The Morphological Spectrum of Papillary Renal Cell Carcinoma and Prevalence of Provisional/Emerging Renal Tumor Entities with Papillary Growth

Renal cell carcinoma (RCC) represents a heterogeneous disease, encompassing an increasing number of tumor subtypes. Post-2016, the World Health Organization (WHO) classification recognized that the spectrum of papillary renal cell carcinoma is evolving and has long surpassed the dichotomic simplistic "type 1 versus type 2" classification. The differential diagnosis of pRCC includes several new provisional/emerging entities with papillary growth. Type 2 tumors have been cleared out of several confounding entities, now regarded as independent tumors with specific clinical and molecular backgrounds. In this work we describe the prevalence and characteristics of emerging papillary tumor entities in two renal tumor cohorts (one consisting of consecutive papillary tumors from a single institute, the other consisting of consultation cases from several centers). After a review of 154 consecutive pRCC cases, 58% remained type 1 pRCC, and 34% type 2 pRCC. Papillary renal neoplasm with reversed polarity (1.3%), biphasic hyalinizing psammomatous RCC (1.3%), and biphasic squamoid/alveolar RCC (4.5%) were rare. Among 281 consultation cases, 121 (43%) tumors had a dominant papillary growth (most frequently MiT family translocation RCCs, mucinous tubular and spindle cell carcinoma and clear cell papillary RCC). Our data confirm that the spectrum of RCCs with papillary growth represents a major diagnostical challenge, frequently requiring a second expert opinion. Papillary renal neoplasm with reversed polarity, biphasic hyalinizing psammomatous RCC, and biphasic squamoid/alveolar RCC are rarely sent out for a second opinion, but correct classification and knowledge of these variants will improve our understanding of the clinical behavior of renal tumors with papillary growth

Targeting ALK in Neuroendocrine Tumors of the Lung.

Background Anaplastic lymphoma kinase (ALK) rearrangements are known oncogenic drivers in non-small cell lung cancer (NSCLC). Few case reports described the occurrence of such rearrangements in large cell neuroendocrine carcinomas (LCNECs) of the lung without information on clinical responses to ALK tyrosine kinase inhibitors (TKIs) in these cases. Currently, neuroendocrine tumors of the lungs are not screened for ALK rearrangements. Methods To illustrate the clinical impact of molecular characterization in LCNECs, we report the disease course in three patients with ALK-rearranged metastatic LCNEC from our clinical routine, as well as their treatment response to ALK TKIs (index cases). To gain insight into the prevalence of ALK rearrangements in neuroendocrine tumors of the lung, we analyzed a retrospective cohort of 436 tumor biopsies including LCNEC (n = 61), small cell lung cancer (SCLC) (n = 206), typical (n = 91) and atypical (n = 69) carcinoids, and mixed histology (n = 9) for the presence of ALK rearrangements using a sequential diagnostic algorithm. ALK immunohistochemistry (IHC) was evaluable in 362 cases; fluorescence in situ hybridization (FISH) was evaluable in 28 out of the 35 IHC-positive cases, followed by next-generation sequencing (NGS) that was available in 12 cases. Results Within the retrospective cohort, ALK IHC was positive in 35 out of 362 (9.7%) evaluable samples. FISH was positive in 3 out of the 28 (10.7%) evaluable cases: 2 with atypical carcinoids and 1 with LCNEC. Additionally, the 3 index cases showed positive ALK IHC, which was confirmed by NGS. Within the retrospective cohort, NGS confirmed the presence of an ALK genomic rearrangement in one FISH-positive atypical carcinoid where material was sufficient for sequencing. Two out of three patients with metastatic ALK-rearranged LCNEC received up-front treatment with the ALK TKI alectinib and showed rapid tumor response at all metastatic sites, including multiple brain metastases. Conclusions ALK rearrangements represent rare but targetable oncogenic driver alterations in LCNEC. Contrarily to NSCLC, the detection of ALK rearrangements in neuroendocrine tumors of the lung is challenging, since ALK IHC can lead to false-positive results and therefore needs confirmation by FISH or NGS. Up-front comprehensive molecular profiling with NGS should be performed in metastatic LCNEC in order not to miss actionable genomic alterations

