Pathways Case Studies: October 2022

A 45-year-old Caucasian woman with a history of drug allergies (barbiturates and sulfonamides), depression, as well as recurrent acute episodes of generalized fatigue, dyspnea, and significant abdominal pain, was referred to Mayo Clinic for biochemical and molecular testing. Family history (Figure 1) revealed a maternal grandmother who died unexpectedly at the age of 36 after receiving barbiturates in preparation for an appendectomy. Even though no molecular investigations were performed, acute intermittent porphyria (AIP) was listed as the cause of death on the death certificate. Molecular genetic testing of the proband’s maternal aunt, however, was reportedly suggestive of variegate porphyria (VP) instead, while urinary porphyria testing (specimens not collected during attack) of the patient along with relevant family members were all negative and/or indeterminate (Table 1). Of note, multiple maternal family members including this aunt were reported to have symptoms and skin manifestations possibly consistent with lupus.

Despite the clinical presentation and family history of the patient, biochemical testing requested by the referring clinician only encompassed erythrocyte porphobilinogen deaminase (PBGD) activity as a means to establish/rule out AIP. No additional testing was ordered to investigate the possibility of VP and/or other acute hepatic porphyrias. Erythrocyte PBGD results were unremarkable (Table 1), and a subsequent molecular acute porphyria panel was reluctantly ordered by the clinician at the insistence of the patient. This later revealed that the patient was heterozygous for a well-known autosomal dominant pathogenic variant (c.503G>T, p.R168L) within the PPOX gene (Figure 2), which is consistent with a diagnosis of VP.

Figure 1: Family tree and relevant medical history
Figure 2: Sanger sequencing result for the PPOX gene
Table 1: Biochemical testing

Considering the clinical presentation and family history of this patient, which biochemical test/s should have been ordered for a more timely diagnosis prior to molecular testing?

  • Erythrocyte protoporphyrin fractionation; samples collected during an acute neurovisceral attack.
  • Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack.
  • Urinary porphobilinogen only; samples collected after an acute neurovisceral attack.
  • Erythrocyte porphobilinogen deaminase enzyme activity; samples collected during an acute neurovisceral attack.

The correct answer is ...

Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack.


Although the clinical presentation and medical history of the patient is consistent with an acute hepatic porphyria, inappropriate porphyrin testing and sample collection conditions impair the proper differentiation of acute porphyria subtypes, subsequently leading to a delay in and/or incorrect diagnosis. A timely differentiation of these subtypes is imperative, as some acute porphyrias such as VP and hereditary coproporphyria (HCP) have the propensity to lead to photosensitivity and ensuing cutaneous lesions. The following brief discussion of the aforementioned testing options are based on the Mayo Clinic acute porphyria testing algorithm,1 and depict some of the most important pitfalls and advantages to keep in mind when ordering biochemical testing for these diseases: 

OptionUrinary porphobilinogen (PBG) only; samples after an acute neurovisceral attack

While random porphobilinogen testing is valuable in the identification of an acute porphyria, PBG may be elevated in acute subtypes other than AIP, i.e., VP and HCP.2 This may be ascribed to the idea that aberrant accumulation of heme biosynthesis pathway intermediates activate delta-aminolevulinate synthase 1 enzyme activity, subsequently leading to induced elevations of PBG and aminolevulinic acid.3 Therefore, positive PBG measurements alone cannot be used for differentiation of acute subtypes, and additional testing is required for further classification.2 Furthermore, since porphobilinogen levels typically normalize within a few days after an acute neurovisceral episode, it is imperative that samples are collected during such an episode.4 This could explain the uninformative/normal urine porphyrin results obtained in the presented case and the relevant family members, as all specimens were reportedly collected in between attacks.

Option – Erythrocyte PBG enzyme activity; samples collected during an acute neurovisceral attack

PBGD catalyses the third enzymatic step of the heme biosynthesis pathway which encompasses the combination of four porphobilinogen molecules to form hydroxymethylbilane.3 Although a deficient PBGD enzyme activity is characteristic of AIP, unaffected erythrocyte activity does not rule out AIP, as 5%–10% of affected individuals only present with deficient activity in nonerythroid cells.5 A deficiency in this enzyme may also be masked by artificial stimulation via excessive alcohol ingestion,6 as well as by the collection of specimens during an attack.7 In addition, a normal PBGD activity alone will neither confirm nor rule out other acute hepatic porphyrias, subsequently being uninformative in the presented case. 

Option – Protoporphyrin fractionation; samples collected during an acute neurovisceral attack 

Erythrocyte protoporphyrins (metal free and zinc-complexed) concentrations are generally unaffected in acute porphyrias2 and are therefore not a relevant testing option in the current situation. 

