Programmed Death Ligand-1 and Tumor Mutation Burden Testing of Patients With Lung Cancer for Selection of Immune Checkpoint Inhibitor Therapies: Guideline From the College of American Pathologists, Association for Molecular Pathology, International Association for the Study of Lung Cancer, Pulmonary Pathology Society, and LUNGevity Foundation.

CONTEXT.­: Rapid advancements in the understanding and manipulation of tumor-immune interactions have led to the approval of immune therapies for patients with non-small cell lung cancer. Certain immune checkpoint inhibitor therapies require the use of companion diagnostics, but methodologic variability has led to uncertainty around test selection and implementation in practice. OBJECTIVE.­: To develop evidence-based guideline recommendations for the testing of immunotherapy/immunomodulatory biomarkers, including programmed death ligand-1 (PD-L1) and tumor mutation burden (TMB), in patients with lung cancer. DESIGN.­: The College of American Pathologists convened a panel of experts in non-small cell lung cancer and biomarker testing to develop evidence-based recommendations in accordance with the standards for trustworthy clinical practice guidelines established by the National Academy of Medicine. A systematic literature review was conducted to address 8 key questions. Using the Grading of Recommendations Assessment, Development, and Evaluation (GRADE) approach, recommendations were created from the available evidence, certainty of that evidence, and key judgments as defined in the GRADE Evidence to Decision framework. RESULTS.­: Six recommendation statements were developed. CONCLUSIONS.­: This guideline summarizes the current understanding and hurdles associated with the use of PD-L1 expression and TMB testing for immune checkpoint inhibitor therapy selection in patients with advanced non-small cell lung cancer and presents evidence-based recommendations for PD-L1 and TMB testing in the clinical setting.

Combining Panitumumab With Anti-PD-1 Antibody in Cutaneous Squamous Cell Carcinoma of the Head and Neck After Inadequate Response to Anti-PD-1 Antibody Alone.

Anti-epidermal growth factor receptor (EGFR) antibodies and anti-programmed cell death 1 protein (PD-1) antibodies have been used separately to treat metastatic cutaneous squamous cell carcinoma (cSCC). While two anti-EGFR antibodies have similar clinical activity, cetuximab is administered weekly, whereas panitumumab is administered every two weeks. This report details findings using panitumumab in combination with anti-PD-1 antibody in patients with relapsed refractory cSCC. Three consecutive patients with poor performance status and rapidly progressive recurrent cutaneous squamous cell carcinoma (cSCC) of the face or scalp signed informed consent to receive an anti-PD-1 antibody with the option to add panitumumab were there inadequate response. After 2, 5, and 7 cycles of anti-PD-1 antibody treatment, respectively, panitumumab was added and the combination was continued for 27, 7, and 5 cycles, respectively. Fatigue, rash, and hypomagnesemia were reported, consistent with expectations for either agent alone. All three patients achieved durable complete response. The favorable clinical outcomes support further evaluation of the combination of anti-PD1 and anti-EGFR antibodies to control refractory cSCC of the face or scalp. J Drugs Dermatol. 2021;20(8):901-904. doi:10.36849/JDD.6175.

Utility and Limitations of Albumin mRNA In Situ Hybridization Detection in the Diagnosis of Hepatobiliary Lesions and Metastatic Carcinoma to the Liver.

Albumin messenger RNA (mRNA) in situ hybridization is a sensitive and specific biomarker for hepatocellular carcinoma (HCC). Intrahepatic cholangiocarcinoma (ICC) shows variable sensitivity, whereas extrahepatic cholangiocarcinoma (ECC) and metastatic carcinoma are generally negative. We studied the clinical utility and limitations of albumin mRNA detection in a cohort of HCCs, ICCs, ECCs, bile duct adenomas, bile duct hamartomas, and metastatic carcinomas to the liver; and investigated the variability in sensitivity observed for this biomarker in ICCs. We identified 122 cases of hepatobiliary lesions and metastatic carcinomas. Albumin mRNA detection was performed using RNAscope run on formalin-fixed, paraffin-embedded tissue sections. ICCs were categorized according to the classification proposed by Hayashi and colleagues into the small duct, large duct, and indeterminate subtypes. Albumin mRNA was detected in all 17 HCCs and focally in 6/8 (75%) of bile duct adenomas. All 28 nonhepatic carcinomas, 13 bile duct hamartomas, and 9 ECCs were negative. Albumin mRNA was found in 38/47 (80.9%) of ICC with 35/37 (94.6%) in the small duct subtype, 2/3 (66.7%) in the indeterminate subtype, and 1/7 (14.3%) of the large duct subtype (P<0.003). Albumin mRNA detection is a sensitive and specific biomarker for HCCs. It is highly sensitive and moderately specific in the diagnosis of ICC with small gland morphology, but not ICCs with large duct morphology and in metastatic carcinoma. The variability in the sensitivity of albumin mRNA expression in ICCs may depend on the subtypes of ICC.

