Comprehensive Genomic Profiling of Carcinoma of Unknown Primary Origin: Retrospective Molecular Classification Considering the CUPISCO Study Design.

BACKGROUND: Carcinoma of unknown primary origin (CUP) accounts for 2%-5% of newly diagnosed advanced malignancies, with chemotherapy as the standard of care. CUPISCO (NCT03498521) is an ongoing randomized trial using comprehensive genomic profiling (CGP) to assign patients with CUP to targeted or immunotherapy treatment arms based on genomic profiling. We performed a retrospective analysis of CUP cases referred for CGP to determine how many were potentially eligible for enrollment into an experimental CUPISCO arm. MATERIALS AND METHODS: Centrally reviewed adenocarcinoma and undifferentiated CUP specimens in the FoundationCore database were analyzed using the hybrid capture-based FoundationOne CDx assay (mean coverage, >600×). Presence of genomic alterations, microsatellite instability (MSI), tumor mutational burden (TMB), genomic loss of heterozygosity (gLOH), and programmed death-ligand 1 (PD-L1) positivity were determined. RESULTS: A total of 96 of 303 patients (31.7%) could be matched to an experimental CUPISCO arm. Key genomic alterations included ERBB2 (7.3%), PIK3CA (6.3%), NF1 (5.6%), NF2 (4.6%), BRAF (4.3%), IDH1 (3.3%), PTEN, FGFR2, EGFR (3.6% each), MET (4.3%), CDK6 (3.0%), FBXW7, CDK4 (2.3% each), IDH2, RET, ROS1, NTRK (1.0% each), and ALK (0.7%). Median TMB was 3.75 mutations per megabase of DNA; 34 patients (11.6%) had a TMB ≥16 mutations per megabase. Three patients (1%) had high MSI, and 42 (14%) displayed high PD-L1 expression (tumor proportion score ≥50%). gLOH could be assessed in 199 of 303 specimens; 19.6% had a score of >16%. CONCLUSIONS: Thirty-two percent of patients would have been eligible for targeted therapy in CUPISCO. Future studies, including additional biomarkers such as PD-L1 positivity and gLOH, may identify a greater proportion potentially benefiting from CGP-informed treatment. Clinical trial identification number. NCT03498521 IMPLICATIONS FOR PRACTICE: The findings of this retrospective analysis of carcinoma of unknown primary origin (CUP) cases validate the experimental treatment arms being used in the CUPISCO study (NCT03498521), an ongoing randomized trial using comprehensive genomic profiling to assign patients with CUP to targeted or immunotherapy treatment arms based on the presence of pathogenic genomic alterations. The findings also suggest that future studies including additional biomarkers and treatment arms, such as programmed death-ligand 1 positivity and genomic loss of heterozygosity, may identify a greater proportion of patients with CUP potentially benefiting from comprehensive genomic profiling-informed treatment.

Yield and Utility of Germline Testing Following Tumor Sequencing in Patients With Cancer.

Importance: Both germline genetic testing and tumor DNA sequencing are increasingly used in cancer care. The indications for testing and utility of these 2 tests differ, and guidelines recommend that germline analysis follow tumor sequencing in certain patients to determine whether particular variants are of somatic or germline origin. Broad clinical experience with such follow-up testing has not yet been thoroughly described. Objective: To examine the yield and utility of germline testing following tumor DNA sequencing in a large, diverse patient population. Design, Setting, and Participants: A retrospective cohort study examined germline testing through a laboratory supporting multiple academic and community clinics. Participants included 2023 patients with cancer who received germline testing and previously underwent tumor DNA sequencing. These patients received germline testing between January 5, 2015, and January 31, 2020, although most (81% of patients) received testing between January 2, 2018, and January 31, 2020. Main Outcomes and Measures: The prevalence of pathogenic germline variants (PGVs) was calculated by gene, cancer type, and age at diagnosis. Potential actionability of these findings was determined based on current management guidelines, precision therapy labels, and clinical trial eligibility criteria. Patient records were reviewed to determine whether germline follow-up testing would have been recommended by current guidelines. Results: Among 2023 eligible patients, 1085 were female (53.6%), and the median age at cancer diagnosis was 56 (range, 0-92) years. Pathogenic germline variants were detected in 617 patients (30.5%; 95% CI, 28.5%-32.6%) and were prevalent across patient ages (1-85 years) and cancer types, including cancers known to be strongly associated with germline variance (eg, breast, colorectal) as well as others (eg, renal, lung, and bladder). Many patients (78%-82%) with PGVs met criteria for germline follow-up testing, and 8.1% of PGVs were missed by tumor sequencing. Among those with germline-positive findings, 69 patients (11.2%) had PGVs identified only after presenting with a second primary cancer that possibly could have been detected earlier or prevented given current gene-specific surveillance and risk-reduction recommendations. Conclusions and Relevance: The findings of this study suggest that germline analysis following tumor sequencing often produces findings that may impact patient care by influencing systemic therapy choices, surgical decisions, additional cancer screening, and genetic counseling in families. Current guidelines and tumor testing approaches appear to capture many, but not all, of these germline findings, reinforcing the utility of both expanded germline follow-up testing as well as germline analysis independent of tumor sequencing in appropriate patients.