Option – Urine (including porphobilinogen) and fecal porphyrins; samples collected during an acute neurovisceral attack

Besides the valuable contribution of PBG, urinary porphyrins may provide supplemental information about alternative porphyrin-intermediates (uroporphyrin to coproporphyrin) that could be auxiliary in the differentiation between acute porphyria subtypes. However, since VP and HCP present with nearly indistinguishable urinary profiles, fecal porphyrin testing is required to differentiate between these subtypes and is subsequently invaluable in the biochemical diagnosis of VP.7 Some of these characteristic/distinguishable findings are based on the observation that while the fecal coproporphyrin III/coproporphyrin I ratio is elevated in VP, it is even higher in HCP, and typically normal in AIP. Moreover, elevated fecal protoporphyrin excretion is seen only in VP cases and generally remains unaffected in other acute hepatic porphyrias. Consequently, fecal testing is a key component that should have been considered in the presented individual. However, as in the case of PBG measurements, the diagnostic success of these tests rely on sample collection timing to avoid false negatives.4,7


Based on some of these caveats, it is clear that no single biochemical test can differentiate between acute porphyria subtypes, and a comprehensive view of these (as described1) are required for an accurate and timely diagnosis of these patients.


  1. Mayo Clinic Laboratories (2022). Porphyria (acute) testing algorithm,
  2. Tortorelli S, White AL, Raymond K. Disorders of porphyrin metabolism. In: Biochemical and molecular basis of pediatric disease. 5th ed. PA: Academic Press; 2021: 503-528
  3. Bonkovsky HL, Guo JT, Hou W, Li T, Narang T, Thapar M. Porphyrin and heme metabolism and the porphyrias. Compr Physiol. 2013;3(1):365-401. doi:10.1002/cphy.c120006. PMID: 23720291
  4. Gounden V, Jialal I. Acute Porphyria. In StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2022 Jan-.
  5. Sassa S. The porphyrias. Photodermatol Photoimmunol Photomed. 2002;18(2):56-67. doi:10.1034/j.1600-0781.2002.180202
  6. McColl KE, Thompson GG, Moore MR, Goldberg A. Acute ethanol ingestion and haem biosynthesis in healthy subjects. Eur J Clin Invest. 1980;10:107-112. doi:10.1111/j.1365-2362.1980.tb02068
  7. Trier H, Krishnasamy VP, Kasi PM. Clinical manifestations and diagnostic challenges in acute porphyrias. Case Rep Hematol. 2013;628602. doi:10.1155/2013/628602. 

Zinandré (Zin) Stander, Ph.D.

Fellow, Clinical Biochemical Genetics
Mayo Clinic

Silvia Tortorelli, M.D., Ph.D.

Consultant, Laboratory Genetics and Genomics
Mayo Clinic

Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

Linda Hasadsri, M.D., Ph.D.

Consultant, Laboratory Genetics and Genomics
Mayo Clinic

Assistant Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

A 50-year-old man undergoes resection of a neuroendocrine tumor (NET). Three years later, he develops exertional dyspnea and lower extremity edema. An echocardiogram reveals severe tricuspid and pulmonary valve regurgitation, as well as an enlarged right ventricle.

Figure 1: Gross valve
Figure 2: HE 20X
Figure 3: 100x
Figure 4: VVG 100x

Given the clinical presentation, which of the following is the most likely scenario regarding this patient's current tumor status? 

  • Residual NET of the stomach
  • Recurrent NET of the left testicle
  • Recurrent NET of the small intestine
  • Primary NET of the right lung

The correct answer is ...

Recurrent NET of the left testicle.

This case is that of a patient with carcinoid syndrome resulting in valvular dysfunction. Carcinoid tumors are neuroendocrine tumors that commonly arise in the lungs and gastrointestinal tract, but have been known to arise in other less common primary sites. They may secrete substances such as serotonin, histamine, tachykinins, and prostaglandins, resulting in various clinical manifestations. Carcinoid heart disease (CHD), or Hedinger syndrome, occurs when serotonin, through the activation of TGF-beta, acts on heart valves by inducing myofibroblastic metaplasia. Histologically, this results in plaques with a “stuck on” appearance composed of fibroblasts, smooth muscle cells, and extracellular matrix components such as collagen and myxoid ground substance. Grossly, the valves and chordae are thickened with a pearly appearance.