Quantitative assessment of PD-L1 as an analyte in immunohistochemistry diagnostic assays using a standardized cell line tissue microarray.

Programmed death 1 ligand 1 (PD-L1) Immunohistochemistry (IHC) is the key FDA-approved predictive marker to identify responders to anti-PD1 axis drugs. Multiple PD-L1 IHC assays with various antibodies and cut points have been used in clinical trials across tumor types. Comparative performance characteristics of these assays have been extensively studied qualitatively but not quantitatively. Here we evaluate the use of a standardized PD-L1 Index tissue microarray (TMA) to objectively determine agreement between antibody assays for PD-L1 applying quantitative digital image analysis. Using a specially constructed Index TMA containing a panel of ten isogenic cell lines in triplicate, we tested identical but independently grown batches of isogenic cells to prove Index TMAs can be produced in large quantities and hence serve as a standardization tool. Then the Index TMAs were evaluated using quantitative immunofluorescence (QIF) to validate the TMA itself and also to compare antibodies including E1L3N, SP142 and SP263. Next, an inter-laboratory and inter-assay comparison of 5 PD-L1 chromogenic IHC assays (US Food and Drug Administration (FDA) approved and lab developed test (LDT)) were performed at 12 sites around the USA. As previously reported, the SP142 FDA assay failed to detect low levels of PD-L1 in cell lines distinguished by the other four assays. The assays for 22C3 FDA, 28-8-FDA, SP263 FDA, and E1L3N LDT were highly similar across sites and all laboratories showed a high consistency over time for all assays using this Index TMA. In conclusion, we were able to objectively quantify PD-L1 expression on a standardized Index TMA using digital image analysis and we confirmed previous subjective assessments of these assays, but now in a multi-institutional setting. We envision commercial use of this Index TMA or similar smaller version as a useful standardization mechanism to compare results between institutions and to identify abnormalities while running routine clinical samples.

Concurrent Eosinophilic Solid and Cystic Renal Cell Carcinoma and Angiomyolipoma With Epithelial Cysts in the Setting of Tuberous Sclerosis Complex: A Rare Synchronous Occurrence of 2 Distinct Entities.

Eosinophilic solid and cystic renal cell carcinoma (ESCRCC) is a recently described distinct renal neoplasm known to occur almost exclusively in female patients with or without tuberous sclerosis complex (TSC). We report a case of ESCRCC with 2 synchronous angiomyolipomas, including 1 angiomyolipoma with epithelial cysts (AMLEC), a rare cystic variant of AML that typically arises sporadically in the absence of TSC, in a 46-year-old woman with TSC. Besides additional copy number alterations identified in ESCRCC via molecular karyotyping, we also report a unique histologic feature of TSC-associated ESCRCC previously not described in detail, with formation of semicircular multinucleated neoplastic giant cells engulfing an additional intact neoplastic cell, simulating emperipolesis. To the best of our knowledge, this is the first reported case of ESCRCC with concurrent AMLEC in a patient with TSC, confirmed through additional genetic testing showing a germline heterozygous mutation in TSC1. Awareness of ESCRCC helps avoid the pitfall of a diagnosis of unclassified renal cell carcinoma, a typically much more aggressive tumor.

IMP3 Immunoreactivity is More Sensitive Than AMACR in Detecting Dysplastic Epithelium and Early Adenocarcinoma in Barrett Esophagus.