Comprehensive Assessment of Immuno-oncology Biomarkers in Adenocarcinoma, Urothelial Carcinoma, and Squamous-cell Carcinoma of the Bladder.

BACKGROUND: In patients with rare histologies of bladder cancer, including adenocarcinoma of the bladder (ACB) and squamous-cell carcinoma (SCC), there are limited standard therapy options, defining an unmet medical need. OBJECTIVE: In this comparative comprehensive genomic profiling (CGP) study, genomic alterations (GAs), and immuno-oncology (IO) biomarkers have been analyzed. DESIGN, SETTING, AND PARTICIPANTS: Within the Foundation Medicine database, 143 cases with centrally reviewed pure ACB, 2142 with pure urothelial carcinoma (UC), and 83 with pure SCC were subjected to CGP. All patients developed advanced disease following a primary diagnosis of bladder cancer. INTERVENTION: CGP using a hybrid capture-based assay and immunohistochemistry (IHC). OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) was determined on 114 loci. Programmed cell-death ligand-1 (PD-L1) expression was determined by IHC (Ventana SP-142 assay), with >1% tumor cells (TCs) or tumor-infiltrating lymphocytes (TILs) scoring positive. RESULTS AND LIMITATIONS: Pure ACB patients were younger and more often female than pure UC and pure SCC patients. UC and SCC had a significantly higher median TMB than ACB (p < 0.001). Rare CD274 (PD-L1) amplification cases were more frequently seen in SCC than in UC (5% vs 1%), and were not seen in ACB. MSI high status was very uncommon in all tumor types (0-1%). The frequencies of PD-L1 expression in both TCs and TILs was higher in UC and SCC (both 30%) than in ACB (18%). The results are limited by their retrospective nature and lack of clinical data annotation. CONCLUSIONS: Deep sequencing revealed significant differences in IO biomarkers among the three major subtypes of bladder carcinomas. UC and SCC revealed higher frequencies of PD-L1 expression and higher TMB than ACB, and SCC has the highest frequency of CD274 amplification. The presence of pure SCC features should not disqualify patients for inclusion in IO trials. PATIENT SUMMARY: Tumor samples from patients diagnosed with advanced pure adenocarcinoma of the bladder (ACB) or pure squamous-cell carcinoma (SCC) have been analyzed in terms of frequency of putative immunotherapy biomarkers. The results indicated that pure SCC of the bladder was characterized by genomic features that portend similar response possibilities to immunotherapy compared with the classical pure urothelial carcinoma. Conversely, for pure ACB there might be different therapeutic opportunities, such as targeted therapies against peculiar genomic alterations in selected patients.

Genomic Features of Metastatic Testicular Sex Cord Stromal Tumors.