The pathogenicity of serotonin with regards to valvular heart disease is highly dependent on the location of the primary tumor as well as any possible metastases. Tumors with venous drainage into the portal circulation are subjected to the hepatic metabolism of serotonin. However, a tumor location with drainage to the systemic circulation bypasses this inactivation. Likewise, serotonin can be deactivated in the lungs.

As such, the correct answer for this question is “recurrent NET of the left testicle,” as the venous drainage of the testes occurs via the gonadal veins to the inferior vena cava and the systemic circulation. This allows unmetabolized serotonin to reach the right side of the heart, affecting the tricuspid and pulmonary valves, as seen in this case example. Two of the answer choices (“residual NET of the stomach” and “recurrent NET of the small intestine”) both describe scenarios whereby tumors would drain into the portal circulation. Of note, in either of those choices, metastatic disease or cirrhosis could also allow for the bypassing of hepatic serotonin metabolism.

Though this question describes right-sided carcinoid heart disease, there are other situations in which the left-sided valves can be affected. One of the answer choices (“primary NET of the right lung”) describes a scenario in which serotonin could be secreted into the pulmonary venous drainage and subsequently exposed to the mitral and aortic valves. Another possibility would be a patient with a patent foramen ovale or other septal defect. Under the proper hemodynamic conditions, a right-to-left shunt would allow for secreted serotonin to bypass the lungs (where it would otherwise be metabolized) and affect the left-sided valves. 

Of note, there are also a few medications that can produce similar valvular findings to those seen in patients with carcinoid heart disease, including fenfluramine and ergot alkaloids. 


  1. Grozinsky-Glasberg, S., Grossman, A.B., Gross, D.J., 2015. Carcinoid heart disease: from pathophysiology to treatment - ‘Something in the way it moves'. Neuroendocrinology 2015;101(4)263–273. doi:10.1159/000381930
  2. Ram P, Penalver JL, Lo KBU, Rangaswami J, Pressman GS. Carcinoid heart disease: review of current knowledge. Tex Heart Inst J. 2019 Feb 1;46(1):21-27. doi:10.14503/THIJ-17-6562. PMID: 30833833; PMCID: PMC6378997
  3. Clement D, Ramage J, Srirajaskanthan R. Update on pathophysiology, treatment, and complications of carcinoid syndrome. J Oncol. 2020 Jan 21;2020:8341426. doi:10.1155/2020/8341426. PMID: 32322270; PMCID: PMC7160731
Square photo of Philip Hurst

Philip Hurst, M.D.

Fellow, Cardiovascular Pathology
Mayo Clinic

Andrew Layman portrait square

Andrew Layman, M.D.

Senior Associate Consultant, Anatomic Pathology
Mayo Clinic

A 15-month-old boy presents with vomiting and weight loss. A large, heterogeneous brain lesion is found by MRI (see Figure 1), and the patient undergoes resection. Representative cytopathologic (Figure 2) and histopathologic (Figure 3) photomicrographs of the tumor are shown.

Figure 1: Heterogeneous brain lesion
Figure 2: Representative cytopathologic photomicrograph
Figure 3: Representative histopathologic photomicrograph

Which of the following immunohistochemical findings is characteristic of this entity?

  • Absence of membranous EMA expression
  • Positivity for GFAP
  • Positivity for synaptophysin
  • Loss of INI1 expression

The correct answer is ...

Loss of INI1 expression.

The lumbar puncture cytology shows tumor cells with a striking rhabdoid morphology, characterized by enlarged eccentrically located nuclei, and dense cytoplasm with intracytoplasmic globular inclusions. The histopathologic sections reveal a polymorphic tumor, including areas with clear cell and spindled morphologies, as well as few rhabdoid cells. A radiologically and histologically heterogeneous tumor, coupled with the presence of rhabdoid cells, in a child in the first three years of life raises concern for an atypical teratoid/rhabdoid tumor (AT/RT). The hallmark molecular feature of this entity is an inactivating mutation in either SMARCB1 (approximately 95% of AT/RTs) or, less frequently, SMARCA4. Both of these genes are part of the SWI/SNF complex which regulates chromatin remodeling and cellular differentiation. The immunohistochemical surrogates for inactivating SMARCB1 or SMARCA4 alterations are loss of expression of INI1 or BRG1, respectively. Interestingly, WHO diagnostic criteria do not require rhabdoid morphology for classification as AT/RT; an embryonal CNS tumor with SMARCB1/INI1 or SMARCA4/BRG1 loss is sufficient for diagnosis.

Reflective of the morphologic polymorphism, AT/RTs can display a wide range of immunoreactivity and mimic numerous other tumors. 

AT/RTs may express GFAP and thereby resemble a high-grade glioma. 