Mutational Landscape and Expression of PD-L1 in Patients with Non-Small Cell Lung Cancer Harboring Genomic Alterations of the MET gene

BACKGROUND Mesenchymal-to-epithelial transition (MET) exon 14 skipping mutations and MET gene amplification occur in 3-5% of non-small cell lung cancer (NSCLC) patients. Tyrosine kinase inhibitors (TKIs) targeting MET alterations have shown promising results in these patients. OBJECTIVE The aim of this study was to describe the genomic profile, PD-L1 expression and clinicopathological features of MET dysregulated NSCLC. PATIENTS AND METHODS We identified 188 patients with advanced-stage NSCLC with data on MET expression by immunohistochemistry (IHC). IHC for PD-L1 expression was performed in 131 patient samples, and next-generation sequencing (NGS) analysis was performed in 109 patient samples. RESULTS MET exon 14 skipping alterations were identified in 16 (14.7%) samples, MET amplifications with cut-off ≥4 copy number variations were identified in 11 (10.1%) samples, and an oncogenic MET mutation (MET p.D1228N) was identified in 1 (0.9%) sample. 12/15 tumors (80.0%) harboring MET exon 14 alterations and 7/11 (63.6%) MET-amplified tumors expressed PD-L1 in ≥1% of tumor cells. Tumors harboring MET exon 14 skipping alterations expressed PD-L1 more frequently than MET wild-type IHC-positive tumors (p = 0.045). Twenty-five percent of MET exon 14-altered cases and 33% of MET-amplified cases harbored potentially targetable oncogenic co-mutations in KRAS, BRAF, and EGFR. The most frequent co-occurring mutations in all MET-altered tumors were TP53, KRAS, BRAF, and CDK4. CONCLUSIONS We demonstrated that MET exon 14 skipping alterations and MET amplification are not mutually exclusive to other oncogenic co-mutations, and report the association of genomic MET alterations with PD-L1 expression. Since genomic MET alterations are emerging targets requiring upfront treatment, optimal understanding of the co-mutational landscape for this patient population is needed

Targeting ALK in Neuroendocrine Tumors of the Lung

Background Anaplastic lymphoma kinase (ALK) rearrangements are known oncogenic drivers in non-small cell lung cancer (NSCLC). Few case reports described the occurrence of such rearrangements in large cell neuroendocrine carcinomas (LCNECs) of the lung without information on clinical responses to ALK tyrosine kinase inhibitors (TKIs) in these cases. Currently, neuroendocrine tumors of the lungs are not screened for ALK rearrangements. Methods To illustrate the clinical impact of molecular characterization in LCNECs, we report the disease course in three patients with ALK-rearranged metastatic LCNEC from our clinical routine, as well as their treatment response to ALK TKIs (index cases). To gain insight into the prevalence of ALK rearrangements in neuroendocrine tumors of the lung, we analyzed a retrospective cohort of 436 tumor biopsies including LCNEC (n = 61), small cell lung cancer (SCLC) (n = 206), typical (n = 91) and atypical (n = 69) carcinoids, and mixed histology (n = 9) for the presence of ALK rearrangements using a sequential diagnostic algorithm. ALK immunohistochemistry (IHC) was evaluable in 362 cases; fluorescence in situ hybridization (FISH) was evaluable in 28 out of the 35 IHC-positive cases, followed by next-generation sequencing (NGS) that was available in 12 cases. Results Within the retrospective cohort, ALK IHC was positive in 35 out of 362 (9.7%) evaluable samples. FISH was positive in 3 out of the 28 (10.7%) evaluable cases: 2 with atypical carcinoids and 1 with LCNEC. Additionally, the 3 index cases showed positive ALK IHC, which was confirmed by NGS. Within the retrospective cohort, NGS confirmed the presence of an ALK genomic rearrangement in one FISH-positive atypical carcinoid where material was sufficient for sequencing. Two out of three patients with metastatic ALK-rearranged LCNEC received up-front treatment with the ALK TKI alectinib and showed rapid tumor response at all metastatic sites, including multiple brain metastases. Conclusions ALK rearrangements represent rare but targetable oncogenic driver alterations in LCNEC. Contrarily to NSCLC, the detection of ALK rearrangements in neuroendocrine tumors of the lung is challenging, since ALK IHC can lead to false-positive results and therefore needs confirmation by FISH or NGS. Up-front comprehensive molecular profiling with NGS should be performed in metastatic LCNEC in order not to miss actionable genomic alterations

