High-Grade Appendiceal Mucinous Neoplasm: Clinicopathologic Findings in 35 Cases

With Institutional Review Board approval (Dana Farber Cancer Institute Office for Human Research Studies, IRB No. 18-480, approval date: September 24, 2018), we searched the pathology departmental archives of Basingstoke and North Hampshire Hospital (Basingstoke, United Kingdom), Beth Israel Deaconess Medical Center (Boston, Massachusetts), Massachusetts General Hospital (Boston), the University of Pittsburgh Medical Center (Pittsburgh, Pennsylvania), and the University of Rochester Medical Center (Rochester, New York) from January 2005 to March 2021 for cases signed out as HAMN or that might represent HAMNs, including terms such as high-grade appendiceal adenoma or mucinous cystadenocarcinoma. Additionally, 15 cases were from the consult files of several of the authors; as most of the authors routinely review appendiceal mucinous neoplasms, some of these cases had been prospectively flagged. We included cases as based on PSOGI criteria, namely appendiceal neoplasms architecturally resembling LAMN but displaying any amount of high-grade cytologic atypia (described below).³  Tumors were included if they were confined to the mucosa, provided they had architectural features consistent with LAMN/HAMN; this terminology conforms to the current recommendation to avoid the use of the term adenoma for tumors confined to the mucosa unless they have features that resemble colorectal adenomas.³  Thus, tumors that morphologically resembled colonic tubular or tubulovillous adenomatous polyps (polyp-forming lesions demonstrating intestinal-type dysplastic epithelium with intervening lamina propria), without the architectural features of an appendiceal mucinous neoplasm at least focally, including flat or undulating areas, were excluded. Tumors that showed infiltrative-type invasion, even focally, were excluded, as these qualified as invasive adenocarcinomas.

Thirty-five tumors were identified. For each, we gathered clinical and pathologic information, including patient age and sex, presenting symptoms, imaging findings, surgical procedure performed, gross lesion size, gross findings, perforation, whether the lesion was entirely submitted, additional therapy, time to recurrence (if any), and patient outcome at last follow-up. Some of these data were unavailable for some consultation cases. In addition, we collected histopathologic information on each case, including location in appendix, margin status, whether the high-grade dysplasia was diffuse (≥75% of the neoplasm) or nondiffuse (<75% of the neoplasm), approximate percentage of high-grade dysplasia in nondiffuse cases, architecture of high-grade areas (eg, villous, flat, cribriform, micropapillary), cytologic characteristics of high-grade epithelium, deepest extent of epithelium and mucin in the appendix wall, details of peritoneal disease (if applicable), and node status. When patients had peritoneal mucin, slides were reviewed to determine whether the mucin was cellular (therefore qualifying as PMP), and if the mucin was cellular, the grade of PMP based on Davison criteria.⁹ 

Immunohistochemistry for p53 was performed on 10 cases with the Leica DO-7 clone, RTU (Leica Biosystems, Wetzlar, Germany), with ER2 antigen retrieval on the Leica Bond III platform. Molecular analysis for single nucleotide variants, insertions/deletions, and copy number variations was performed on 8 cases by isolating genomic DNA from paraffin blocks, after identifying high-grade areas as targets, and performing anchored multiplex polymerase chain reaction using the ArcherDx platform and Illumina NextSeq next-generation sequencing as described elsewhere.¹⁰  Archival material for ancillary testing was not available for the remaining cases.

Clinical details for the 35 patients are summarized in Table 1. The patients' ages ranged from 23 to 79 years (mean, 57 years), and 57% of them were female. Presenting symptoms included abdominal or pelvic pain (n = 18, 51%) including 6 that were described as “appendicitis” in clinical notes, and abdominal distention/bloating (n = 6, 17%). Two patients had an abdominal mass and 7 were found to have HAMNs incidentally, including 1 patient who had a mucocele noted on screening colonoscopy. Information on abdominal imaging was available for 24 patients and showed appendiceal dilatation/mucocele in 9 cases. Another patient had what was misinterpreted as a uterine fibroid, but intraoperatively was found to have a large cyst contiguous with the appendix (presumably a cystically dilated appendix), with normal uterus and ovaries. Four patients had imaging consistent with appendicitis. Peritoneal disease/omental thickening was found in 10 patients, of whom 3 also had an enlarged ovary (2 right, 1 left). At initial surgery, 19 patients underwent appendectomy, 3 underwent appendectomy with cecectomy, 11 underwent right hemicolectomy, and 2 underwent total colectomy. Nine patients underwent cytoreductive surgery with hyperthermic intraperitoneal chemotherapy (HIPEC) during their initial surgical procedure. However, in 3 cases, cytoreduction was incomplete; in 1 patient, complete cytoreduction was intended but could not be performed, whereas in the other 2 patients, only tumor debulking for symptom relief was intended.