CONTEXT: α-methylacyl coenzyme A racemase (AMACR) and insulin-like growth factor-II mRNA-binding protein 3 (IMP3) are 2 markers helpful in detecting difficult cases of dysplasia in Barrett esophagus (BE). However, no comparison studies have been performed to assess their performance in the same patient population. OBJECTIVES: The aim of our study was to compare the immunohistochemical expression of IMP3 and AMACR in dysplastic lesions and early adenocarcinoma (EAC) arising in BE and evaluate their sensitivity and specificity. DESIGN: A total of 98 cases [BE negative for dysplasia, n=24; indefinite for dysplasia (BE-IND), n=18; low-grade dysplasia (LGD), n=24; high-grade dysplasia (HGD), n=16; and EAC, n=16] were immunostained for AMACR and IMP3 and evaluated for the degree, the extent, and the intensity of staining. RESULTS: No immunoreactivity for AMACR or IMP3 was observed in all 24 cases of BE negative for dyplasia. One of 18 (5.5%) cases of BE-IND was positive for IMP3, but all were negative for AMACR. AMACR and IMP3 were positive in 16.7% versus 41.7 % of the cases with BE-LGD, 25% versus 62.5% of BE-HGD, and 62.5% versus 93.7% of EAC, respectively. The sensitivity of AMACR and IMP3 for the detection of dysplasia in BE is 16.7% and 41.7% for LGD, 25% and 62.5% for HGD, and 62.5% and 93.7% in EAC, respectively. The specificity is 100% for both markers. In addition, a comparison of the intensity of reactivity shows a better result with IMP3 (36/98, 36.7%) than with AMACR (18/98, 18.4%) (P<0.001). CONCLUSIONS: IMP3 has a similar specificity, but a better sensitivity, intensity, and extent of reactivity in comparison with AMACR, and may be used as an alternative to AMACR, in support of the diagnosis of BE-dysplasia and EAC.

Cutaneous histoplasmosis with prominent parasitization of epidermal keratinocytes: report of a case.

Disseminated histoplasmosis most commonly occurs in immunosuppressed individuals and involves the skin in approximately 6% of patients. Cutaneous histoplasmosis with an intraepithelial-predominant distribution has not been described. A 47-year-old man was admitted to our institution with fever and vancomycin-resistant enterococcal bacteremia. He had been diagnosed with T-cell prolymphocytic leukemia 4 years earlier and had undergone matched-unrelated-donor stem cell transplant 2 years earlier; on admission, he had relapsed disease. His medical history was significant for disseminated histoplasmosis 6 months before admission, controlled with multiple antifungal regimens. During this final hospitalization, the patient developed multiple 2-5 mm erythematous papules, a hemorrhagic crust across the chest, shoulders, forearms, dorsal aspect of the fingers, abdomen and thighs. Skin biopsy revealed clusters of oval yeast forms mostly confined to the cytoplasm of keratinocytes and within the stratum corneum; scattered organisms were present in the underlying superficial dermis without any significant associated inflammatory infiltrate. Special stains and immunohistochemical studies confirmed these to be Histoplasma organisms. We highlight this previously unrecognized pattern of cutaneous histoplasmosis to ensure its prompt recognition and appropriate antifungal therapy.

Expression and utility of IMP3 in the differential diagnosis of atypical glandular cells and adenocarcinoma in liquid-based cervical cytology.

BACKGROUND: Atypical glandular cell (AGC) interpretation in gynecological cytopathology presents many diagnostic challenges. We evaluated the expression of IMP3 in liquid-based cervical cytology and its utility in differentiating premalignant/malignant glandular lesions from benign/reactive processes. Additionally, we tried to determine whether IMP3 may be useful in differentiating among the types of uterine adenocarcinomas. DESIGN: Our cohort included 82 cases; 59 diagnosed with AGC and 23 with adenocarcinoma (Ac). IMP3 immunocytochemical stain was performed on ThinPrep slides and the results correlated with subsequent biopsy findings. IMP3 positivity was assessed by strong (2+ and 3+) granular cytoplasmic staining in at least one group of three epithelial cells. RESULTS: In the AGC group, IMP3 was positive in 14 (73.7%) of 19 cases that on histologic follow-up were confirmed Ac, and 39 (98.6%) of 40 non-glandular lesions/benign cases were negative. In the Ac group, IMP3 was expressed in 16 (69.6%) of 23 cases, of which 16 (72.2%) of 21 were uterine Ac. By combining the two groups, and excluding the 2 extrauterine carcinomas, IMP3 was positive in 30 (75%) of 40 uterine Ac, most of which (86.7%) were in situ/invasive endocervical Ac, and type II endometrial Ac (Papillary Serous and Clear Cell Carcinoma), and only 40% endometrioid Ac. CONCLUSION: In ThinPrep slides with AGC, IMP3 positivity predicts the presence of a significant endocervical or endometrial lesion on subsequent histology, and may also be a potential diagnostic tool useful in differentiating among the types of adenocarcinomas of the female lower genital tract.