Testing for deficient mismatch repair and microsatellite instability : A focused update

Testing to detect mismatch repair deficiency (dMMR) and high-grade microsatellite instability (MSI-H) has become an integral part of the routine diagnostic workup for colorectal cancer (CRC). While MSI was initially considered to be a possible indicator of a hereditary disposition to cancer (Lynch syndrome, LS), today the prediction of the therapy response to immune checkpoint inhibitors (ICI) is in the foreground. Corresponding recommendations and testing algorithms are available for use in primary diagnosis (reviewed in: Rüschoff et al. 2021). Given the increasing importance for routine use and the expanding indication spectrum of ICI therapies for non-CRCs, such as endometrial, small intestinal, gastric, and biliary tract cancers, an updated review of dMMR/MSI testing is presented. The focus is on the challenges in the assessment of immunohistochemical stains and the value of PCR-based procedures, considering the expanded ICI indication spectrum. A practice-oriented flowchart for everyday diagnostic decision-making is provided that considers new data on the frequency and type of discordances between MMR-IHC and MSI-PCR findings, and the possible role of Next Generation Sequencing in clarifying them. Reference is made to the significance of systematic quality assurance measures (e.g., QuIP MSI portal and multicenter proficiency testing), including regular continued training and education. // Der Nachweis der Mismatch-Reparatur-Defizienz (dMMR) mit konsekutiver hochgradiger Mikrosatelliteninstabilität (MSI-H) ist inzwischen fester Bestandteil der Diagnostik des kolorektalen Karzinoms (KRK). Galt MSI anfänglich als möglicher Indikator einer erblichen Krebsdisposition (Lynch-Syndrom, LS) steht heute die Vorhersage des Therapieansprechens auf Immuncheckpoint-Inhibitoren (ICI) im Vordergrund. Entsprechende Empfehlungen und Testalgorithmen liegen für den Einsatz in der Primärdiagnostik vor (Übersicht in: Rüschoff et al. 2021). Aufgrund des damit verbundenen routinemäßigen Einsatzes und des sich erweiternden Indikationsspektrums von ICI-Therapien für Nicht-KRK wie Endometrium‑, Dünndarm‑, Magen- und Gallenwegskarzinome wird eine aktualisierte Übersicht zur dMMR/MSI-Testung vorgelegt. Fokus sind die Herausforderungen bei der Beurteilung immunhistochemischer Färbungen und die Wertigkeit PCR-basierter Verfahren unter Berücksichtigung des erweiterten ICI-Indikationsspektrums. Anhand neuer Daten zur Häufigkeit und Art von Diskordanzen zwischen dMMR- und MSI-Befund und der möglichen Rolle von Next Generation Sequencing zu deren Aufklärung wird ein praxisorientiertes Diagramm zur Entscheidungsfindung im diagnostischen Alltag vorgestellt. Wir weisen zudem auf die Bedeutung systematischer Qualitätssicherungsmaßnahmen (z. B. QuIP MSI-Portal und Ringversuche) einschließlich einer regelmäßigen Fortbildung hin

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF V600E mutations in colorectal carcinoma

The activating BRAF V600 mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF V600E mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF V600E mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF V600E mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7%) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5%) and 15 IHC-positive cases (15/39; 38.5%) showed a BRAF wild-type by SaS. UDS detected a BRAF V600E mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100% and 63% and specificities of 95 and 100% for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAFV600E (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF V600E mutations in colorectal carcinom

Prospective observational study of the role of the microbiome in BCG responsiveness prediction (SILENT-EMPIRE): a study protocol

