusions and most commonly arise in the extremities of young adults. Whether MLSs develop primarily in the retroperitoneum is controversial, and a recent retrospective study found no molecularly confirmed examples. Because MLSs tend to metastasize to deep soft tissues, purported examples of primary retroperitoneal lesions might represent distant metastasis, most commonly from extremities. In addition, well-differentiated or dedifferentiated liposarcomas, which are characterized by MDM2 amplifications, may exhibit prominent myxoid changes and mimic MLSs. Here, we document 5 cases of MLSs that originated in the retroperitoneum that were identified through critical clinicopathologic reevaluation. These cases accounted for 2.3% of 214 primary retroperitoneal liposarcomas and 3.2% of 156 MLSs in our database. They occurred in 3 men and 2 women with a median age of 32 years. All tumors were localized to the retroperitoneum at presentation, and no patient developed extra-abdominal recurrences during the clinical course (median, 50 mo). All 5 cases exhibited at least focal classic histologic findings. All harbored DDIT3 gene rearrangements, and none harbored MDM2 amplifications according to fluorescence in situ hybridization. This study demonstrates that primary MLSs can occur in the retroperitoneum, albeit rarely, and can be accurately diagnosed through combined clinicopathologic and molecular analysis.

INSM1 expression and its diagnostic significance in extraskeletal myxoid chondrosarcoma

Extraskeletal myxoid chondrosarcoma is a rare subtype of sarcoma that affects the soft tissue and bones in middle-aged and elderly adults. Its diagnosis can be challenging, with the differential diagnoses including a wide variety of mesenchymal tumors. The line of differentiation of extraskeletal myxoid chondrosarcoma has been controversial, but recent evidence suggests a neuroendocrine phenotype. INSM1 is a zinc-finger transcription factor that plays a pivotal role in neuroendocrine differentiation, and has been proposed as a promising immunohistochemical marker of neuroendocrine carcinoma. The aim of this study was to determine the prevalence of INSM1 expression in extraskeletal myxoid chondrosarcoma and to understand its significance in sarcoma diagnosis. We immunostained the representative sections of 31 NR4A3-rearranged extraskeletal myxoid chondrosarcomas and 187 histological mimics. Nuclear staining of moderate or higher intensity in at least 5% of tumor cells was considered positive. Twenty-eight of the 31 extraskeletal myxoid chondrosarcomas (90%) were positive for INSM1, providing strong evidence for neuroendocrine differentiation. The staining was diffuse (>50%) in 17 cases, with most immunopositive tumors showing at least focal strong expression. The INSM1 staining extent was not correlated with cytomorphology, synaptophysin expression, or fusion types (EWSR1 vs non-EWSR1). In contrast, INSM1 expression was negative in 94% of the 187 other mesenchymal tumors. INSM1-positive mimics comprised a small subset of chordoma (1 of 10), soft tissue myoepithelioma (1 of 20), ossifying fibromyxoid tumor (3 of 10), and Ewing sarcoma (3 of 10), among other tumor types. The majority of these cases showed labeling in <25% of the tumor cells. Although not entirely sensitive or specific, INSM1 could be a potential marker for the diagnosis of extraskeletal myxoid chondrosarcoma when molecular genetic access is limited.

Clarifying the Distinction Between Malignant Peripheral Nerve Sheath Tumor and Dedifferentiated Liposarcoma: A Critical Reappraisal of the Diagnostic Utility of MDM2 and H3K27me3 Status

Malignant peripheral nerve sheath tumor (MPNST) and dedifferentiated liposarcoma (DDLPS) are 2 major types of pleomorphic spindle cell sarcoma. The differentiation of MPNST and DDLPS by histomorphology alone can be problematic. Although MDM2 amplification and PRC2 alteration leading to H3K27me3 deficiency are genetic hallmarks of DDLPS and MPNST, respectively, a small number of MDM2-amplified MPNSTs and H3K27me3-deficient DDLPSs have been reported in the literature. We systematically compared MDM2 and H3K27me3 status in 68 MPNSTs and 47 DDLPSs. Of the 62 MPNSTs, 22 were immunopositive for MDM2, mostly in a weak and/or focal manner. Of the 21 MDM2-positive MPNSTs successfully tested by fluorescence in situ hybridization, high-level MDM2 amplification was observed in 1 case. In contrast, MDM2 staining and high-level MDM2 amplification were positive in all the DDLPS tested (28/28 and 20/20). Of the 68 MPNSTs, 42 cases (62%) exhibited complete loss of H3K27me3. All the 13 MPNSTs that showed heterologous differentiation were deficient in H3K27me3. Of the 47 DDLPSs, 3 cases (6%) had complete loss of H3K27me3, all of which exhibited heterologous differentiation. One case of H3K27me3-deficient DDLPS exhibited homozygous loss of EED according to targeted next-generation sequencing, whereas there were no alterations in NF1 and CDKN2A. In conclusion, high-level MDM2 amplification strongly suggests DDLPS over MPNST. Although a good marker for MPNST, H3K27me3 deficiency also uncommonly occurs in DDLPS in association with PRC2 mutational inactivation. Because both markers are imperfectly specific, rare sarcomas with dual features could be encountered, and their classification should integrate other parameters.

PAX7 immunohistochemical evaluation of Ewing sarcoma and other small round cell tumours

Ewing sarcoma is a small round cell tumour that affects bone and soft tissues. Although the detection of the specific fusion gene is a robust method of its diagnosis, immunohistochemistry may serve as a practical surrogate. Recent tissue microarray studies suggested that PAX7 is a novel marker, because it was expressed consistently in Ewing sarcoma, in addition to rhabdomyosarcoma and synovial sarcoma. Here, we evaluated the utility of PAX7 immunohistochemistry in whole‐tissue sections of an expanded array of round cell malignancies with adequate molecular characterisation.

Recycling and Long-Term Storage of Fluorescence In Situ Hybridization Slides

OBJECTIVESnAlthough fluorescence in situ hybridization (FISH) technology is adequate, demand exists for additional recycling and long-term storage of FISH slides.nnnMETHODSnFormalin-fixed paraffin-embedded slides derived from breast cancer cases were used for this study. Each slide was probed, and then procedures for removing probes were performed, such as removing the fluorescent probe and diamidino-2-phenylindole signals. Formamide was used for removing probes, and then slides were stored dry at room temperature (22°C), 4°C, -20°C, or -80°C for 101 days. Following storage, each slide was probed in a similar manner to the initial probing. Evaluation was performed using automatic signal count software. Tiles and spots were counted immediately after the initial probing. Reprobed spots for each slide were then compared with the initial probing.nnnRESULTSnSlides stored at -20°C and -80°C for 101 days showed the best recovery of probing.nnnCONCLUSIONSnOur approach for probe removal and recycling allows repeated examination of even a limited number of slides.

Extraskeletal osteosarcoma: MDM2 and H3K27me3 analysis of 19 cases suggest disease heterogeneity

Extraskeletal osteosarcoma (ESOS) is a sarcoma in the non‐skeletal tissue that directly produces neoplastic osteoid or bone. De‐differentiated liposarcoma (DDLPS) and malignant peripheral nerve sheath tumour (MPNST) are the two most common types of sarcoma that can harbour heterologous osteosarcomatous differentiation. We aimed to determine the potential relationship of ESOS to DDLPS and MPNST.