The pathologic findings are summarized in Table 2. Gross findings were available for all cases. In 14 cases, the gross description documented perforation, and in another case, the prosector did not specifically note perforation but described serosal mucin or fibrinopurulent exudate on the serosa, indicating perforation. An additional case did not document perforation in the pathology report, but the intraoperative note described in situ rupture of a cystic appendix. Additional gross findings included appendiceal dilatation (n = 16), distortion (n = 4), and luminal mucin (n = 8). Gross lesion size was recorded for 30 cases and averaged 7.9 cm (range, 2.5–19.0 cm). The appendix was entirely submitted in 31 cases (89%).

On histology, 23 appendices (66%) were entirely involved by neoplasm, while in 7 cases (20%) only the distal appendix was involved, and in 2 (6%), only the proximal appendix was involved. Extent and location of involvement were unclear in the remaining 3. Only 2 cases (both appendectomies) had positive surgical margins, both of which had tumor otherwise limited to the appendix (pT1 and pT3).

HAMN generally resembled LAMN on low power (Figure 1, A). By definition, all cases showed high-grade nuclear cytology (Figure 1, B through D). These areas showed nuclear enlargement, vesicular or hyperchromatic nuclei, prominent anisonucleosis, prominent nucleoli in some cases, increased mitoses, and loss of nuclear polarity. In 20 cases (57%), the high-grade dysplasia was diffuse, whereas in the rest, the high-grade portions accounted for approximately 10% to 60% of the tumor; overall mean high-grade percentage was 70%, median percentage was 80%, 14 cases (40%) were entirely high-grade (no low-grade component), and 7 cases (20%) showed less than 50% high-grade epithelium. In 18 cases, the high-grade neoplastic epithelium showed apical cytoplasmic mucin, and in another 15 cases, the high-grade epithelium showed areas in which the cytoplasm was more dense and eosinophilic, without apical mucin caps. In 2 cases, the high-grade neoplastic epithelium showed only the latter appearance throughout. The high-grade areas of the tumors showed some patterns of growth that are similar to LAMN, including flat (21 cases, 60%), undulating (19, 54%), villous (9, 26%), and scalloped (5, 14%) growth. However, in addition to the high-grade cytologic features, many cases showed architectural complexity beyond what is seen in LAMN, including micropapillarity (small clusters of epithelial cells projecting toward the lumen without associated supporting fibrovascular tissue; 15 cases, 43%; Figure 2, A), cribriforming (arching and interconnecting epithelial cells forming multiple lumens; 10 cases, 29%; Figure 2, B), and rarely multilayered/solid epithelial growth (disordered multiple layers of epithelial cells; 2 cases, 6%; Figure 2, C).

The tumor was confined to the appendiceal mucosa in 6 cases (pTis), and 2 had mucin or epithelium extending into the submucosa (pT1). One case had mucin and epithelium extending into the muscular wall of the appendix (pT2), and 4 cases had mucin extending to the subserosa (pT3), with epithelium also extending that deeply in 3 of the cases. The remaining 22 cases had tumor or mucin extending to the serosal surface (pT4a). Seven of these 22 cases had acellular mucin on the appendix serosa, and 6 also had peritoneal acellular mucin (pM1a). Fifteen of the 22 pT4a cases had cellular mucin on the appendix serosa. Two of these 15 cases had no peritoneal mucin, 2 had acellular mucin on peritoneal sites (pM1a), and 11 cases had PMP (pM1b), which was grade 2 in 10 cases (Figure 2, D and E) and grade 1 in the remaining case (Figure 2, F). Sites of involvement by PMP are listed in Table 2; the most common sites were omentum, peritoneum, bowel serosa, and, in women, the ovaries. None of the 19 cases with lymph node examination had lymph node involvement (mean node count: 14).