INTRODUCTION The human microbiota, the community of micro-organisms in different cavities, has been increasingly linked with inflammatory and neoplastic diseases. While investigation into the gut microbiome has been robust, the urinary microbiome has only recently been described. Investigation into the relationship between bladder cancer (BC) and the bladder and the intestinal microbiome may elucidate a pathophysiological relationship between the two. The bladder or the intestinal microbiome or the interplay between both may also act as a non-invasive biomarker for tumour behaviour. While these associations have not yet been fully investigated, urologists have been manipulating the bladder microbiome for treatment of BC for more than 40 years, treating high grade non-muscle invasive BC (NMIBC) with intravesical BCG immunotherapy. Neither the association between the microbiome sampled directly from bladder tissue and the response to BCG-therapy nor the association between response to BCG-therapy with the faecal microbiome has been studied until now. A prognostic tool prior to initiation of BCG-therapy is still needed. METHODS AND ANALYSIS In patients with NMIBC bladder samples will be collected during surgery (bladder microbiome assessment), faecal samples (microbiome assessment), instrumented urine and blood samples (biobank) will also be taken. We will analyse the microbial community by 16S rDNA gene amplicon sequencing. The difference in alpha diversity (diversity of species within each sample) and beta diversity (change in species diversity) between BCG-candidates will be assessed. Subgroup analysis will be performed which will lead to the development of a clinical prediction model estimating risk of BCG-response. ETHICS AND DISSEMINATION The study has been approved by the Cantonal Ethics Committee Zurich (2021-01783) and it is being conducted in accordance with the Declaration of Helsinki and Good Clinical Practice. Study results will be disseminated through peer-reviewed journals and national and international scientific conferences. TRIAL REGISTRATION NUMBER NCT05204199

Infiltrative growth pattern of prostate cancer is associated with lower uptake on PSMA PET and reduced diffusion restriction on mpMRI

Purpose: Recently, a significant association was shown between novel growth patterns on histopathology of prostate cancer (PCa) and prostate-specific membrane antigen (PSMA) uptake on [68Ga]PSMA-PET. It is the aim of this study to evaluate the association between these growth patterns and ADC (mm2/1000 s) values in comparison to [68Ga]PSMA uptake on PET/MRI. Methods: We retrospectively evaluated patients who underwent [68Ga]PSMA PET/MRI for staging or biopsy guidance, followed by radical prostatectomy at our institution between 07/2016 and 01/2020. The dominant lesion per patient was selected based on histopathology and correlated to PET/MRI in a multidisciplinary meeting, and quantified using SUVmax for PSMA uptake and ADCmean for diffusion restriction. PCa growth pattern was classified as expansive (EXP) or infiltrative (INF) according to its properties of forming a tumoral mass or infiltrating diffusely between benign glands by two independent pathologists. Furthermore, the corresponding WHO2016 ISUP tumor grade was evaluated. The t test was used to compare means, Pearson's test for categorical correlation, Cohen's kappa test for interrater agreement, and ROC curve to determine the best cutoff. Results: Sixty-two patients were included (mean PSA 11.7 ± 12.5). The interrater agreement between both pathologists was almost perfect with κ = 0.81. While 25 lesions had an EXP-growth with an ADCmean of 0.777 ± 0.109, 37 showed an INF-growth with a significantly higher ADCmean of 1.079 ± 0.262 (p < 0.001). We also observed a significant difference regarding PSMA SUVmax for the EXP-growth (19.2 ± 10.9) versus the INF-growth (9.4 ± 6.2, p < 0.001). Within the lesions encompassing the EXP- or the INF-growth, no significant correlation between the ISUP groups and ADCmean could be observed (p = 0.982 and p = 0.861, respectively). Conclusion: PCa with INF-growth showed significantly lower SUVmax and higher ADCmean values compared to PCa with EXP-growth. Within the growth groups, ADCmean values were independent from ISUP grading. Keywords: Diffusion-weighted imaging; MRI; PSMA PET/MRI; Prostate cancer; Radical prostatectom