Cokeromyces recurvatus identified in lung biopsy: case report of a non-pathogenic fungus, highlighting its potential histologic mimics.

We report a case of aspiration in a patient with gastric outlet obstruction due to pancreatic adenocarcinoma, in which three large yeasts were identified on tissue biopsy of the lung infiltrate. The histologic sections of the yeasts showed densely eosinophilic, round to oval, thick-walled structures with frayed borders and intra-cystic bluish inclusions. There was a background of mixed neutrophilic and eosinophilic infiltrate along with focal tissue necrosis. Our initial differential diagnoses included the usual large yeasts such as Cryptococcus, Coccidioides, and Blastomyces. Immunohistochemistry revealed reactivity to the Blastomyces antibody. Mycology studies eventually identified the organism as Cokeromyces recurvatus. Anti-fungal treatment was withheld with spontaneous resolution of the infiltrates. This case demonstrates the importance of using culture to speciate organisms identified on tissue, separating pathogens from non-pathogens and non-living artifacts in order for appropriate management.

Role of lymphovascular invasion and immunohistochemical expression of IMP3 in the risk stratification of superficially invasive pT1 esophageal adenocarcinoma.

BACKGROUND: A problem in the management of patients with Barrett's esophagus-related pT1 esophageal adenocarcinoma is to distinguish those who should be treated conservatively (endoscopic mucosal resection and/or radiofrequency ablation) from those who require esophago-gastrectomy. Recently, lymphovascular invasion (LVI) has emerged as one of the best predictors of regional lymph node metastasis (LNM) and recurrence-free survival (RFS) in pT1 EAC. However, LVI may be underestimated, both because of interobserver variability and incomplete sampling. The aim of our study was to correlate the presence of LVI, with the immunohistochemical expression of IMP3 in pT1 EAC and assess their role in further stratifying these lesions into high and low risk groups based on the potential for lymph node metastasis and poor outcome. DESIGN: Depth of invasion, assessed in five sublevels (m2, m3, sm1, sm2, and sm3), LVI, and expression of IMP3 were studied in 30 patients who underwent esophagogastrectomy for pT1 EAC (2001-2010) at Hartford Hospital, and correlated with LNM and RFS. IMP3 was considered positive when expressed in >50% of the malignant cells with an intensity of stain of 2-3+. RESULTS: Ten of 18 (55.5%) cases with IMP3 expression demonstrated LVI and 2/10 (20%) showed LNM and died of disease. In contrast, none of the 12 IMP3 negative cases showed LVI (p<0.004; 2-tailed Fisher exact test) or had LNM/DOD. CONCLUSIONS: In pT1 EAC, (1) based on IMP3 expression, pT1 EAC may be divided into high risk (LVI+/IMP3+) and low risk (LVI-/IMP3-) categories. (2) Absence of IMP3 expression is associated with a significantly reduced risk of LVI (Negative Predictive Value: 100%). (3) Since identifying lymphovascular invasion and other morphological parameters is prone to significant inter-observer variation, IMP3 may be useful as an ancillary marker especially in these pT1 lesions in predicting their clinical behavior, the risk stratification and potentially on the type of treatment.

The utility of PAX8 and IMP3 immunohistochemical stains in the differential diagnosis of benign, premalignant, and malignant endocervical glandular lesions.