Ten cases had available paraffin blocks for immunohistochemical testing of the primary lesion; molecular testing was also performed on 8 of these primary lesions. Detected molecular abnormalities included 7 KRAS mutations (35G>A[Gly12Asp] in 2 tumors; 35G>T[Gly12Val] in 2; 34G>A[Gly12Ser] in 1; 38G>A[Gly13Asp] in 1; 183A>C[Gln61His] in 1), 4 TP53 mutations (403T>A[Cys135Ser], 455C>T[Pro152Leu], 586C>T[Arg196Ter], 637C>T[Arg213Ter]), 2 GNAS mutations (602G>A[Arg201His], p.Arg201His), and 1 mutation each in ARID1A (6070C>T[Arg2024Trp]), ERBB3 (245A>T[Glu82Val]), BRCA1 (3082C>T[Arg1028Cys]), NF1 (1381C>T[Arg461Ter]), and SMAD4 (1448-1G>A). Additionally, 2 cases showed copy number variations (MYC gain in 2, MET gain in 1). The 4 cases with TP53 mutations had abnormal p53 expression patterns by immunohistochemistry (3 increased expression and 1 null pattern). The 4 cases without TP53 mutation had a wild-type pattern of expression (although staining was only focal in 1), as did 2 cases without molecular analysis (Figure 3). The molecular testing was performed on 6 cases with diffuse high-grade cytology and 2 with nondiffuse high-grade cytology; the nondiffuse cases showed 1 KRAS mutation, 2 TP53 mutations, and the ARID1A, ERBB3, NF1, and SMAD4 mutations.

One of the patients who initially had an appendectomy and had HAMN confined to the appendix had a completion right hemicolectomy. Neither of the 2 patients with a positive appendectomy margin had additional surgical intervention. Five patients received adjuvant chemotherapy, always FOLFOX based, including 2 patients with pT4 disease who had only acellular mucin at extra-appendiceal sites; in both of these cases, the use of older terminology of mucinous cystadenocarcinoma in the pathology report presumably led to the decision to offer adjuvant therapy.

Two patients with pT4a disease but no PMP at initial surgery had progression to PMP on follow-up. The first patient had what was described intraoperatively as a large cystic mass contiguous with the appendix (microscopically consistent with a cystically dilated appendix) that ruptured intraoperatively, and microscopically showed low-grade epithelium on the serosal surface but no extra-appendiceal mucin. She reportedly developed grade 1 PMP within 3 months and underwent cytoreductive surgery and HIPEC at that time (operation performed at an outside institution and slides not available to confirm grade). The other patient had a grossly perforated HAMN with mucinous epithelium that extended into subserosa, and acellular mucin on the colonic serosa (but no neoplastic mucinous epithelial cells beyond the subserosa). One year later, he developed grade 2 PMP and underwent cytoreductive surgery and HIPEC.

Outcome data were available for 29 patients, including 1 patient who died perioperatively (<1 month after surgery). Excluding that 1 patient, median follow-up after surgery was 34 months (range, 4–171 months). At last follow-up, 20 patients were alive without disease or had died of other causes (mean, 41 months; range, 4–171 months). All 13 patients with HAMN confined to the appendix were alive without disease, died of other disease, or had no follow-up. Three patients were alive with disease 5, 5, and 32 months after cytoreduction and HIPEC. Five patients died of disease 8, 24, 63, 65, and 106 months after initial surgery.

In total, 13 patients were known to have PMP during their clinical course. Eleven patients initially presented with or developed high-grade PMP in our series; among them, 5 died of disease, 2 were alive with disease, and 2 were alive without disease at last follow-up. Two other patients had low-grade PMP as specified below. Of the 7 patients who presented with PMP and achieved complete cytoreduction, 5 experienced recurrence 12 to 40 months after their initial surgery (including one whose peritoneal disease re-recurred with signet ring cells, warranting designation as Davison grade 3 PMP); 4 died of disease; and the other one was alive with disease. The 1 patient who presented with PMP and did not successfully achieve complete cytoreduction died of disease at 8 months, and the 2 patients who underwent planned palliative debulking (rather than attempted complete cytoreduction) were alive with progressive disease at 5 months (high-grade PMP) and 5 months (low-grade PMP). The 2 patients who developed PMP after initial surgery remained disease-free at last follow-up (44 months [high-grade PMP] and 68 months [low-grade PMP]). No follow-up information was available for 1 recent patient who presented with PMP. No patient in our study developed metastatic disease outside the peritoneal cavity (pM1c).