What's behind 68 Ga-PSMA-11 uptake in primary prostate cancer PET? Investigation of histopathological parameters and immunohistochemical PSMA expression patterns

Purpose: Prostate-specific membrane antigen (PSMA-) PET has become a promising tool in staging and restaging of prostate carcinoma (PCa). However, specific primary tumour features might impact accuracy of PSMA-PET for PCa detection. We investigated histopathological parameters and immunohistochemical PSMA expression patterns on radical prostatectomy (RPE) specimens and correlated them to the corresponding 68Ga-PSMA-11-PET examinations. Methods: RPE specimens of 62 patients with preoperative 68Ga-PSMA-11-PET between 2016 and 2018 were analysed. WHO/ISUP grade groups, growth pattern (expansive vs. infiltrative), tumour area and diameter as well as immunohistochemical PSMA heterogeneity, intensity and negative tumour area (PSMA%neg) were correlated with spatially corresponding SUVmax on 68Ga-PSMA-11-PET in a multidisciplinary analysis. Results: All tumours showed medium to strong membranous (2-3 +) and weak to strong cytoplasmic (1-3 +) PSMA expression. Heterogeneously expressed PSMA was found in 38 cases (61%). Twenty-five cases (40%) showed at least 5% and up to 80% PSMA%neg. PSMA%neg, infiltrative growth pattern, smaller tumour area and diameter and WHO/ISUP grade group 2 significantly correlated with lower SUVmax values. A ROC curve analysis revealed 20% PSMA%neg as an optimal cutoff with the highest sensitivity and specificity (89% and 86%, AUC 0.923) for a negative PSMA-PET scan. A multiple logistic regression model revealed tumoural PSMA%neg (p < 0.01, OR = 9.629) and growth pattern (p = 0.0497, OR = 306.537) as significant predictors for a negative PSMA-PET scan. Conclusions: We describe PSMA%neg, infiltrative growth pattern, smaller tumour size and WHO/ISUP grade group 2 as parameters associated with a lower 68Ga-PSMA-11 uptake in prostate cancer. These findings can serve as fundament for future biopsy-based biomarker development to enable an individualized, tumour-adapted imaging approach. Keywords: Glutamate carboxypeptidase II; Immunohistochemistry; Neoplasm staging; Positron emission tomography; Prostatic neoplasm

Ultra-deep sequencing confirms immunohistochemistry as a highly sensitive and specific method for detecting BRAF (V600E) mutations in colorectal carcinoma

The activating BRAF (V600) mutation is a well-established negative prognostic biomarker in metastatic colorectal carcinoma (CRC). A recently developed monoclonal mouse antibody (clone VE1) has been shown to detect reliably BRAF (V600E) mutated protein by immunohistochemistry (IHC). In this study, we aimed to compare the detection of BRAF (V600E) mutations by IHC, Sanger sequencing (SaS), and ultra-deep sequencing (UDS) in CRC. VE1-IHC was established in a cohort of 68 KRAS wild-type CRCs. The VE1-IHC was only positive in the three patients with a known BRAF (V600E) mutation as assessed by SaS and UDS. The test cohort consisted of 265 non-selected, consecutive CRC samples. Thirty-nine out of 265 cases (14.7 %) were positive by VE1-IHC. SaS of 20 randomly selected IHC negative tumors showed BRAF wild-type (20/20). Twenty-four IHC-positive cases were confirmed by SaS (24/39; 61.5 %) and 15 IHC-positive cases (15/39; 38.5 %) showed a BRAF wild-type by SaS. UDS detected a BRAF (V600E) mutation in 13 of these 15 discordant cases. In one tumor, the mutation frequency was below our threshold for UDS positivity, while in another case, UDS could not be performed due to low DNA amount. Statistical analysis showed sensitivities of 100 % and 63 % and specificities of 95 and 100 % for VE1-IHC and SaS, respectively, compared to combined results of SaS and UDS. Our data suggests that there is high concordance between UDS and IHC using the anti-BRAF(V600E) (VE1) antibody. Thus, VE1 immunohistochemistry is a highly sensitive and specific method in detecting BRAF (V600E) mutations in colorectal carcinoma