UNLABELLED: Glandular lesions of the endocervix can be diagnostically challenging and occasionally the differential diagnosis includes endocervical adenocarcinoma in situ (EC AIS) and well-differentiated endocervical adenocarcinoma (ECA). PAX8 and IMP3 are two markers which have not been well studied in the endocervix. Our aim was to evaluate their immunohistochemical (IHC) expression in benign and malignant endocervical glandular lesions as well as to compare them to the traditionally used panel (Ki-67, p16, CEA). DESIGN: We searched our surgical pathology files for a cohort of benign endocervical glandular lesions as well as premalignant and malignant groups including EC AIS and ECA. An IHC panel consisting of PAX8, IMP3, Ki-67, p16, and CEA was performed on all cases. Immunoreactivity was scored on a degree of positivity (S0=no immunoreactivity, S1=up to 10% cells, S2=between 10 and 50% cells, S3=>50% cells) and intensity (Int0 - absent, Int1 - mild/faint, Int2 - moderate, Int3 - strong). RESULTS: PAX8 showed diffuse positivity (S3) with at least a moderate intensity of staining (Int2) in the benign group. PAX8 was focal (S1) in ECA and faint (Int1), compared to EC AIS, which was moderate (S2) and faint (Int1). IMP3 expression was focal in the benign group (S1), moderate (S2) in EC AIS and moderate-to-diffuse (S2-3) in ECA. IMP3 intensity was faint (Int1) in benign lesions, moderate (Int2) in EC AIS, and strong (Int3) in ECA. Significant Ki-67, p16, and CEA expression was noted in the premalignant/malignant cohort. CONCLUSION: PAX8 and IMP3 can be helpful in the differential diagnosis of benign vs. malignant endocervical glandular lesions. Our study, however, shows that there is some degree of overlap of staining in both the benign and malignant group. As such, PAX8 and IMP3 should always be interpreted with caution and in combination with the histomorphology.

Immune evasion and recognition of the syphilis spirochete in blood and skin of secondary syphilis patients: two immunologically distinct compartments.

BACKGROUND: The clinical syndrome associated with secondary syphilis (SS) reflects the propensity of Treponema pallidum (Tp) to escape immune recognition while simultaneously inducing inflammation. METHODS: To better understand the duality of immune evasion and immune recognition in human syphilis, herein we used a combination of flow cytometry, immunohistochemistry (IHC), and transcriptional profiling to study the immune response in the blood and skin of 27 HIV(-) SS patients in relation to spirochetal burdens. Ex vivo opsonophagocytosis assays using human syphilitic sera (HSS) were performed to model spirochete-monocyte/macrophage interactions in vivo. RESULTS: Despite the presence of low-level spirochetemia, as well as immunophenotypic changes suggestive of monocyte activation, we did not detect systemic cytokine production. SS subjects had substantial decreases in circulating DCs and in IFNγ-producing and cytotoxic NK-cells, along with an emergent CD56-/CD16+ NK-cell subset in blood. Skin lesions, which had visible Tp by IHC and substantial amounts of Tp-DNA, had large numbers of macrophages (CD68+), a relative increase in CD8+ T-cells over CD4+ T-cells and were enriched for CD56+ NK-cells. Skin lesions contained transcripts for cytokines (IFN-γ, TNF-α), chemokines (CCL2, CXCL10), macrophage and DC activation markers (CD40, CD86), Fc-mediated phagocytosis receptors (FcγRI, FcγR3), IFN-ß and effector molecules associated with CD8 and NK-cell cytotoxic responses. While HSS promoted uptake of Tp in conjunction with monocyte activation, most spirochetes were not internalized. CONCLUSIONS: Our findings support the importance of macrophage driven opsonophagocytosis and cell mediated immunity in treponemal clearance, while suggesting that the balance between phagocytic uptake and evasion is influenced by the relative burdens of bacteria in blood and skin and the presence of Tp subpopulations with differential capacities for binding opsonic antibodies. They also bring to light the extent of the systemic innate and adaptive immunologic abnormalities that define the secondary stage of the disease, which in the skin of patients trends towards a T-cell cytolytic response.

IMP3 immunocytochemical staining increases sensitivity in the routine cytologic evaluation of biliary brush specimens.

Biliary brush cytology is an important diagnostic tool in the evaluation of biliary strictures. Here, we evaluated 64 patients with biliary strictures who underwent endoscopic retrograde cholangiopancreatography with bile duct brushings. We assessed the utility of combining routine Papanicolaou-stained cytologic evaluation with immunocytochemical expression of insulin-like growth factor mRNA-binding protein-3 (IMP3). Definitive diagnoses were obtained via tissue resection/autopsy, biopsy, fine needle aspiration, or clinical progression of disease. Thirty-nine of the 64 patients were ultimately diagnosed with malignancy. The sensitivity of routine cytology for the detection of malignancy was 33.3%, immunocytochemical-IMP3 expression was 64.1%, and the combined sensitivity was 71.8%. The specificity of each method was 100%. The sensitivity of IMP3 immunocytochemical staining in the detection of malignancy in biliary brushings was superior to routine PAP-stained cytologic evaluation. Moreover, the combined use of biliary brushing cytology and IMP3 immunohistochemistry proved superior to the use of either method alone.