In our series, we found that HAMNs generally had gross features identical to LAMNs, with appendiceal dilatation, and occasional perforation and serosal mucin. Histologically, HAMNs and LAMNs can have similar architectural features, appearing as flat, scalloped, undulating, or occasionally villous neoplastic epithelial proliferations. However, high-grade areas often had additional architectural complexity not seen in LAMNs, particularly micropapillary or cribriform architecture. This is similar to the findings of Liao et al,¹¹  who described papillary fronds and cribriform structures in their series of 9 cases of HAMN. We observed that, unlike the neoplastic epithelial cells in LAMN that have tall mucin vacuoles, the neoplastic cells in HAMN often have reduced mucin, with columnar cells that have eosinophilic cytoplasm and lack apical cytoplasmic mucin, although most cases had at least some areas in which the high-grade cells had small apical mucin caps. We found that nearly half of HAMNs had focal or segmental involvement of the appendix, similar to Liao et al¹¹  who reported that most of their HAMNs had 20% to 80% low-grade components. This suggests progression of a LAMN to HAMN. However, in our series, most cases were diffusely high-grade, suggesting that the high-grade component overtakes the low-grade precursor. In cases that are entirely high-grade, the possibility that some tumors arise de novo as high-grade cannot be excluded.

Fifty-four percent of the HAMNs in our series showed disseminated peritoneal disease, including 7 (20% overall) with acellular mucin and 12 (34% overall) with PMP. This is similar to the higher end of reported rates for LAMN, which range from 5% to 38% for PMP development depending on the series.^1,2,12–15  We found that, as with LAMN, neoplastic mucinous epithelium on the appendiceal serosa was the key risk factor for progression to PMP. In our series, 2 patients with pT4 disease and mucinous epithelium on the appendix serosa (one of whom also had acellular mucin on the colonic serosa) had progression to PMP (pM1b disease). Nine other patients had pT4 disease and none developed PMP, including 3 with serosal mucinous epithelium. Six of these patients also had acellular mucin at extra-appendiceal sites. Prior studies on appendiceal mucinous neoplasms (mostly LAMNs) have shown that acellular mucin on the appendix serosa carries a low risk of recurrence as PMP, whereas cellular mucinous tumor on the appendix serosa is associated with increased risk of progression to PMP.^2,16  The number of patients in our series who fit into this category is small, but our data do not support the contention that HAMN is more aggressive than LAMN in this regard. Others have found that patients with LAMNs and disease limited to the right lower quadrant or remote acellular mucin deposits have a better outcome than patients with cellular peritoneal mucinous tumor deposits, and that many of these patients can be followed up conservatively.^12–20  Our data, while limited, suggest that a similar approach may be reasonable for patients with HAMN. Additionally, none of our patients developed nodal or extraperitoneal disease. This suggests that HAMNs are unlikely to spread via lymphatic or hematogenous routes, similar to LAMNs.

In contrast to perforated HAMNs with serosal mucinous epithelial cells, tumors confined to the appendix did not recur or progress. In our series, 4 cases had mucin and/or epithelial cells in the subserosa (pT3) and another 9 had epithelium or mucin limited to the mucosa or submucosa, and none had recurrence. This is similar to the behavior of LAMNs, which generally do not recur if the appendix is excised with an intact serosa.^2,12  Although our numbers are small, we found no support for staging LAMNs and HAMNs differently, and we advocate that the same AJCC staging system should be applied to LAMN and HAMN. We are not aware of another human cancer that is staged differently depending on its grade, and appendiceal mucinous neoplasms should not be an exception. We also had 2 cases in which the proximal margin was positive, yet no additional surgical intervention was performed, and the patients were alive without disease on follow-up. Prior studies of LAMNs with a positive appendiceal margin have shown that additional resection specimens generally do not have residual neoplasia, and even without cecal resection, patients have a benign course.^2,13,21  The biology of HAMN may be similar in this regard, although case numbers remain too small to draw definitive conclusions.