BACKGROUND: Metastatic testicular sex cord stromal tumors of the testis (MSCSTs) comprise an extremely uncommon form of genitourinary malignancy. OBJECTIVE: To perform comprehensive genomic profiling (CGP) to enable the search for potential therapy targets. DESIGN, SETTING, AND PARTICIPANTS: Ten patients with testicular Leydig cell tumors (LCTs), six with Sertoli cell tumors (SCTs), and three with undifferentiated sex cord stromal tumors (USCSTs) and a comparison group of 366 patients with ovarian sex cord stromal tumors (SCSTs) underwent hybrid-capture-based CGP to evaluate all classes of genomic alterations (GAs). The tumor mutational burden (TMB) was determined on 1.1 Mbp of sequenced DNA, and microsatellite instability (MSI) was determined on 114 loci. INTERVENTION: CGP on tumor samples. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Descriptive analyses and differences between histological subgroups were reported. RESULTS AND LIMITATIONS: In these patients, all of whom had metastatic disease at the time of sequencing, the primary testis tumor was sequenced in six (32%) patients and a metastatic site in 13 (68%) patients. The overall frequencies of GAs were similar in LCTs, SCTs, and USCSTs, ranging from 3.0 to 3.5 GAs/tumor. The most frequent untargetable GAs included CTNNB1 and CDKN2A/B, both ranging from 20% to 33% of cases. Targetable GAs were uncommon in all MSCST subgroups, but several tumors showed potential for cell-cycle inhibitors (CDK4 in LCTs), mTOR inhibitors (RICTOR, NF2, and PTEN in all three tumor types), hedgehog inhibitors (PTCH1 in LCTs), and poly(ADP-ribose) polymerase inhibitors (BAP1 in SCTs). No MSI-high status was identified. The TMB was also low in all MSCST groups, and tumors featuring a TMB of ≥10 mutations/Mb were not identified. GA findings from ovarian SCSTs largely recapitulated those from MSCSTs. A lack of clinical outcome correlation is a limitation of the present analyses. CONCLUSIONS: Rare cases of testicular MSCSTs have GAs linked to potential targeted therapy benefits on CGP. In contrast, the lack of MSI-high status and an overall low TMB indicate a likely lack of benefit for immunotherapies. PATIENT SUMMARY: Genomic profiling can guide clinical research and disclose therapeutic opportunities for patients with rare testicular cancers for which standard therapies are lacking.

SMARCA4 inactivation defines a subset of undifferentiated uterine sarcomas with rhabdoid and small cell features and germline mutation association.

A rare subset of aggressive SMARCA4-deficient uterine sarcomas has been recently proposed, with only a limited number of cases having been previously described. Here, we identify 16 additional cases of SMARCA4-deficient uterine sarcoma from the database of a large, CLIA-certified and CAP-accredited, reference molecular laboratory, and we expand on their clinicopathological and genomic features. Median patient's age was 49 years (range 32-70). Most tumors were aggressive with distant metastasis. SMARCA4-deficient uterine sarcoma demonstrated predominantly rhabdoid or large epithelioid cells with abundant cytoplasm, but also had varying degrees of small cell and spindle cell morphology. Tumors were microsatellite stable and exhibited no other or only few co-occurring genomic alterations by comprehensive genomic profiling. We discovered one patient, who developed SMARCA4-deficient uterine sarcoma at the age of 55, had a germline SMARCA4 mutation, whose daughter had previously died of small cell carcinoma of the ovary, hypercalcemic type, at the age of 32. Our data support the notion that SMARCA4 inactivation is the driver oncogenic event of a morphologically and molecularly distinct form of uterine sarcoma. Identification of SMARCA4-deficient uterine sarcomas may be clinically important due to their aggressive behavior, germline association, and emerging targeted therapies.

Metaplastic breast cancers: Genomic profiling, mutational burden and tumor-infiltrating lymphocytes.

Metaplastic breast cancer (MPBC) is a rare subtype that accounts for <1% of all breast cancers. Although these are typically "triple negative," they are relatively chemotherapy-refractory compared to conventional triple negative invasive breast cancers with more aggressive features and an overall poor prognosis. MPBC is a heterogeneous group of tumors that are enriched for TP53 and PIK3CA mutations, and have been found to have high PD-L1 expression though the mechanisms underlying its immunogenicity remain unclear. We perform comprehensive genomic profiling in the largest MPBC dataset (n = 192) to date and assess for other potential biomarkers of immune response.

EML4-ALK Rearrangement and Its Therapeutic Implications in Inflammatory Myofibroblastic Tumors.