Positivity for synaptophysin can be suggestive of medulloblastoma (especially the anaplastic variant) or CNS Ewing sarcoma (if more monomorphic areas happen to be sampled).

AT/RTs can also entrap normal choroid plexus, and the juxtaposition of choroid plexus with a poorly-differentiated high-grade malignancy in a pediatric patient can lead to consideration of choroid plexus carcinoma. Both entities may show variable or absent expression of EMA, further confounding evaluation.

Above all, it is the absence of INI1 (or BRG1) staining that distinguishes AT/RT from nearly all other entities on the differential diagnosis. AT/RT should be at least considered when approaching a poorly-differentiated CNS tumor in a very young child, making evaluation of SMARCB1 and SMARCA4 critical in diagnosing this entity.


  1. Chan V, Marro A, Findlay JM, Schmitt LM, Das S. A systematic review of atypical teratoid rhabdoid tumor in adults. Front Oncol. 2018 Nov 28;8:567. doi:10.3389/fonc.2018.00567. PMID:30547013; PMCID: PMC6279935
  2. Meyers SP, Khademian ZP, Biegel JA, Chuang SH, Korones DN, Zimmerman RA. Primary intracranial atypical teratoid/rhabdoid tumors of infancy and childhood: MRI features and patient outcomes. AJNR Am J Neuroradiol. 2006 May;27(5):962-71. PMID: 16687525; PMCID: PMC7975730.
  3. Haberler C, Wesseling P, Huang A, et al. Atypical teratoid/rhabdoid tumor. In: WHO Classification of Tumours Editorial Board, Central Nervous System Tumours, 5th ed, 2021.
Photo of Ryan W. Kendziora, M.D.

Ryan Kendziora, M.D.

Resident, Anatomic and Clinical Pathology
Mayo Clinic

Aditya Raghunathan, M.D., M.P.H.

Consultant, Anatomic Pathology
Mayo Clinic

Associate Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

A 48-year-old man with a history of smoking presents with a mass involving the lung and anterior mediastinum. 

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5
Figure 6
Figure 7

What is your preferred diagnosis?

  • Malignant rhabdoid tumor
  • NUT midline carcinoma
  • Thoracic SMARCA4-deficient undifferentiated tumor
  • SMARCA4-deficient non-small cell lung carcinoma (NSCLC)

The correct answer is ...

Thoracic SMARCA4-deficient undifferentiated tumor.

Thoracic SMARCA4-deficient undifferentiated tumor is an aggressive malignancy that may arise in the lung, pulmonary hilum, mediastinum, or pleura.1,2 It predominantly occurs in middle-aged adults, but may occur in a wide age range. The vast majority patients with this tumor have a history of smoking.3 Metastases are frequently present at the time of diagnosis. SMARCA4-deficient undifferentiated tumors may also be primary to extra-thoracic organs (e.g., stomach, pancreas, kidney, ovary, and uterus), such that clinical correlation is critical for determination of the primary site.

On histology, these tumors are typically composed of sheets of relatively monotonous epithelioid cells with focal rhabdoid morphology, large round nuclei, vesicular chromatin, and prominent nucleoli. Mitotic figures are abundant and necrosis is often present, in keeping with a high-grade malignancy. The cells may appear discohesive, raising suspicion for a hematolymphoid neoplasm. On morphology, the differential diagnosis may also include pulmonary and thymic carcinoma, NUT midline carcinoma, melanoma, germ cell tumor, malignant rhabdoid tumor, epithelioid sarcoma, and other sarcomas. 

On immunohistochemistry, these tumors are negative for NUT and typically do not express melanoma or lymphoid markers. Keratin expression is typically weak and focal but may be negative.3 Diffuse strong expression of keratin is not in keeping with this entity. A negative claudin-4 may also be helpful in ruling out carcinoma. TTF1 and p40 may rarely be focally positive.3 CD34 and SALL4 are often expressed in these tumors,2,4 but they may be distinguished from germ cell tumors and sarcomas by loss of BRG1 immunoreactivity. BRG1 loss is a defining feature of these tumors, reflecting biallelic inactivation of the chromatin remodeling gene SMARCA4.5 Approximately one quarter of cases show a severe reduction rather than full loss of BRG1 immunostaining.2,3  Immunoreactivity for INI1, encoded by SMARCB1, is retained in these tumors, helping to distinguish them from malignant rhabdoid tumors and epithelioid sarcomas.