Human herpesvirus-6 in patients with Crohn's disease.

Human herpesvirus-6 (HHV-6) infections are usually asymptomatic reactivations in immunocompetent persons, but may be severe in immunocompromised individuals. Although primary HHV-6 infection is mainly associated with roseola infantum, it has also been associated with gastroenteritis, diarrhea, and nausea in children. In this study, we investigated the potential role of HHV-6 in Crohn's disease (CD). Evidence of HHV-6 infection in CD patients and controls was determined by immunohistochemistry (IHC), polymerase chain reaction (PCR), and quantitative real-time PCR (qPCR). Fifty-one tissue blocks from 23 CD patients and 20 tissue blocks from 20 controls were examined. Quantitativereal-time PCR was used to assess HHV-6 viral loads. IHC, PCR and qPCR indicated the presence of HHV-6 in both CD patients and controls. Immunohistochemistry of tissues revealed an almost equal frequency and distribution of positive cells; however, non-specific immunostaining confounded interpretation. HHV-6 DNA was detected in 52% (12/23) of CD and 55% (11/20) of control patients by PCR and in 69.5% (16/23) of CD cases and 65% (13/20) of controls by qPCR. Mean viral load in intestinal tissues was similar in CD and controls (33.4 and 57.9 copies microg(-1) DNA, respectively). Finding equal evidence of HHV-6 in patients and controls by multiple methods suggests that this virus is ubiquitous and probably not a cause of CD.

Consensus recommendations on estrogen receptor testing in breast cancer by immunohistochemistry.

Estrogen receptor (ER) status in breast cancer is currently the most important predictive biomarker that determines breast cancer prognosis after treatment with endocrine therapy. Although immunohistochemistry has been widely viewed as the gold standard methodology for ER testing in breast cancer, lack of standardized procedures, and lack of regulatory adherence to testing guidelines has resulted in high rates of "false-negative" results worldwide. Standardized testing is only possible after all aspects of ER testing--preanalytical, analytical, and postanalytical, have been closely controlled. A meeting of the "ad-hoc committee" of expert pathologists, technologists, and scientists, representing academic centers, reference laboratories, and various agencies, issued standardization testing recommendations, aimed at optimization of clinical ER testing environment, as a step toward improved standardized testing.

KOC (K homology domain containing protein overexpressed in cancer) and S100A4-protein immunoreactivity improves the diagnostic sensitivity of biliary brushing cytology for diagnosing pancreaticobiliary malignancies.

Biliary tract brush cytology is one of the favored methods of evaluating lesions of the pancreatobiliary tract. However, although its specificity has been reported to be high (91-100%), the sensitivity is lower (30-88%). In this study we applied KOC and S100A4 protein immunocytochemistry to assess their potential use as adjunct markers in differentiating benign from malignant cells, and improve the diagnostic sensitivity of this method for pancreatobiliary malignancies. The authors examined KOC and S100A4 protein expression in 44 alcohol-fixed cytology specimens obtained by biliary brushings. Diagnoses included: (1) benign/atypical favor reactive (20 cases), (2) atypical/not diagnostic of malignancy (3 cases), and (3) suspicious for malignancy/malignant (21 cases). Alcohol-fixed Papanicolaou-stained slides (PAP) were stained with monoclonal antibody to KOC/L523S and polyclonal antibody to S100A4 protein. Results were recorded as negative or positive. Twenty-four cases were confirmed positive for adenocarcinoma and 20 cases were negative. The sensitivity and specificity of cytology was 83 and 95%, KOC showed a sensitivity of 92% and specificity of 95%. S100A4 protein showed a sensitivity of 79% and a specificity of 95%. The combined use of KOC and S100A4 protein showed a sensitivity of 100% and a specificity of 95%, respectively. The concurrent use of KOC and S100A4 protein improves the diagnostic sensitivity of biliary brushings cytology and demonstrates similar specificity as cytology alone in the diagnosis of pancreatobiliary malignancy.