With the advent of precision medicine, medical oncology is undergoing a transcendental change. These molecular studies have allowed us to learn about potential targeted therapies for patients with advanced cancers. Perhaps the best-known example of success in precision medicine is chronic myeloid leukemia and its response to tyrosine kinase inhibitors targeting the BCR-ABL kinase. Since that original discovery, the role of molecular therapeutics has expanded, and it now presents us with treatment options for common malignancies and rare atypical tumors. In this article, we present a case of a 61-year-old female with a recurrent pulmonary inflammatory myofibroblastic tumor. Subsequent molecular studies revealed an ALK rearrangement. The significance of this alteration in this tumor type and its therapeutic implications are discussed herein. KEY POINTS: This case exemplifies the heterogeneous behavior of inflammatory myofibroblastic tumors (IMTs) and the current role of targeted therapy in the therapeutic armamentarium of neoplastic processes.As evidenced by the different mutations found in IMTs, it is of great importance to perform next-generation sequencing in uncommon neoplasms.These studies can find different potential targets and therapeutic options for patients devoid of standard effective therapies.

Seronegative Autoimmune Hepatitis A Clinically Challenging Difficult Diagnosis.

Autoimmune hepatitis (AIH) is a complex liver disease of unknown cause which results in immune-mediated liver injury with varied clinical presentations. Seronegative AIH follows a similar course to autoantibody-positive disease and diagnosis may be challenging. There are no single serologic tests of sufficient diagnostic specificity, and delay in appropriate treatment may lead to progression of the liver disease and liver failure. The revised conventional diagnostic criteria (RDC) scoring for AIH is complex and not routinely used in the clinical practice. The more recent simplified diagnostic criteria (SDC) scoring proposed by International Autoimmune Hepatitis Group in 2008 has wider application in routine practice facilitating the diagnosis of AIH with a specificity and sensitivity of ~90%. In this report, we describe a case of seronegative autoimmune hepatitis diagnosed using RDC. SDC score calculated in our case was 4 and was not diagnostic for AIH. We subsequently used the complex revised diagnostic criteria for definitive diagnosis. Some of the patients previously diagnosed as cryptogenic active hepatitis of unknown etiology probably had an unrecognized diagnosis of seronegative autoimmune hepatitis. SDC scoring may not be applicable in patients with seronegative autoimmune hepatitis. These patients should be reassessed by using RDC.

Cryptococcal lymphadenitis: an unusual initial presentation of HIV infection.

Lymphadenopathy can occur at any stage of HIV infection, with multiple aetiologies including reactive, infectious and malignant. An accurate and timely diagnosis has obvious implications for treatment. We report cryptococcal lymphadenitis as the presenting manifestation of HIV infection. The diagnosis in our patient was eventually confirmed with a lymph node biopsy. Fine needle aspiration cytology has been shown to be a rapid and cost-effective method, which has been used in the diagnosis of lymphadenopathy in HIV infection, and could have been used to make an earlier diagnosis in our patient.

Cancerous 'floater': a lesson learned about tissue identity testing, endometrial cancer and microsatellite instability.

A 46-year-old woman presented with endometrial cells on a pap smear and underwent endometrial curettage. The specimen revealed secretory endometrium and a possible endometrial polyp. In addition, a single 4 mm fragment of well-differentiated adenocarcinoma was found. Tissue identity DNA genotyping was performed and the adenocarcinoma tissue fragment showed a drastically different allelic pattern from that of the background endometrium. To confirm tissue contamination, genotyping of three other tumor specimens-probable sources for a contaminant-was performed but failed to identify a match. Without confirmation of contamination, a second endometrial curettage was obtained from the patient, in which similar adenocarcinoma tissue was once again found. Further workup demonstrated that the patient had a microsatellite unstable (MSI) endometrial adenocarcinoma by immunohistochemistry and molecular testing. The patient subsequently underwent staging surgery, which revealed an early-stage, well-differentiated endometrioid adenocarcinoma. This case study illustrates an uncommon, yet important caveat of tissue identity testing by DNA genotyping, where MSI instability can significantly alter the allelic pattern of DNA polymorphisms in the tumor genome, leading to erroneous conclusion regarding the tissue identity. Awareness of this phenomenon is crucial for a molecular pathologist to avoid interpretation errors of tissue identity testing in a cancer diagnostic workup.