Importantly, the presence of any morphologic features definitive for carcinoma (e.g., gland formation, papillary architecture, or keratinization) excludes this diagnosis. However, approximately 5% of thoracic SMARCA4-deficient undifferentiated tumors are associated with adjacent areas of non-small cell lung carcinoma (NSCLC), raising the possibility that these tumors are undifferentiated or poorly differentiated carcinomas arising from epithelial precursors.3 In keeping with this notion, both thoracic SMARCA4-deficient undifferentiated tumors and pulmonary carcinomas tend to have a genomic smoking signature. Cases with definitive morphologic features of carcinoma in the SMARCA4-deficient areas are best classified as SMARCA4-deficient NSCLC, and represent about 5% of conventional NSCLC.6-8 The prognosis of SMARCA4-deficient undifferentiated tumor is worse than that of SMARCA4-deficient NSCLC, with a median survival of only 4-7 months.3,5  


  1. Sauter JL, Graham RP, Larsen BT, Jenkins SM, Roden AC, Boland JM. SMARCA4-deficient thoracic sarcoma: a distinctive clinicopathological entity with undifferentiated rhabdoid morphology and aggressive behavior. Mod Pathol.2017;30:1422-32.
  2. Yoshida A, Kobayashi E, Kubo T, Kodaira M, Motoi T, Motoi N, Yonemori K, Ohe Y, Watanabe SI, Kawai A, Kohno T, Kishimoto H, Ichikawa H, Hiraoka N. Clinicopathological and molecular characterization of SMARCA4-deficient thoracic sarcomas with comparison to potentially related entities. Mod Pathol. 2017;30:797-809.
  3. Rekhtman N, Montecalvo J, Chang JC, Alex D, Ptashkin RN, Ai N, Sauter JL, Kezlarian B, Jungbluth A, Desmeules P, Beras A, Bishop JA, Plodkowski AJ, Gounder MM, Schoenfeld AJ, Namakydoust A, Li BT, Rudin CM, Riely GJ, Jones DR, Ladanyi M, Travis WD. SMARCA4-deficient thoracic sarcomatoid tumors represent primarily smoking-related undifferentiated carcinomas rather than primary thoracic sarcomas. J Thorac Oncol.2020;15:231-47.
  4. Perret R, Chalabreysse L, Watson S, Serre I, Garcia S, Forest F, Yvorel V, Pissaloux D, Thomas de Montpreville V, Masliah-Planchon J, Lantuejoul S, Brevet M, Blay JY, Coindre JM, Tirode F, Le Loarer F. SMARCA4-deficient thoracic sarcomas: clinicopathologic study of 30 cases with an emphasis on their nosology and differential diagnoses. Am J Surg Pathol. 2019;43:455-65.
  5. Le Loarer F, Watson S, Pierron G, de Montpreville VT, Ballet S, Firmin N, Auguste A, Pissaloux D, Boyault S, Paindavoine S, Dechelotte PJ, Besse B, Vignaud JM, Brevet M, Fadel E, Richer W, Treilleux I, Masliah-Planchon J, Devouassoux-Shisheboran M, Zalcman G, Allory Y, Bourdeaut F, Thivolet-Bejui F, Ranchere-Vince D, Girard N, Lantuejoul S, Galateau-Sallé F, Coindre JM, Leary A, Delattre O, Blay JY, Tirode F. SMARCA4 inactivation defines a group of undifferentiated thoracic malignancies transcriptionally related to BAF-deficient sarcomas. Nat Genet.2015;47:1200-5.
  6. Dagogo-Jack I, Schrock AB, Kem M, Jessop N, Lee J, Ali SM, Ross JS, Lennerz JK, Shaw AT, Mino-Kenudson M. Clinicopathologic Characteristics of BRG1-Deficient NSCLC. J Thorac Oncol. 2020;15:766-76.
  7. Herpel E, Rieker RJ, Dienemann H, Muley T, Meister M, Hartmann A, Warth A, Agaimy A. SMARCA4 and SMARCA2 deficiency in non-small cell lung cancer: immunohistochemical survey of 316 consecutive specimens. Ann Diagn Pathol. 2017;26:47-51.
  8. Nambirajan A, Singh V, Bhardwaj N, Mittal S, Kumar S, Jain D. SMARCA4/BRG1-deficient non-small cell lung carcinomas: a case series and review of the literature. Arch Pathol Lab Med. 2021;145:90-8.

Julia Naso, M.D., Ph.D.

Fellow, Pulmonary Pathology
Mayo Clinic

Marie-Christine Aubry, M.D.

Consultant, Anatomic Pathology
Mayo Clinic

Professor of Laboratory Medicine and Pathology
Mayo Clinic College of Medicine and Science

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