The grade of the peritoneal tumor in our cases was usually concordant with the grade of the primary tumor (HAMN with grade 2 PMP), although discordant peritoneal tumor grade was seen in 2 cases (HAMN with grade 1 PMP). In the series by Yantiss et al,¹⁶  dealing with localized PMP, 5 appendiceal mucinous neoplasms with high-grade atypia (presumably HAMN) had low-grade mucinous epithelial cells on the appendix serosa. Discordance between primary and peritoneal tumor occurs uncommonly in cases of appendiceal mucinous neoplasm with PMP, including HAMN,¹⁹  but our data suggest that most HAMNs, as expected, spread to the peritoneum as grade 2 PMP, while LAMNs spread as grade 1 PMP. However, others have also noted isolated cases of appendiceal mucinous neoplasms with high-grade features associated with low-grade PMP.⁹  Perhaps, HAMNs with both low- and high-grade areas might disseminate as either grade 2 or grade 1 PMP, depending on which component spreads to the peritoneal cavity, which is itself dependent upon the location of the rupture site in the appendix relative to the areas of high-grade atypia. Determining the grade of tumor at the exact point of rupture was not possible in many of our cases owing to the disrupted nature of these specimens.

In our series, 11 patients either presented with or developed high-grade (grade 2) PMP. Only 2 of those patients were alive without disease on follow-up (44 and 62 months after cytoreductive surgery), and most others either died of their disease (5 patients) or still had disease (2 patients); 1 recent patient had no follow-up, and 1 died perioperatively. Although this is a small number of patients, this suggests a worse prognosis than disseminated LAMN, which usually results in grade 1 PMP that, after cytoreductive surgery and HIPEC, is associated with a median disease-free survival of 170.5 months.²²  This is not surprising, as several studies have shown that grade 2 PMP carries a worse prognosis than grade 1 PMP.^9,22,23 

Molecular characterization of HAMN has been reported in previous small series. We were able to perform molecular analysis on 8 HAMNs (3 purely mucinous and 5 mixed mucinous/intestinal-type), finding KRAS mutations in 7 cases (88%) and GNAS mutations in 2 mixed cases (25%). Our limited data are similar to those of other groups, who found KRAS mutations in nearly all of their HAMNs and GNAS mutations in 20% to 56%.^11,24–26  These data are similar to LAMN, in which KRAS and GNAS are frequently mutated, although GNAS mutations are perhaps more frequent in LAMNs (reported in greater than 50%).^2,27–29  In fact, other studies have noted that GNAS mutations occur more frequently in LAMNs and low-grade PMP than in mucinous adenocarcinomas and high-grade PMP,^27,29–31  also suggesting the possibility that a subset of HAMNs might arise de novo rather than progress from LAMN. TP53 mutations were detected in 4 of 8 HAMNs, similar to the study by Liao et al,¹¹  which supports the high-grade nature of the tumors. Immunohistochemistry for p53 predicted the presence or absence of a TP53 mutation correctly in 8 cases with molecular data (though staining was challenging to interpret in 1). Whether p53 can serve as a biomarker for HAMN requires additional study; we caution that p53 staining should not supersede the assessment of the degree of cytologic atypia to arrive at a diagnosis of HAMN, since p53 overexpression and TP53 mutations have been described rarely in LAMNs.^7,32  We found isolated mutations in other genes, such as ERBB3, ARID1A, BRCA1, and SMAD4, and others have found mutations in APC, RNF43, and ATM in occasional HAMNs.^11,24–26  Additionally, we detected MYC gain in 2 cases and MET gain in 1 case; to our knowledge, copy number variations have not previously been reported in HAMN.

We recognize that in this retrospective study, we included 4 consultation cases that were not submitted entirely for microscopic examination. All 4 were pT4a with peritoneal disease; on follow-up, 2 patients were alive without disease (44 and 62 months) and 2 survived for years before dying of disease (65 and 106 months). Therefore, the inclusion of these cases did not skew the data. However, in clinical practice, any HAMN should be submitted entirely to exclude focal invasive adenocarcinoma, which may affect patient management.

In conclusion, we found that HAMNs are rare tumors that often have architectural features of LAMNs, but have high-grade cytologic and, often, architectural features. HAMNs may be diffusely high-grade or show a mixture of low- and high-grade areas, and often show reduced mucin in the neoplastic epithelial cells as compared to LAMN. Based on our data, HAMNs and LAMNs exhibit similar biologic behavior, with a comparable low risk of recurrence if the tumor is confined to the appendix without perforation of the serosa. Tumors with serosal perforation, particularly those with cellular serosal mucin deposits, carry a risk of progressing to PMP. When they spread to the peritoneum, they often produce grade 2 PMP, which may result in a more aggressive course than classic grade 1 PMP. As with LAMN, most are associated with KRAS mutations and a subset have TP53 mutations. From our data, we recommend that the AJCC staging system developed for LAMN should also be applied to HAMN.